Molybdoenzymes: The Role of Electrons, Protons, and Dihydrogen

20 Molybdoenzymes: The Role of Electrons, Protons, and Dihydrogen

E D W A R D I. S T I E F E L , W I L L I A M E . N E W T O N , G E R A L D K. L A M O N T H A D F I E L D , and W I L L I A M A. B U L E N

D. W A T T ,

Bioinorganic Chemistry—II Downloaded from pubs.acs.org by UNIV LAVAL on 04/09/16. For personal use only.

1

Charles F . Kettering Research Laboratory, Yellow Springs, Ohio 45387

The biochemistry of molybdenum enzymes and the coordi nation chemistry of molybdenum are each discussed as background for understanding the role of molybdenum in enzymes. Electron transfer pathways and spectroscopic data implicate the molybdenum site in substrate reactions. For xanthine oxidase there is evidence for involvement of proton transfer in substrate oxidation. For nitrogenase, data on dihydrogen inhibition of nitrogen fixation and HD formation (under dideuterium and dinitrogen) can be inter preted in terms of a bound diimide-level intermediate. Sev eral mechanistic schemes are possible for ATP utilization, dihydrogen evolution, and substrate reduction by nitrogen ase. For other molybdoenzymes, oxo transfer and coupled proton-electron transfer processes are alternative mechanis tic possibilities. Molybdenum may be uniquely suited for its biochemical role.

Molybdenum

is t h e o n l y element of t h e s e c o n d t r a n s i t i o n r o w k n o w n

to h a v e a n a t u r a l b i o l o g i c a l f u n c t i o n . I t is also c o n s i d e r a b l y less a b u n d a n t i n t h e earth's crust t h a n t h e first t r a n s i t i o n - r o w elements w h i c h p l a y k e y b i o l o g i c a l r o l e s — i r o n , cobalt, c o p p e r , a n d m a n g a n e s e .

The

r e l a t i v e s c a r c i t y of m o l y b d e n u m , c o u p l e d w i t h t h e great i m p o r t a n c e of the b i o l o g i c a l processes f o r w h i c h i t is essential, has l e d to c o n s i d e r a t i o n of t h e p o t e n t i a l i n s i g h t w h i c h this m a y g i v e c o n c e r n i n g t h e o r i g i n of l i f e o n e a r t h . I n p a r t i c u l a r , C r i c k a n d O r g e l ( 1 ) h a v e s u g g e s t e d that t h e u s e of m o l y b d e n u m b y terrestrial m i c r o o r g a n i s m s m a y ( w e a k l y ) s u p p o r t a 1

Deceased 353

354

BIOINORGANIC C H E M I S T R Y

d i r e c t e d p a n s p e r m i a h y p o t h e s i s f o r t h e o r i g i n of l i f e o n e a r t h .

II

This

h y p o t h e s i s c l a i m s that l i f e d i d n o t s p o n t a n e o u s l y o r i g i n a t e o n e a r t h b u t r a t h e r was sent to e a r t h f r o m s o m e w h e r e else i n the u n i v e r s e . C r i c k a n d O r g e l r e a s o n e d that i f l i f e o r i g i n a t e d o n e a r t h , i t is u n l i k e l y t h a t a n elem e n t as rare as m o l y b d e n u m w o u l d h a v e b e e n c h o s e n f o r s u c h a n i m p o r tant task as n i t r o g e n

fixation.

O n the other h a n d , i f l i f e o r i g i n a t e d else-

w h e r e , w h e r e m o l y b d e n u m w a s a b u n d a n t , t h e n the use of this e l e m e n t w o u l d not b e at a l l u n u s u a l . Bioinorganic Chemistry—II Downloaded from pubs.acs.org by UNIV LAVAL on 04/09/16. For personal use only.

These interesting arguments can be faulted on two grounds.

First,

w h i l e m o l y b d e n u m is i n d e e d r e l a t i v e l y r a r e i n the earth's c r u s t or i n the e a r t h as a w h o l e , this is not the case i n seawater (2,3,4). ing

to

recent

estimates, the

c o m p a r a b l e w i t h (2,3)

concentration

of

I n fact, a c c o r d -

molybdenum

is e i t h e r

o r p e r h a p s s l i g h t l y exceeds ( 4 ) that of m a n g a n e s e ,

i r o n , a n d c o p p e r . W h i l e this s i t u a t i o n m a y result f r o m the p r e s e n c e of l i f e a n d / o r f r o m a n o x i d i z i n g a t m o s p h e r e of m o r e recent o r i g i n ( 5 ) , at the v e r y least i t opens the p o s s i b i l i t y t h a t m o l y b d e n u m m a y h a v e b e e n r e a s o n a b l y a b u n d a n t i n the a n c i e n t waters w h e r e l i f e s u p p o s e d l y arose. A s e c o n d a r g u m e n t against the extraterrestrial o r i g i n of l i f e w o u l d b e v a l i d if m o l y b d e n u m w e r e the o n l y a v a i l a b l e m e t a l w h i c h , w h e n i n c o r p o r a t e d i n t o a p r o t e i n s y s t e m , c o u l d c a t a l y z e c e r t a i n reactions. If this w e r e the case, t h e n e v e n i f m o l y b d e n u m w e r e r e l a t i v e l y r a r e , i t w o u l d b e w o r t h t h e effort for the m i c r o o r g a n i s m s to extract i t f r o m the e n v i r o n m e n t .

The

o r g a n i s m s w h i c h l e a r n e d h o w to use m o l y b d e n u m ( f o r e x a m p l e , to fix n i t r o g e n ) w o u l d t h e n h a v e a n e v o l u t i o n a r y a d v a n t a g e o v e r organisms w h i c h d i d not. T h e selective s u r v i v a l of the m o l y b d e n u m - u s i n g ( n i t r o g e n fixing)

species w o u l d ensure the c o n t i n u e d use of m o l y b d e n u m b y f u t u r e

generations. T h e q u e s t i o n t h e n arises as to w h a t c h e m i c a l features of m o l y b d e n u m m a k e i t u n i q u e l y s u i t a b l e for t h e b i o l o g i c a l reactions i n w h i c h i t p a r t i c i pates. I n this c h a p t e r w e first discuss s o m e of the i n f o r m a t i o n g a i n e d f r o m b i o l o g i c a l studies of m o l y b d e n u m e n z y m e s p a y i n g p a r t i c u l a r a t t e n t i o n to n i t r o g e n a s e a n d to x a n t h i n e oxidase. F o r nitrogenase, w e focus o n t h e relationship between dihydrogen, dinitrogen, a n d the enzyme w h e r e there is e v i d e n c e for s e q u e n t i a l t w o - e l e c t r o n - t w o - p r o t o n processes i n the p r o d u c t i o n of a m m o n i a f r o m d i n i t r o g e n .

F o r x a n t h i n e oxidase, w e

sum-

m a r i z e the d a t a w h i c h i m p l i c a t e p r o t o n transfer ( i n a d d i t i o n to e l e c t r o n t r a n s f e r ) as a feature of the m o l y b d e n u m site. S o m e of o u r recent results a n d some trends i n m o l y b d e n u m c o o r d i n a t i o n c h e m i s t r y h e l p to d e t e r m i n e r e a s o n a b l e p o s s i b i l i t i e s for the a c t i o n of m o l y b d e n u m i n these systems.

F i n a l l y , some of t h e m e c h a n i s t i c p r o p o s a l s are c o n s i d e r e d

and

e v a l u a t e d i n terms of the most recent results f r o m b i o l o g i c a l a n d i n o r g a n i c systems.

20.

STIEFEL E T A L .

Molybdoenzymes:

355

Molybdoenzymes

Occurrence

and Biological

Importance

N i t r o g e n a s e is t h e e n z y m e w h i c h catalyzes t h e r e d u c -

Nitrogenase.

t i o n of d i n i t r o g e n to a m m o n i a . T h e process of b i o l o g i c a l n i t r o g e n

fixation

is r e s p o n s i b l e f o r m o s t of t h e fixed n i t r o g e n i n p u t i n t o t h e b i o s p h e r e , o u t w e i g h i n g t h e e n o r m o u s a m o u n t of d i n i t r o g e n that is fixed ( c o n v e r t e d to a m m o n i a ) b y t h e H a b e r process (6).

T h e contrast b e t w e e n t h e H a b e r

process a n d t h e b i o l o g i c a l process is s t a r t l i n g . T h e f o r m e r r e q u i r e s h i g h


t e m p e r a t u r e , h i g h pressure,

a n d dihydrogen

as a feedstock f o r b o t h

energy a n d r e d u c i n g p o w e r ( 7 ) . T h e latter process w o r k s ( o f t e n i n a i r i n vitro) under ambient conditions a t m of d i n i t r o g e n )

(i.e., r o o m t e m p e r a t u r e a n d at 0.8

a n d uses ( u l t i m a t e l y ) solar energy as i n p u t e i t h e r

d i r e c t l y , o r i n d i r e c t l y t h r o u g h t h e c a r b o h y d r a t e s w h i c h are p r o d u c e d b y photosynthesis.

T h u s , t h e o v e r a l l b i o l o g i c a l process m a k e s u s e of r e a d i l y

a v a i l a b l e a i r , w a t e r , a n d s u n l i g h t to effect t h e d e s i r e d t r a n s f o r m a t i o n . T h e a b i l i t y to fix n i t r o g e n is e x c l u s i v e l y a p r o p e r t y o f p r o k a r y o t i c organisms—bacteria

a n d blue-green

algae

(8, 9 ) .

T h e nitrogen-fixing

b a c t e r i a i n c l u d e b o t h f r e e - l i v i n g species a n d those w h i c h l i v e s y m b i otically w i t h higher plants. strict aerobes

(such

( s u c h as Klebsiella

pneumonae),

vinelandii),

f a c u l t a t i v e anaerobes

a n d strict anaerobes ( s u c h as Clostri-

A d d i t i o n a l l y , some p h o t o s y n t h e t i c b a t c t e r i a (e.g.,

dium pasteurianum). Rhodospirullum

A m o n g t h e free l i v i n g species there are

as Azotobacter

rubrum

a n d Chromatium)

are k n o w n to fix n i t r o g e n .

T h e most p r o m i n e n t of t h e s y m b i o t i c fixing species are m e m b e r s of t h e genus Rhizobium w h i c h fix n i t r o g e n w h e n l i v i n g as b a c t e r o i d s i n t h e r o o t n o d u l e s of l e g u m i n o u s p l a n t s ( s o y b e a n s , peas, a l f a l f a ) . R e c e n t l y , some species of r h i z o b i a h a v e b e e n i n d u c e d to fix n i t r o g e n i n a f r e e - l i v i n g state (10-15),

s h o w i n g c o n c l u s i v e l y that t h e genes f o r n i t r o g e n

fixation

are

i n t h e m i c r o o r g a n i s m s a n d n o t i n t h e p l a n t . T h e r a t h e r stringent c o n d i tions r e q u i r e d to observe t h e n i t r o g e n fixation b y f r e e - l i v i n g r h i z o b i a i n c u l t u r e suggests that a d d i t i o n a l organisms m a y b e f o u n d to fix n i t r o g e n under more precisely controlled conditions. nitrogen filamentous

fixation

A m o n g t h e b l u e - g r e e n algae,

occurs i n both unicellular (such

( s u c h as Anabaena)

both photosynthetic

as Gleocapsa)

and

species ( 9 ) . T h e b l u e - g r e e n algae a r e

a n d n i t r o g e n - f i x i n g a n d are thus r e m a r k a b l y

self-

sufficient organisms. Nitrogenase

has b e e n successfully

isolated from

several bacterial

species (8) a n d seems to b e r e m a r k a b l y s i m i l a r i n a l l cases. T o date, n o p u r e p r e p a r a t i o n s of nitrogenase

have been obtained from

blue-green

algae ( 9 ) . Nitrate Reductase.

N i t r a t e r e d u c t a s e is f o u n d w i d e l y d i s t r i b u t e d

a m o n g p l a n t s a n d m i c r o o r g a n i s m s a n d catalyzes t h e r e d u c t i o n of N 0 " 3

to N 0 " (16,17,18,19). 2

T h e p h y s i o l o g i c a l r o l e of this e n z y m e d e p e n d s

356


o n the o r g a n i s m .

II

O f t e n t h e e n z y m e n i t r i t e reductase, w h i c h catalyzes

the r e d u c t i o n of N 0 " to N H , is f o u n d i n a d d i t i o n to the n i t r a t e r e d u c t a s e . 2

3

I n this case, n i t r a t e reductase p l a y s a n a s s i m i l a t o r y r o l e , b e i n g r e s p o n s i b l e for the first step i n the c o n v e r s i o n of N 0 " to N H . I n other o r g a n i s m s , 3

3

the n i t r a t e serves as the t e r m i n a l e l e c t r o n a c c e p t o r i n a n e l e c t r o n t r a n s p o r t system, s i m i l a r to 0

2

or S 0 ~ , a n d the n i t r a t e reductase p l a y s a r e s p i r a t o r y 4

2

or d i s s i m i l a t o r y r o l e . U n l i k e t h e nitrogenases, w h i c h d o n o t v a r y m u c h i n c o m p o s i t i o n


a n d p h y s i c a l p r o p e r t i e s w i t h the source, n i t r a t e reductases

vary

con-

s i d e r a b l y f r o m one o r g a n i s m to the next. T h e o n l y feature w h i c h t h e y a l l seem to h a v e i n c o m m o n is a n absolute r e q u i r e m e n t f o r the p r e s e n c e of molybdenum. Xanthine Dehydrogenase.

T h i s e n z y m e catalyzes the o x i d a t i o n of

x a n t h i n e to u r i c a c i d a n d is f o u n d i n v a r i o u s m i c r o o r g a n i s m s ( i n c l u d i n g b a c t e r i a a n d f u n g i ) a n d a n i m a l s ( i n c l u d i n g insects, fish, b i r d s , a n d m a m m a l s ) (19, 20,21).

I n some b a c t e r i a a n d f u n g i , x a n t h i n e c a n serve as t h e

sole n i t r o g e n source. C o u p l i n g this fact w i t h t h e presence of m o l y b d e n u m i n nitrogenase a n d n i t r a t e r e d u c t a s e , w e find that i n e a c h case a m o l y b denum enzyme

p l a y s a r o l e i n n i t r o g e n a s s i m i l a t i o n a n d i s , i n fact,

r e s p o n s i b l e for t h e first step i n this process.

M o l y b d e n u m appears

p l a y a role i n the m e t a b o l i s m of n i t r o g e n s i m i l a r to t h a t p l a y e d by

to first

t r a n s i t i o n - r o w elements ( i r o n , m a n g a n e s e , a n d c o p p e r ) i n the m e t a b o l i s m of o x y g e n . T h i s e n z y m e is v e r y closely r e l a t e d to t h e x a n -

Xanthine Oxidase.

t h i n e d e h y d r o g e n a s e systems (19, 20, 21),

a n d i n some cases, t h e oxidase

a n d d e h y d r o g e n a s e are i n t e r c o n v e r t i b l e forms of the same e n z y m e

(22).

X a n t h i n e oxidase is f o u n d i n a v a r i e t y of m a m m a l i a n systems i n c l u d i n g m a n . I n most organisms, the o x i d a t i o n of x a n t h i n e to u r i c a c i d is f o l l o w e d b y f u r t h e r d e g r a d a t i o n of the u r i c a c i d . H o w e v e r , i n m a n a n d some other p r i m a t e s , u r i c a c i d is the t e r m i n a l species i n p u r i n e c a t a b o l i s m a n d is e x c r e t e d t h r o u g h the k i d n e y s . E x c e s s a c c u m u l a t i o n of u r i c a c i d leads to t h e s y n d r o m e c a l l e d gout. N o w a d a y s , gout is often t r e a t e d w i t h i n h i b i t o r s of x a n t h i n e oxidase, a n d the n a t u r e of these i n h i b i t o r s a n d t h e i r r e a c t i o n w i t h x a n t h i n e oxidase e n z y m e (23,

has g i v e n i n s i g h t i n t o t h e f u n c t i o n i n g of

the

24).

Aldehyde Oxidase.

T h i s e n z y m e is u s u a l l y f o u n d i n s i m i l a r l o c a -

tions to x a n t h i n e oxidase or d e h y d r o g e n a s e insects, b i r d s , a n d m a m m a l s (20, 21).

a n d has b e e n i s o l a t e d f r o m

A l d e h y d e oxidase seems to b e a

p o o r c h o i c e of n a m e for this e n z y m e because, w h i l e it o x i d i z e s a l d e h y d e s to c a r b o x y l i c a c i d s , i t also accepts a v a r i e t y of p u r i n e s a n d p y r i m i d i n e s as o x i d i z a b l e substrates.

F o r e x a m p l e , a l d e h y d e oxidase catalyzes the

c o n v e r s i o n of 2 - h y d r o x y p y r i m i d i n e to u r a c i l a n d of a d e n i n e to 8 - h y d r o x y a d e n i n e (25).

It appears that x a n t h i n e oxidase a n d a l d e h y d e oxidase are

20.

STiEFEL

357

Molybdoenzymes

ETAL.

a set o f p u r i n e a n d p y r i m i d i n e h y d r o x y l a s e s w i t h a r a t h e r b r o a d r a n g e of substrate specificity. Sulfite Oxidase.

This enzyme, isolated from bovine

( 2 6 , 27) a n d

c h i c k e n fiver (28), catalyzes t h e o x i d a t i o n o f sulfite t o sulfate. T h i s i s p o s s i b l y a c r u c i a l f u n c t i o n i n a n i m a l s as S 0 ~ ( o r S 0 , i t s gaseous p r e 3

c u r s o r ) i s toxic w h i l e S 0

4

2

2

2

" is r e l a t i v e l y i n n o c u o u s . F o r e x a m p l e , o n e o f

t h e first signs o f m o l y b d e n u m d e f i c i e n c y i n rats is a g r e a t l y i n c r e a s e d susceptibility to S 0 poisoning ( 2 8 ) . I n addition, a h u m a n child b o r n 2


w i t h o u t sulfite oxidase a c t i v i t y d i d n o t s u r v i v e f o r v e r y l o n g ( 2 9 ) . O t h e r E n z y m e s . M o l y b d e n u m has b e e n suggested as a c o m p o n e n t of a n e n z y m e possessing C 0

2

reductase or formate dehydrogenase activity

I n t h e latter case, t h e u n i q u e o b s e r v a t i o n h a s b e e n

(30,31,32).

made

t h a t t u n g s t e n c a n substitute f o r m o l y b d e n u m w h i l e m a i n t a i n i n g a c t i v i t y (33).

This enzyme

NADPH

is also p o s t u l a t e d t o c o n t a i n s e l e n i u m (32). A

dehydrogenase

from

a mitochondrial fraction m a y contain

m o l y b d e n u m b a s e d u p o n t h e o b s e r v a t i o n of a M o ( V ) E P R s i g n a l (34). A u t h e n t i c a t i o n o f these

findings

m a y l e n g t h e n t h e list o f m o l y b d e n u m

enzymes. The Molybdenum Cofactor.

B a s e d o n genetic evidence, C o v e a n d

P a t e m a n ( 3 5 ) suggested t h a t x a n t h i n e d e h y d r o g e n a s e a n d n i t r a t e r e d u c -

Cyt+

Cyt*

3

2

NADPH

FAD-*-Cyt -*b

NADP

"Mo

cofactor"

Figure 1. Nitrate reductase from Neurospora crassa —composition and presumed electron transfer sequence (16, 18) tase o f t h e f u n g u s Aspergillus

nidulans h a v e a c o m m o n

molybdenum-

c o n t a i n i n g u n i t . T h e w o r k of N a s o n a n d c o - w o r k e r s w i t h t h e nit-1 m u t a n t of Neurospora

crassa also p o i n t s t o a " m o l y b d e n u m c o f a c t o r " c o m m o n t o

a l l m o l y b d e n u m - c o n t a i n i n g e n z y m e s (36, 37, 38). T h e Neurospora n i t r a t e r e d u c t a s e , as s h o w n s c h e m a t i c a l l y i n F i g u r e 1, catalyzes t h e r e d u c t i o n of N 0 " b y N A D P H 3

a n d contains F A D a n d a b - t y p e c y t o c h r o m e i n

a d d i t i o n t o m o l y b d e n u m . T h e f u l l e n z y m e also has N A D P H : c y t o c h r o m e c r e d u c t a s e a c t i v i t y . T h e nit-1 m u t a n t p r o d u c e s a n e n z y m e t h a t c a n n o t

358


reduce activity.

N 0 " b u t s t i l l possesses the N A D P H : c y t o c h r o m e c 3

II

reductase

S i g n i f i c a n t l y , the f u l l e n z y m e a c t i v i t y t o w a r d s n i t r a t e (as w e l l

as other properties of the e n z y m e ) c a n b e r e s t o r e d b y t r e a t m e n t of nit-1 extracts w i t h the n e u t r a l i z e d , a c i d - h y d r o l y s i s p r o d u c t of a n y of the a b o v e m e n t i o n e d m o l y b d e n u m e n z y m e s . T h e s e e n z y m e s donate a m o l y b d e n u m c o n t a i n i n g g r o u p (38)

w h i c h leads to the i n v i t r o a s s e m b l y of the i n t a c t

a n d a c t i v e n i t r a t e reductase. S i m p l e m o l y b d e n u m complexes are u n a b l e to activate the nit-1 extracts n o r are n e u t r a l i z e d a c i d - h y d r o l y s i s p r o d u c t s


of n o n - m o l y b d e n u m e n z y m e s . T h e d o n a t e d m o l y b d e n u m - c o n t a i n i n g f a c tor c a n arise f r o m e n z y m e s i s o l a t e d f r o m m a m m a l s o r b a c t e r i a . I n studies u s i n g b a c t e r i a l extracts, the f a c t o r w a s

d i a l y z a b l e (39)

p r e s u m e d to b e a l o w m o l e c u l a r w e i g h t m o l y b d e n u m

a n d is t h u s

compound.

R u s s i a n w o r k e r s c l a i m to h a v e i s o l a t e d l o w m o l e c u l a r w e i g h t m o l y b d e n u m - c o n t a i n i n g p e p t i d e s f r o m nitrogenase a n d x a n t h i n e oxidase w h i c h are a c t i v e i n the r e c o n s t i t u t i o n of the nit-1 m u t a n t (40, 41).

Zumft

(42)

also c l a i m s to h a v e s e p a r a t e d t w o l o w m o l e c u l a r w e i g h t m o l y b d e n u m c o n t a i n i n g fractions f r o m nitrogenase w h i c h also s h o w r e c o n s t i t u t i n g activity.

U n f o r t u n a t e l y , at present, the d e t a i l e d n a t u r e of the m o l y b -

d e n u m - c o n t a i n i n g fractions is v i r t u a l l y u n k n o w n , a l t h o u g h t h e R u s s i a n w o r k e r s c l a i m t h a t i t is ( a t least p r e d o m i n a n t l y ) a s m a l l p e p t i d e . theless, the m e r e existence of a c o m m o n cofactor i n d i c a t e s a s t r u c t u r a l feature i n a l l m o l y b d o e n z y m e s

None-

common

a n d opens the p o s s i b i l i t y t h a t

t h e m o l y b d e n u m sites i n t h e v a r i o u s e n z y m e s operate i n a m e c h a n i s t i c a l l y similar manner. R e c e n t l y , B r i l l a n d c o - w o r k e r s (43,44) h a v e i s o l a t e d m u t a n t strains of Azotobacter ponent.

vinelandii

This component

w h i c h produce a n inactive nitrogenase can be

reactivated b y

treatment w i t h

comthe

n e u t r a l i z e d a c i d - h y d r o l y s i s p r o d u c t s of other nitrogenases ( w h i c h t h e m selves b e c o m e i n a c t i v e o n s u c h a t r e a t m e n t ) b u t n o t a p p a r e n t l y w i t h any

other m o l y b d e n u m e n z y m e s .

T h i s m a y e i t h e r reflect a

difference

b e t w e e n the c o f a c t o r i n nitrogenase a n d other m o l y b d e n u m e n z y m e s or m a y b e c a u s e d b y the r e c o n s t i t u t i o n c o n d i t i o n s u s e d w h i c h m a y n o t h a v e been

sufficiently v a r i e d to a l l o w f o r different m o l y b d e n u m

oxidation

states to b e a t t a i n e d . I n a n y event, the c h e m i c a l c h a r a c t e r i z a t i o n a n d a u t h e n t i c a t i o n of t h e m o l y b d e n u m c o f a c t o r s h o u l d r e v e a l s o m e of the i n t i m a t e details of the m o l y b d e n u m site. Biochemistry

of

Nitrogenase

General Considerations.

T h e nitrogenase enzyme

consists of

separately isolable proteins—the m o l y b d e n u m - i r o n protein I, F r a c t i o n I, molybdoferredoxin)

two

(Component

a n d the i r o n p r o t e i n ( C o m p o n e n t I I ,

F r a c t i o n I I , a z o f e r r e d o x i n ). T h e m o s t recent w o r k o n nitrogenase c o m -

20.

STiEFEL

359

Molybdoenzymes

ET AL.

ponents f r o m a v a r i e t y of organisms indicates great s i m i l a r i t y i n m o l e c u l a r w e i g h t , n u m b e r of s u b u n i t s , a n d m o l y b d e n u m , i r o n a n d i n o r g a n i c sulfide contents.

F o r Azotobacter vinelandii, the o r g a n i s m f o r w h i c h w e present

o u r e x p e r i m e n t a l results, the m o l y b d e n u m - i r o n p r o t e i n has a m o l e c u l a r w e i g h t of 226,000, f o u r s u b u n i t s , t w o m o l y b d e n u m , r o u g h l y 24 i r o n , a n d 22 l a b i l e sulfide ions p e r m o l e c u l e ( 4 5 ) . T h e i r o n p r o t e i n has a m o l e c u l a r w e i g h t of a r o u n d 65,000 w i t h f o u r i r o n a n d f o u r l a b i l e sulfide ions p e r m o l e . W i t h Azotobacter vinehndii

( a n d to date o n l y for this o r g a n i s m ) ,


it is possible to isolate a 1:1 c o m p l e x of the i r o n a n d m o l y b d e n u m - i r o n p r o t e i n s b y a v o i d i n g t h e use of D E A E c e l l u l o s e d u r i n g t h e p r e p a r a t i o n . T h i s nitrogenase

complex

studies d i s c u s s e d b e l o w .

has b e e n

e x c l u s i v e l y u s e d f o r the r e a c t i o n

Its p r e p a r a t i o n has b e e n d e s c r i b e d i n d e t a i l

elsewhere (46, 4 7 ) . A n i n p u t - o u t p u t scheme f o r nitrogenase is s h o w n i n F i g u r e 2.

The

m a t e r i a l i n the b o x represents the c a t a l y t i c e n t i t i e s — t h e i r o n p r o t e i n , the m o l y b d e n u m - i r o n p r o t e i n , a n d M g

Figure 2.

2 +

ions. I n p u t consists of a r e d u c -

Nitrogenase—input-output diagram (7, 8, 9, 52)

i n g agent, A T P , a n d a source of protons ( H 0 ) . 2

b e f e r r e d o x i n or nite ( S 0 2

4

2

flavodoxin

T h e r e d u c i n g agent c a n

i n v i v o , b u t i n assay systems i n v i t r o , d i t h i o -

" ) i n v a r i a b l y serves this f u n c t i o n , b e i n g o x i d i z e d i n the process

b y t w o electrons to S 0 " . 3

2

T h e A T P is h y d r o l y z e d d u r i n g nitrogenase

t u r n o v e r to A D P a n d P j . U n d e r o p t i m a l ( i n v i t r o ) c o n d i t i o n s , 4 - 5

moles

of A T P are h y d r o l y z e d p e r m o l e of e l e c t r o n p a i r s p a s s i n g t h r o u g h t h e enzyme

(48).

A n u n u s u a l feature of nitrogenase w h i c h c o n t r i b u t e s to

t h e d i f f i c u l t y i n its s t u d y is t h e f a c t that i t does not r e q u i r e a r e d u c i b l e substrate. I n the absence of r e d u c i b l e substrate ( v i d e i n f r a ) , t h e e n z y m e system turns over a n d evolves d i h y d r o g e n v i a t h e s o - c a l l e d " A T P - d e p e n d ent h y d r o g e n

e v o l u t i o n " r e a c t i o n w h i c h r e q u i r e s the same i n p u t s as

n i t r o g e n fixation ( 4 9 ) .

T h u s , nitrogenase is n o t e a s i l y s t u d i e d i n a f u l l y

360


II

r e d u c e d state b e c a u s e this state w i l l g i v e r i s e t o d i h y d r o g e n e v o l u t i o n . P e r h a p s i n t h e f u t u r e , r a p i d d e t e c t i o n t e c h n i q u e s w i l l a l l o w some g l i m p s e of this k e y state. T h e o u t p u t for nitrogenase consists o f d i h y d r o g e n a n d ( as a p p r o p r i a t e ) r e d u c e d substrate. T h e presence o f r e d u c i b l e substrates c u r t a i l s the d i h y d r o g e n e v o l u t i o n r e a c t i o n ( a l t h o u g h often not c o m p l e t e l y )

(50,51,

52, 53 ) a n d d i v e r t s electrons f r o m d i h y d r o g e n p r o d u c t i o n t o substrate reduction.

T h e n a t u r a l substrate i s d i n i t r o g e n w h i c h undergoes

a six-


e l e c t r o n r e d u c t i o n t o a m m o n i a . O t h e r substrates h a v e also b e e n f o u n d , a n d t h e i r reactions are l i s t e d i n T a b l e I . T h e r e d u c i b l e substrates (52, 53)

i n c l u d e m o l e c u l e s w i t h t r i p l e b o n d s (acetylenes, n i t r i l e s , i s o n i t r i l e s ,

and

cyanide)

or reactive double

nitrous oxide, aliène).

( o r potentially triple) bonds

(azide,

S i g n i f i c a n t l y , t h e acetylenes are r e d u c e d b y a

t w o - e l e c t r o n process t o ethylenes ( w i t h n o trace o f e t h a n e ) .

If D 0 2

replaces H 0 as the p r o t o n source, t h e n d i d e u t e r i u m is e v o l v e d , N D is 2

3

f o r m e d f r o m d i n i t r o g e n , a n d acetylene dideuteroethylene

is r e d u c e d

e x c l u s i v e l y t o cis-

(54).

Table I.

Substrate N

2

N H 2

4

Half-Reactions

+6H

+

+ 6e--*2NH

3

+2H

+

+ 2e"->2NH

3

H C N + 6 H + 6e"

CH + N H

+

N 0

+2H

+

HN

+2H

+

2

3

RNC

f o r Nitrogenase

4

3

+ 2e--*N + H 0 2

+ 2e--+N

+ 6 H + 6e"

2

+ N H

2

3

RNH

2

+ CH

4

R C N + 6 H + 6e" -> R C H

3

+ N H

3

+

+

C H 2

2

+2H 2H

+

+

+

2e-^C H

+ 2e - ^ H

2

Mg ->

4

2

2 +

[ATP + H 0 2

A D P + Pi] 2nd International Conference on Chemistry and Uses of Molybdenum

Dihydrogen Reactions of Nitrogenase.

O n e o f the p u z z l e s w h i c h

n i t r o g e n a s e has p r e s e n t e d lies i n its reactions i n v o l v i n g d i h y d r o g e n . I n e a r l y studies, d i h y d r o g e n w a s c o n s i d e r e d a r e d u c i n g agent for d i n i t r o g e n , a n d t h e nitrogenase e n z y m e w a s t h o u g h t t o c a t a l y z e the H a b e r process r e a c t i o n ( R e a c t i o n 1 ) . F o r e x a m p l e , c r u d e cell-free extracts o f 3H

2

+ N

2

-» 2NH

3

Clostri(1)

20.

STIEFEL E T A L .

361

Molybdoenzymes

c o u l d i n d e e d use d i h y d r o g e n as t h e r e d u c t a n t f o r

dium pasteurianum

d i n i t r o g e n ( 5 5 ) . H o w e v e r , i t is n o w clear that this process d e p e n d s u p o n the presence of t h e e n z y m e h y d r o g e n a s e H

2

+ 2Fd

o x

->2H

+

( 5 6 ) w h i c h catalyzes R e a c t i o n + 2Fd

(2)

r e d

2. H e r e , d i h y d r o g e n reduces o x i d i z e d f e r r e d o x i n ( F d ) o x

to F d

r e d

which

c a n t h e n serve as t h e e l e c t r o n d o n o r f o r t h e n i t r o g e n a s e - c a t a l y z e d r e d u c -


t i o n of d i n i t r o g e n to a m m o n i a ( F i g u r e 2 ). T h u s , R e a c t i o n 1 is c a t a l y z e d only w h e n hydrogenase,

nitrogenase, a n d f e r r e d o x i n a r e present a n d

r e q u i r e s A T P h y d r o l y s i s as a c o - r e a c t i o n . W h e n p u r i f i e d nitrogenase is i n c u b a t e d u n d e r d i n i t r o g e n a n d d i hydrogen, the dihydrogen surprisingly inhibits nitrogen

fixation

( 57, 58,

59, 60 ). K i n e t i c studies seem to d i s p l a y a c o m p e t i t i v e i n h i b i t i o n p a t t e r n , a l t h o u g h m o r e d e t a i l e d studies i n progress i n o u r l a b o r a t o r y i n d i c a t e that t r u e c o m p e t i t i v e i n h i b i t i o n is n o t present h e r e (57, 58, 59, 60). the appearance

of H D i n t h e gas

phase w h e n nitrogenase t u r n s over i n t h e presence

A r e l a t e d o b s e r v a t i o n concerns

of d i n i t r o g e n a n d

dideuterium i n H

2

0 (58, 59, 60). L i k e w i s e , nitrogenase t u r n o v e r i n D

2

0

w i t h d i h y d r o g e n a n d d i n i t r o g e n i n t h e gas phase causes H D f o r m a t i o n . T h i s process has u n i f o r m l y b e e n c a l l e d " H D e x c h a n g e " a l t h o u g h there is n o e v i d e n c e f o r t h e f o r m a t i o n of H D O i n t h e aqueous phase ( 5 8 , 5 9 , 61 ). T h e f o r m a t i o n of H D a b s o l u t e l y d e p e n d s o n the presence of d i n i t r o gen, w i t h l o w levels of d i n i t r o g e n sufficing to g i v e m o d e r a t e amounts of H D . I t w o u l d a p p e a r f r o m the k i n e t i c d a t a (58, 59, 60) that d i n i t r o g e n i s , i n a sense, a catalyst f o r t h e H D f o r m a t i o n r e a c t i o n . N o o t h e r substrate leads to H D f o r m a t i o n n o r does H D f o r m a t i o n o c c u r i n t h e absence of r e d u c i b l e substrates. Electron Balance Studies on the Dihydrogen Reactions of N i t r o genase. A c o n t i n u i n g project at t h e K e t t e r i n g L a b o r a t o r y is t h e d e t a i l e d analysis of t h e i n p u t s a n d t h e outputs f o r nitrogenase.

A remarkable

o b s e r v a t i o n a b o u t nitrogenase is that its t u r n o v e r rate is i n d e p e n d e n t of the d e t a i l e d n a t u r e of t h e o u t p u t of t h e e n z y m e

( 5 0 ) . A s discussed

a b o v e , electrons m o v i n g t h r o u g h nitrogenase c a n cause d i h y d r o g e n e v o l u tion,

acetylene

reduction, nitrogen

fixation,

or, d e p e n d i n g

upon the

c o n d i t i o n s , v a r i o u s c o m b i n a t i o n s of these a c t i v i t i e s . H o w e v e r , t h e u t i l i z a t i o n rate of S 0 ~ ( r e d u c t a n t ) a n d t h e h y d r o l y s i s rate of A T P a r e e a c h 2

4

2

t o t a l l y i n d e p e n d e n t of t h e d i s t r i b u t i o n of electrons i n these

products.

F u r t h e r m o r e , e v e n i n a d i h y d r o g e n - i n h i b i t e d n i t r o g e n - f i x i n g system, t h e t u r n o v e r rate ( a s m e a s u r e d b y S 0 " o r A T P u t i l i z a t i o n ) is unaffected. 2

4

2

T h e s e d a t a s t r o n g l y suggest t h a t the r a t e - d e t e r m i n i n g step f o r nitrogenase t u r n o v e r occurs p r i o r to substrate r e d u c t i o n . D i h y d r o g e n i n h i b i t i o n t h e r e fore affects t h e d i s t r i b u t i o n of p r o d u c t s b u t n o t t h e t u r n o v e r rate of t h e

362


enzyme.

II

I n order to p r o b e the n a t u r e of the d i h y d r o g e n i n h i b i t i o n r e a c -

t i o n , c a r e f u l e l e c t r o n b a l a n c e studies h a v e b e e n p e r f o r m e d o n the n i t r o genase c o m p l e x

f r o m A.

T h e p r e l i m i n a r y results of

vinefondii.

studies h a v e b e e n b r i e f l y d i s c u s s e d (46, 47, 58, 59),

such

a n d the e x p e r i m e n t a l

details a n d b u l k of the d a t a w i l l a p p e a r elsewhere

(60).

T h e e x p e r i m e n t a l d e s i g n is s i m p l e . A g i v e n s a m p l e of

nitrogenase

e q u i p p e d w i t h a n A T P - g e n e r a t i n g system, M g , a n d r e d u c t a n t is a l l o w e d 2 +

to t u r n over w i t h o u t substrate (case I ) , a n d the d i h y d r o g e n p r o d u c t i o n is


monitored.

T h e a m o u n t of d i h y d r o g e n p r o d u c e d is f o u n d to b e e q u a l

( w i t h i n e x p e r i m e n t a l e r r o r ) to the d i t h i o n i t e o x i d i z e d ( 5 0 ) .

Therefore,

the e l e c t r o n b a l a n c e e q u a t i o n is : 2[H ]=2[S 0 2

2

4

2

"]

(3)

w h e r e the b r a c k e t s enclose the v a r y i n g n u m b e r of moles of i n d i c a t e d p r o d u c t f o r m e d or r e d u c t a n t o x i d i z e d p e r u n i t t i m e . U s i n g the same nitrogenase p r e p a r a t i o n , d i n i t r o g e n is a d d e d to the r e a c t i o n flask, a n d d i h y d r o g e n e v o l u t i o n a n d a m m o n i a p r o d u c t i o n m e a s u r e d i n the same r e a c t i o n vessel. U n d e r these c i r c u m s t a n c e s

are

(case

2 ) , the e l e c t r o n b a l a n c e E q u a t i o n 4 o b t a i n s : (4)

3[NH ] + 2[H ] = 2[S 0 1 3

2

2

4

2

T h i s is i n f u l l agreement w i t h the n e e d for six electrons to r e d u c e

each

d i n i t r o g e n , i.e., three p e r a m m o n i a f o r m e d . I n case 3, a g a i n w i t h the same p r e p a r a t i o n , a g i v e n a m o u n t

of

d i h y d r o g e n is i n t r o d u c e d i n t o the r e a c t i o n flask i n a d d i t i o n to the d i n i t r o g e n . W h e n d i h y d r o g e n is present, the e q u a t i o n for e l e c t r o n b a l a n c e r e m a i n s the same as a b o v e [ E q u a t i o n 4 ] . H o w e v e r , at the same d i n i t r o g e n l e v e l , less a m m o n i a a n d m o r e d i h y d r o g e n are p r o d u c e d p e r u n i t t i m e c o m p a r e d w i t h case 2 above. C a s e 3 is, of course, the d i h y d r o g e n i n h i b i t i o n r e a c t i o n , a n d as expected, it is f o u n d to shift electrons f r o m a m m o n i a to the f o r m a t i o n of d i h y d r o g e n . T h e c l u e to w h a t is h a p p e n i n g comes i n case 4, w h e n the r e a c t i o n is a n a l y z e d at a l e v e l of d i d e u t e r i u m e q u a l to that of d i h y d r o g e n u s e d i n case 3. H e r e , the H D , d i h y d r o g e n , a n d a m m o n i a p r o d u c e d are m e a s u r e d i n the flask. It is f o u n d that t h e a m m o n i a l e v e l i n case 4 is the same as i n case 3. T h u s , as expected, d i d e u t e r i u m a n d d i h y d r o g e n are e q u i v a l e n t i n t h e i r a b i l i t y to i n h i b i t r e d u c t i o n ( a m m o n i a f o r m a t i o n ) .

However,

d i h y d r o g e n p r o d u c e d is f o u n d to b e the same as i n case 2, w h e n

the no

d i h y d r o g e n or d i d e u t e r i u m is present. T h u s , the presence of d i d e u t e r i u m a n d b y i n f e r e n c e d i h y d r o g e n does not effect the A T P - d e p e n d e n t d i h y d r o g e n e v o l u t i o n r e a c t i o n , a n d o n l y d i n i t r o g e n r e d u c t i o n is effected.

How-

20.

STIEFEL

ET AL.

363

Molybdoenzymes

ever, for case 4, w e find t h a t E q u a t i o n 4 does n o t b a l a n c e .

However,

E q u a t i o n 5 does b a l a n c e , m e a n i n g that one e l e c t r o n is r e q u i r e d for t h e 3[NH ] + 2[H ] + 3

2

1[HD] -

2[S 0 "] 2

4

(5)

2

f o r m a t i o n of e a c h m o l e c u l e of H D . O v e r a w i d e r a n g e of d i d e u t e r i u m a n d d i n i t r o g e n pressures, e l e c t r o n b a l a n c e c a n b e a c h i e v e d o n l y b y a d d i n g this t e r m i n H D .


I n s u m m a r y , the k e y e x p e r i m e n t a l findings a r e : 1. N i t r o g e n a s e t u r n o v e r rate ( e l e c t r o n flow) is i n d e p e n d e n t of r e d u c i b l e substrate, e l e c t r o n d i s t r i b u t i o n a m o n g a m i x t u r e of substrates, or the p r e s e n c e of d i h y d r o g e n . 2. O n l y d i n i t r o g e n r e d u c t i o n is i n h i b i t e d b y d i h y d r o g e n or d i d e u terium. 3. I n the presence of d i d e u t e r i u m a n d d i n i t r o g e n , H D is p r o d u c e d i n a r e a c t i o n w h i c h uses one e l e c t r o n to f o r m e a c h H D . 4. D e u t e r i u m does not affect d i h y d r o g e n e v o l u t i o n i n either t h e p r e s e n c e or absence of d i n i t r o g e n . T h e s e results t a k e n together suggest s t r o n g l y that d i h y d r o g e n a n d d i d e u t e r i u m d i v e r t electrons f r o m d i n i t r o g e n r e d u c t i o n , w h i c h i n t h e f o r m e r case leads to d i h y d r o g e n p r o d u c t i o n , b u t i n the latter case leads to H D f o r m a t i o n .

T h u s , i t appears that the d i h y d r o g e n i n h i b i t i o n

of

d i n i t r o g e n r e d u c t i o n a n d the d i n i t r o g e n - d e p e n d e n t H D f o r m a t i o n r e a c tions of nitrogenase are manifestations of the same p h e n o m e n o n . finding

This

is i n t e r p r e t e d o n a m o l e c u l a r l e v e l i n the f o l l o w i n g section.

Intermediates in the Fixation of Dinitrogen.

T h e r e are s t i l l

no

s p e c t r o s c o p i c a l l y d e t e c t e d i n t e r m e d i a t e s i n the r e d u c t i o n of d i n i t r o g e n to ammonia. nitrogenase

W e b e l i e v e , h o w e v e r , that the e l e c t r o n b a l a n c e studies w i t h under

dinitrogen/dihydrogen

and

dinitrogen/dideuterium

atmospheres p o i n t to the existence of s u c h i n t e r m e d i a t e s . T h e s t o i c h i o m e t r y for the H D f o r m a t i o n r e a c t i o n as d e t e r m i n e d e x p e r i m e n t a l l y is s h o w n i n R e a c t i o n 6.

A t first glance, this resembles 2H

+

+ 2e" +

D

2

the substrate

-> 2 H D

( T a b l e I ) , a n d at one t i m e i t w a s t h o u g h t (62)

reactions (6)

that d i d e u t e r i u m w a s i n

fact a nitrogenase substrate. H o w e v e r , the k e y fact r e m a i n s that d i n i t r o g e n , at least i n c a t a l y t i c a m o u n t s , is r e q u i r e d for H D f o r m a t i o n a n d t h a t i n the process, the r e d u c t i o n of d i n i t r o g e n to a m m o n i a is i n h i b i t e d ( 5 7 61, 63).

T o e x p l a i n these features, w e suggest that a t w o - e l e c t r o n r e d u c -

t i o n p r o d u c t of d i n i t r o g e n is r e a c t i v e t o w a r d s d i h y d r o g e n or d i d e u t e r i u m . I n a n a l o g y to the r e d u c t i o n of acetylene i n D 0 to d s - C H D , this p r o d 2

2

2

2

u c t is p o s t u l a t e d to b e a b o u n d c i s - d i i m i d e species ( 4 5 ) . A s s h o w n i n F i g u r e 3, t h e first step i n d i n i t r o g e n r e d u c t i o n c o u l d i n v o l v e b i n d i n g of d i n i t r o g e n to the e n z y m e , w i t h the m o d e of b i n d i n g

364

BIOINORGANIC CHEMISTRY

left u n s p e c i f i e d .

II

I n a n a l o g y to the r e d u c t i o n of acetylene to e t h y l e n e ,

r e d u c t i o n b y t w o electrons a n d t w o protons is p o s t u l a t e d to p r o d u c e bound

d s - d i i m i d e species

w i t h somewhat

exposed

N - H bonds.

a

The

b o u n d d i i m i d e c o u l d t h e n react w i t h d i h y d r o g e n i n a six-center r e a c t i o n to r e f o r m d i n i t r o g e n ( b o u n d or u n b o u n d ) dihydrogen molecule.

a n d generate a n a d d i t i o n a l

T h e r e a c t i o n , as expressed i n R e a c t i o n 7, is effec-

E-N H 2

2

+ H

2

-» Ε + N

2

+ 2H

t i v e l y the d e c o m p o s i t i o n of b o u n d d i i m i d e to its elements. Bioinorganic Chemistry—II Downloaded from pubs.acs.org by UNIV LAVAL on 04/09/16. For personal use only.

(7)

2

T h i s process

is e x o t h e r m i c for free d i i m i d e b y r o u g h l y 35 k c a l / m o l e . T h e r e a c t i o n of dihydrogen with d s - N H 2

is a l l o w e d b y o r b i t a l s y m m e t r y considerations.

2

T h e r e g e n e r a t i o n of d i n i t r o g e n is significant i n t w o respects.

First, it

shows that d i n i t r o g e n is n o t r e d u c e d to a m m o n i a , a n d so a m m o n i a p r o d u c t i o n is i n h i b i t e d .

S e c o n d , i t means that d i n i t r o g e n is effectively

a

catalyst i n the p r o d u c t i o n of d i h y d r o g e n b y a route w h i c h is not i d e n t i c a l to the s i m p l e A T P - d e p e n d e n t d i h y d r o g e n e v o l u t i o n r e a c t i o n .

Figure 3.

Nitrogenase—scheme of H and HD production reactions

inhibition

2

T h e r e a c t i o n w i t h d i d e u t e r i u m is s h o w n i n the l o w e r p a r t of F i g u r e 3. H e r e , the i n h i b i t i o n of n i t r o g e n fixation leads to the p r o d u c t i o n of

HD

a c c o r d i n g to R e a c t i o n s 8 a n d 9. C l e a r l y , the o v e r a l l process agrees p r e E-N

E-N H 2

+ 2e" + 2 H -> E - N H +

2

2

+

D

2

Ε +

2

N

2

2

+ 2HD

c i s e l y w i t h t h e e l e c t r o n b a l a n c e studies w h i c h i m p l i c a t e t w o p e r p a i r of H D m o l e c u l e s f o r m e d .

(8) (9) electrons

T h u s , the m e c h a n i s m i n v o l v e s t h e

20.

STiEFEL E T AL.

365

Molybdoenzymes

d i v e r s i o n of electrons f r o m N H 2

to d i h y d r o g e n or H D i n agreement w i t h

2

experiment. T h e p o s t u l a t i o n of the N H - l e v e l i n t e r m e d i a t e c l e a r l y i m p l i e s that 2

2

nitrogenase f u n c t i o n s i n a stepwise m a n n e r , w i t h h y d r a z i n e

therefore

i m p l i c a t e d as t h e s e c o n d e n z y m e - b o u n d i n t e r m e d i a t e . T h e q u e s t i o n t h e n arises as to w h e t h e r h y d r a z i n e is a r e d u c i b l e substrate f o r nitrogenase. A t p H 7.2-7.4, t h e n o r m a l

Reduction of Hydrazine by Nitrogenase. assay c o n d i t i o n s , nitrogenase


at o n l y a v e r y s l o w rate.

c a n c a t a l y z e t h e r e d u c t i o n of h y d r a z i n e

H o w e v e r , at these p H v a l u e s , h y d r a z i n e is

present l a r g e l y as t h e h y d r a z i n i u m i o n , N H 2

+

5

, a n d i t is possible t h a t this

c a t i o n i c species cannot serve as a substrate. T o test this i d e a , t h e p H of t h e s o l u t i o n was r a i s e d i n steps to p H 8 w h e r e t h e e n z y m e r e m a i n s a c t i v e , and

s u b s t a n t i a l amounts of n e u t r a l N H 2

4

are present.

T h e production

of a m m o n i a closely p a r a l l e l e d t h e increase i n p H . A t p H 8, h y d r a z i n e is r e d u c e d at ~ 2 0 % of t h e rate of d i h y d r o g e n e v o l u t i o n . I n a l l respects, the r e d u c t i o n of h y d r a z i n e b e h a v e s l i k e that of other substrates, w i t h A T P and S 0 2

4

2

" b e i n g r e q u i r e d a n d d i h y d r o g e n e v o l u t i o n b e i n g decreased b y

an a m o u n t c o m m e n s u r a t e w i t h t h e a m o u n t of h y d r a z i n e r e d u c e d ( 4 5 ) . T h u s , h y d r a z i n e is r e d u c i b l e b y nitrogenase, a n d a l t h o u g h there is s t i l l n o d i r e c t e v i d e n c e , this result establishes t h e p o t e n t i a l of a b o u n d h y d r a z i n e i n t e r m e d i a t e i n t h e o v e r a l l process of d i n i t r o g e n r e d u c t i o n . F i n a l l y , t h e h y d r a z i n e r e d u c t i o n r e a c t i o n is unaffected

b y either

d i h y d r o g e n or d i d e u t e r i u m , i.e., u n d e r either, there is n o i n h i b i t i o n of a m m o n i a f o r m a t i o n , a n d u n d e r d i d e u t e r i u m , there is n o H D p r o d u c t i o n . A s s u m i n g that b o u n d h y d r a z i n e reacts i n t h e same m a n n e r as a d d e d h y d r a z i n e , t h e n t h e d i h y d r o g e n a n d d i d e u t e r i u m effects m u s t o c c u r p r i o r to t h e f o r m a t i o n of b o u n d h y d r a z i n e , a n d a g a i n a d i i m i d e - l e v e l species is i m p l i c a t e d . In

short, t h e e l e c t r o n

balance

strongly implicate b o u n d N H 2

of d i n i t r o g e n b y nitrogenase.

2

and hydrazine reduction

and bound N H 2

4

studies

i n the catalytic reduction

I n this respect, t h e k e y site of nitrogenase

is s h o w n to b e a t w o - e l e c t r o n t w o - p r o t o n reagent. A s e l a b o r a t e d b e l o w , this

finding

points to a p o s s i b l y greater s i m i l a r i t y b e t w e e n

nitrogenase

a n d other m o l y b d e n u m enzymes t h a n m i g h t b e o t h e r w i s e t h o u g h t . Biochemistry

of Xanthine

Oxidase and Other Molybdenum

General Considerations.

Much

Oxidases

e x p e r i m e n t a l i n f o r m a t i o n is a v a i l -

a b l e c o n c e r n i n g t h e r o l e of m o l y b d e n u m i n x a n t h i n e oxidase ( 1 9 , 2 0 ) . I n early w o r k ( p r i o r to 1 9 7 0 ) , there w a s m u c h c o n f u s i o n i n t h e l i t e r a t u r e b e c a u s e of t h e presence of v a r i o u s i n a c t i v e forms of t h e e n z y m e .

I t is

n o w k n o w n that b o t h d e m o l y b d o a n d desulfo forms of x a n t h i n e oxidase w e r e present i n most e a r l y p r e p a r a t i o n s a n d r e m a i n present i n m a n y c u r r e n t p r e p a r a t i o n s as w e l l ( 2 0 , 6 4 ) .

366

BIOINORGANIC

CHEMISTRY

II

A m a j o r a d v a n c e i n t h e e l i m i n a t i o n o f c a t a l y t i c site i n h o m o g e n e i t y i n n e w p r e p a r a t i o n s c a m e w i t h t h e d e v e l o p m e n t of a n affinity c h r o m a t o g r a p h i c m e t h o d ( 6 5 ) for p u r i f y i n g t h e e n z y m e .

T h i s m e t h o d m a d e use

of t h e k n o w n h i g h affinity of x a n t h i n e oxidase f o r a l l o x a n t h i n e w h e n t h e e n z y m e is i n a f u l l y r e d u c e d state. B y a t t a c h i n g a l l o x a n t h i n e to a p o l y m e r i c m a t r i x , a selective a b s o r p t i o n of active e n z y m e w a s a c h i e v e d . Evidence Concerning the Molybdenum Site.

Despite

considerable

debate, there is at present a g o o d d e a l of a g r e e m e n t as to t h e o v e r a l l


m o d e of f u n c t i o n i n g of x a n t h i n e oxidase (20, 65-70). EPR

spectroscopic

properties

Furthermore, the

i n d i c a t e that t h e m o l y b d e n u m

a l d e h y d e oxidase a n d ( t o a s o m e w h a t lesser extent)

sites i n

sulfite oxidase a r e

v e r y s i m i l a r i n n a t u r e to t h a t i n x a n t h i n e oxidase. S e v e r a l lines of e v i d e n c e i n d i c a t e that t h e m o l y b d e n u m oxidases u s e m o l y b d e n u m i n t h e o x i d a t i o n states V I , V , a n d I V . I n t h e o x i d i z e d ( o r resting i n oxygen)

e n z y m e , there is g e n e r a l l y n o E P R s i g n a l .

Upon

r e d u c t i o n w i t h less t h a n s t o i c h i o m e t r i c amounts of substrate ( o r r e d u c t a n t ) , a M o ( V ) E P R s i g n a l appears w h i c h disappears r e d u c t a n t is a d d e d

(20).

when

A reasonable i n t e r p r e t a t i o n i n v o k e s

further Mo(VI)

as b e i n g present i n t h e r e s t i n g oxidase a n d M o ( V ) as a n i n t e r m e d i a t e state i n t h e r e d u c t i o n process.

A s e c o n d l i n e of e v i d e n c e f o r M o ( V I )

concerns t h e w e l l k n o w n a n t a g o n i s m w h i c h t u n g s t e n d i s p l a y s f o r m o l y b d e n u m i n a v a r i e t y of systems. W 0

4

2

" , w h e n u s e d i n p l a c e of M o 0

i n c u l t u r e m e d i a , p l a n t f o o d , o r a n i m a l f e e d , causes either a d e m o l y b d o e n z y m e

4

2

~

the formation of

or a t u n g s t e n - s u b s t i t u t e d p r o t e i n

(71,72).

I n sulfite oxidase, t h e t u n g s t e n - s u b s t i t u t e d p r o t e i n has b e e n c h a r a c t e r i z e d (72).

I t t o t a l l y lacks e n z y m a t i c a c t i v i t y a n d , i n v i e w of t h e greater

difficulty i n r e d u c i n g W ( V I ) c o m p a r e d w i t h M o ( V I ) , u n d o u b t e d l y c o n tains W ( V I ) .

T h e t u n g s t e n p r o t e i n is i m m u n o l o g i c a l l y i d e n t i c a l to its

m o l y b d e n u m a n a l o g . Substrate ( S 0 sten to a n E P R - a c t i v e state b u t S 0 2

3

4

2

2

" ) cannot s i g n i f i c a n t l y r e d u c e t u n g " ( a more powerful reductant) can

p r o d u c e a f u l l y E P R - a c t i v e W ( V ) state. S i g n i f i c a n t l y , t h e W ( V ) E P R s i g n a l w i l l n o t d i s a p p e a r w h e n excess r e d u c t a n t is present. T h e i n a b i l i t y to a c h i e v e t h e ( I V ) state m a y b e r e s p o n s i b l e f o r t h e i n a b i l i t y of t h e t u n g s t e n - p r o t e i n to t u r n over c a t a l y t i c a l l y , w h i c h i n t u r n i m p l i c a t e s a M o ( I V ) state i n t h e c a t a l y t i c c y c l e . T h e r e is, h o w e v e r , m o r e d i r e c t e v i d e n c e f o r t h e presence of M o ( I V ) i n t h e c y c l e of x a n t h i n e oxidase. m e n t s of M a s s e y a n d c o - w o r k e r s

T h i s e v i d e n c e comes f r o m t h e e x p e r i (24) w h o u s e d a l l o x a n t h i n e ( l ) t o

t r a p t h e e n z y m e i n its r e d u c e d state. A s t r o n g c o m p l e x is f o r m e d b e t w e e n t h e r e d u c e d e n z y m e a n d a l l o x a n t h i n e , a n d excess a l l o x a n t h i n e a n d r e d u c tant c a n be removed.

T h e e n z y m e is t h e n r e o x i d i z e d w i t h F e ( C N )

6

3

",

a n d t w o electrons p e r m o l y b d e n u m center a r e f o u n d after t h e electrons r e q u i r e d f o r t h e r e o x i d a t i o n o f t h e i r o n - s u l f u r a n d flavin g r o u p i n g s are

20.

STIEFEL

E T

a c c o u n t e d for.

Molybdoenzymes

AL.

367

If the o x i d i z e d m o l y b d e n u m state is M o ( V I ) , t h e n M o

( I V ) is i m p l i c a t e d as the r e d u c e d state. A n a d d i t i o n a l c r u c i a l p i e c e of i n f o r m a t i o n emerges f r o m t h e a l l o x a n t h i n e s t u d y (24).

T h u s , i t w a s s h o w n t h a t one a l l o x a n t h i n e b i n d s to the

e n z y m e p e r a c t i v e m o l y b d e n u m site. T h i s result c l e a r l y i m p l i e s t h a t the m o l y b d e n u m site is m o n o n u c l e a r . I f a d i n u c l e a r site w e r e i n v o l v e d , t h e n it w o u l d b e u n l i k e l y to r e q u i r e t w o a l l o x a n t h i n e m o l e c u l e s for i n h i b i t i o n a n d w o u l d b e e x p e c t e d to b e at least p a r t i a l l y i n h i b i t e d w i t h one a l l o x a n -


thine/two molybdenum.

A l s o , a difference i n b i n d i n g constant w o u l d

b e e x p e c t e d for the s e c o n d c o m p a r e d w i t h the first b o u n d a l l o x a n t h i n e , b u t n o n e is f o u n d .

T h i s result, c o u p l e d w i t h the l a c k of e v i d e n c e

for

M o ( V ) - M o ( V ) s p i n - s p i n i n t e r a c t i o n s i n the E P R s p e c t r a , c l e a r l y i m p l i cates

a mononuclear

site, a n d it w o u l d seem

that xanthine

oxidase

possesses t w o f u l l c a t a l y t i c u n i t s , e a c h c o n t a i n i n g one m o l y b d e n u m , one flavin, a n d two F e S 2

2

u n i t s (20).

O t h e r m o l y b d e n u m oxidases also c o n -

t a i n p a i r e d p r o s t h e t i c groups a n d s u b u n i t s , a n d i t is l i k e l y t h a t t h e y each have two catalytic units per molecule.

(1)

(2)

E l e c t r o n s p i n resonance s p e c t r o s c o p y has g i v e n t r e m e n d o u s i n s i g h t i n t o t h e n a t u r e of the o v e r a l l x a n t h i n e oxidase reactions as w e l l as i n t o t h e n a t u r e a n d f u n c t i o n of the m o l y b d e n u m site (19, 20).

During turn-

over, t h e E P R s i g n a l f r o m a single M o ( V ) g r o u p appears i n the s p e c t r a of a l l m o l y b d e n u m oxidases. T h e g a n d A values i m p l i c a t e at least one s u l f u r a t o m i n the m o l y b d e n u m c o o r d i n a t i o n sphere, (73, 74)

but until more

definitive m o d e l s b e c o m e a v a i l a b l e , t h e d e t a i l e d n a t u r e of the site m u s t remain obscure.

O n e v e r y i m p o r t a n t feature of the M o ( V ) E P R s i g n a l

f r o m t h e oxidases is t h e n e a r - i s o t r o p i c p r o t o n s u p e r h y p e r f i n e s p l i t t i n g of 1 0 - 1 4 gauss. T h e p r o t o n r e s p o n s i b l e f o r this s p l i t t i n g is e x c h a n g e a b l e as e v i d e n c e d b y t h e r a p i d c o l l a p s e of t h e s p l i t t i n g w h e n D 0 2

H 0 as t h e solvent. T h e p r o t o n has a n a p p a r e n t p K 2

a

replaces

of ~ 8 i n x a n t h i n e

oxidase. F o r sulfite oxidase, w h e r e the E P R s p e c t r a are r e l a t i v e l y s i m p l e , a c l e a r t i t r a t i o n c u r v e is seen w i t h a p K of 8.2. F i n a l l y , B r a y a n d K n o w l e s a

(75)

were able to demonstrate using 8-deuteroxanthine

(2)

that the

BIOINORGANIC

368

CHEMISTRY

II

p r o t o n r e s p o n s i b l e f o r t h e h y p e r f i n e s p l i t t i n g originates i n the 8 - p o s i t i o n of the substrate x a n t h i n e . T h i s p r o t o n seems to b e t r a n s f e r r e d to t h e e n z y m e i n c o n j u n c t i o n w i t h the t w o - e l e c t r o n transfer process.

I n v i e w of the p r o m i n e n c e of t h e

p r o t o n , its p o s s i b l e l o c a t i o n o n the e n z y m e is of c o n s i d e r a b l e i m p o r t a n c e . T h e fact t h a t its s p l i t t i n g of the m o l y b d e n u m s i g n a l is n e a r l y i s o t r o p i c suggests that i t is u n l i k e l y to b e a m o l y b d e n u m h y d r i d e .

Nevertheless,

this p o s s i b i l i t y has not b e e n

as n o t e d

t o t a l l y r u l e d out because,

by


E d m o n d s o n et a l . ( 7 6 ) , i t is p o s s i b l e t h a t this i s o t r o p y is a p p a r e n t a n d not real.

I f the c o m p o n e n t s

of the h y p e r f i n e c o u p l i n g tensor differ i n

s i g n a n d i f t h e i s o t r o p i c s p l i t t i n g is exactly h a l f the a n i s o t r o p i c s p l i t t i n g , the a p p a r e n t i s o t r o p y w o u l d b e e x p l a i n e d .

O n the other h a n d , p r o t o n s

o n p r o t e i n atoms d i r e c t l y b o u n d to m o l y b d e n u m m a y b e r e s p o n s i b l e for the observed

s p l i t t i n g , a n d i n v i e w of m e c h a n i s t i c considerations

recent w o r k o n i n o r g a n i c systems c o n s i d e r e d b e l o w ,

this seems

and most

reasonable.

Electron

Transfer

and Substrate Half-Re actions

E a c h of the m o l y b d e n u m e n z y m e s is a c o m p l e x e n t i t y c o n t a i n i n g m o l y b d e n u m a n d other r e d o x - a c t i v e p r o s t h e t i c groups. are d e s i g n e d

to c a t a l y z e r e d o x

These

enzymes

reactions b y p r o v i d i n g a l o w

p a t h w a y for electrons to transfer f r o m r e d u c t a n t to o x i d a n t .

energy I n most

cases, a n d c e r t a i n l y i n the p h y s i o l o g i c a l r e a c t i o n , the electrons enter a n d leave t h e e n z y m e at different sites. I n the s i m p l e r cases l i k e sulfite o x i dase, a definite s e q u e n c e of e l e c t r o n transfer w i t h i n t h e p r o t e i n c a n formulated.

be

H o w e v e r , m o r e s o p h i s t i c a t e d treatments for the other p r o -

teins r e v e a l that the e l e c t r o n carriers w i t h i n the p r o t e i n a c h i e v e a d i s t r i b u t i o n of

electron occupancy

depending

u p o n their inherent potentials

a n d the t o t a l e l e c t r o n i c c h a r g e of the e n z y m e (69).

T h e inherent redox

p o t e n t i a l of a g i v e n g r o u p m a y b e c h a n g e d b y the presence of substrate or p r e s u m a b l y b y a c o n f o r m a t i o n a l c h a n g e i n the p r o t e i n . T h e q u e s t i o n w h i c h concerns us here is t h e i n t e r a c t i o n of the m o l y b d e n u m site w i t h t h e e x t e r n a l m e d i u m . A l t h o u g h the q u e s t i o n has n o t b e e n a n s w e r e d to t o t a l satisfaction i n a l l cases, i t seems c l e a r t h a t f o r the

molybdenum

oxidases, the m o l y b d e n u m site of the e n z y m e is the one w h i c h interacts w i t h the o x i d i z a b l e substrate. I n contrast, i n the m o l y b d e n u m

reductases

( a l t h o u g h here the e v i d e n c e is not s t r o n g ) , the m o l y b d e n u m site interacts w i t h the r e d u c i b l e substrate. I n e i t h e r event, the m o l y b d e n u m site i n t e r acts w i t h the n a m e d substrate for the r e a c t i o n a n d e i t h e r accepts or donates electrons.

I n a sense, the s i n g l e site of the e n z y m e is l i k e t h e

electrode of a n e l e c t r o c h e m i c a l c e l l w h i c h ( w i t h respect to the m e d i u m ) carries out a c h e m i c a l h a l f - r e a c t i o n . T h u s , to c o m p r e h e n d t h e r o l e w h i c h

20.

STiEFEL

369

Molybdoenzymes

ET AL.

m o l y b d e n u m p l a y s i n e n z y m e s , s c r u t i n y of t h e substrate h a l f - r e a c t i o n s is a p p r o p r i a t e . T h e substrate h a l f - r e a c t i o n s are d i s p l a y e d i n T a b l e s I a n d I I .

In

e a c h case, a t w o - e l e c t r o n process seems to b e i n v o l v e d . O n l y i n n i t r o genase are greater n u m b e r s of electrons t r a n s f e r r e d , a n d the d i s c u s s i o n e a r l i e r i n this p a p e r s u m m a r i z e s the e v i d e n c e t h a t these processes o c c u r i n t w o - e l e c t r o n steps. T h e t w o - e l e c t r o n r e a c t i o n of the m o l y b d e n u m site n e v e r appears to b e s i m p l y a n e l e c t r o n transfer r e a c t i o n . I n the case of


nitrogenase, e a c h substrate takes u p a n e q u a l ( o r g r e a t e r ) n u m b e r of protons to f o r m the p r o d u c t . I n t h e other m o l y b d e n u m e n z y m e s , p r o t o n transfer a n d a d d i t i o n or r e m o v a l of H 0 are also r e q u i r e d . I n e a c h case, 2

h o w e v e r , t h e r e is at least one p r o t o n t r a n s f e r r e d i n the same d i r e c t i o n as the p a i r of electrons.

These data, taken i n conjunction w i t h the E P R

e v i d e n c e for p r o t o n transfer f r o m the substrate to the a c t i v e site i n x a n t h i n e oxidase, suggest t h a t the m o l y b d e n u m site i n a l l the e n z y m e s Table II.

(74)

Substrate Half-Reactions for Molybdoenzymes

N i t r a t e reductase N 0 - + 2 H + 2e"

N0 " +

+

3

2

H 0 2

X a n t h i n e oxidase X a n t h i n e + H 0 - » uric acid + 2 H +

2e"

+

2

0

+

H 0 -> x a n t h i n e + 2 H + 2e~ +

2

H Hypoxanthine A l d e h y d e oxidase RCHO + H 0 2

N ^ N

+

RCOOH + 2H + +

2e"

+

H 0 2

HO

OH

HO'

Sulfite oxidase S0 " + H 0 3

2

2

S0

4

2

" + 2H + +

2e"

2H + +

2e"

370


II

is i n some w a y r e s p o n s i b l e for b o t h p r o t o n a n d e l e c t r o n transfer p r o c esses (66). Molybdenum

Coordination

Chemistry

C o m p a r e d w i t h o t h e r t r a n s i t i o n metals i n b i o l o g i c a l r e d o x systems, the o x i d a t i o n states l i k e l y to b e u s e d b y m o l y b d e n u m are v e r y h i g h

(74).

A s d i s c u s s e d p r e v i o u s l y , the I V , V , a n d V I states are a l i k e l y set of


p a r t i c i p a n t s i n m o l y b d e n u m oxidases, a n d w h i l e the I I a n d I I I states r e m a i n v i a b l e f o r m o l y b d e n u m reductases, i t nevertheless seems l i k e l y t h a t h i g h e r o x i d a t i o n states w i l l b e f o u n d i n these e n z y m e s

as w e l l .

I n d e e d , the s u b s t i t u t i o n of t u n g s t e n for m o l y b d e n u m i n b o t h n i t r a t e r e d u c t a s e a n d n i t r o g e n a s e i n d i c a t e s this l i k e l i h o o d as i t is m u c h m o r e difficult to o b t a i n the l o w e r o x i d a t i o n states of t u n g s t e n . W h e n w e e x a m i n e the o x i d a t i o n states of m o l y b d e n u m , there are s o m e k e y trends w h i c h b e c o m e a p p a r e n t (74).

F i r s t , the h i g h e r o x i d a -

t i o n states are a l w a y s f o u n d to b e c o o r d i n a t e d b y d e p r o t o n a t e d l i g a n d s . I n the most c o m m o n case, these l i g a n d s are w a t e r s , w h i c h w h e n f u l l y T h e compounds

of M o ( I V ) ,

M o ( V ) , a n d M o ( V I ) w i t h d i t h i o c a r b a m a t e s (74, 77, 78, 79)

d e p r o t o n a t e d , are d e s i g n a t e d oxo groups.

nicely illus-

trate the s t r u c t u r a l v a r i e t y as w e l l as the presence of oxo g r o u p s . T h u s t h e complexes

( 3 , 4 , 5 , 6) s h o w the p r e s e n c e of a s i n g l e oxo g r o u p i n t h e

< 7 ) - L

20.

STiEFEL

371

Molybdoenzymes

ET AL.

ΔΗ Values for Reactions of Dithiocarbamate Complexes of Molybdenum"

Table III.

Reaction Mo0 (dtc)

2

+ S0

Mo0 (dtc)

2

+ C H C H 0 - + MoO(dtc)

2

2

MoO(dtc)

MoO(dtc) a

3

2

- - * MoO(dtc)

+ S0

2

3

+ H 0 - » Mo0 (dtc)

2

3MoO(dtc) Bioinorganic Chemistry—II Downloaded from pubs.acs.org by UNIV LAVAL on 04/09/16. For personal use only.

ΔΗ

2

2

2

+ N

2

2

2

3

2

+ N0 "-> Mo0 (dtc) s

2

D a t a f r o m R e f . 87,

-28.5 ±

5.5

-32.0 ±

4.0

+ 3 0 . 1 db 4.2

2

+ 3 H 0 - * 3Mo0 (dtc) 2

"

+ CH C00H

2

+ H

2

4

+ 2NH

2

+51.7 ±

3

+ N0 "

2

(kcal/mol)

12.4

-4.4 ±

2

4.2

d e r i v e d f r o m e x p e r i m e n t a l v a l u e s i n 1,2-dichloroethane

at

25°C.

M o ( I V ) species (3) a n d t w o i n the M o ( V I ) species (6).

T h i s result suggests

the p o s s i b i l i t y of o x y g e n a t o m transfer reactions (80, 81) w i t h M o ( I V )

-

e x t r a c t i n g a n oxo f r o m a substrate (e.g., N 0 ~

[0]

3

> N 0 " ) or M o ( V I ) 2

[O]

d o n a t i n g a n oxo (e.g., S O 3 > S O 4 ). T h e n i t r a t e r e d u c t i o n serves as a d i s t i n c t m o d e l for n i t r a t e r e d u c t a s e , b u t M o ( I I ) ( 8 2 ) , M o ( I I I ) 2

(83), and M o ( V )

2

(84, 85, 86)

compounds

c a n also r e d u c e n i t r a t e to

n i t r i t e . So at present, s u c h m o d e l reactions offer no h e l p i n our d e l i b e r a tions a b o u t m o l y b d e n u m e n z y m e s . Recently,

thermodynamic

studies

have

been

c a r r i e d out

in

our

l a b o r a t o r y ( 8 7 ) to evaluate the p o s s i b l e p a r t i c i p a t i o n of these complexes i n m o d e l reactions.

T h e ΔΗ values for r e l e v a n t reactions are l i s t e d i n

Table III. The M o ( I V ) - M o ( V I )

couple w i t h dithiocarbamate ligands

w o u l d e x o t h e r m i c a l l y execute the S 0 conversions.

3

2

/S0

4

2

- or

CH CHO/CH COOH 3

3

O n the other h a n d , there is a h i g h l y e n d o t h e r m i c r e a c t i o n

w h e n the M o ( I V ) / M o ( V I )

c o u p l e is u s e d to effect the p r o d u c t i o n of

d i h y d r o g e n f r o m w a t e r or the p r o d u c t i o n of a m m o n i a f r o m d i n i t r o g e n . I n t h e case of the N 0 ~ / N 0 ~ c o n v e r s i o n , there is a v e r y s m a l l exother3

2

m i c i t y associated w i t h the r e a c t i o n . T h e s e results s h o w that the d i t h i o c a r b a m a t e complexes c o u l d b e u s e d to m o d e l the sulfite oxidase o r a l d e h y d e oxidase reactions b u t not the nitrogenase r e a c t i o n .

H o w e v e r , the

r e d o x p r o p e r t i e s of the M o ( I V ) / M o ( V I ) c o u p l e v a r y s u b s t a n t i a l l y w i t h l i g a n d ( 8 8 ) , a n d these results therefore d o not v i t i a t e the p o s s i b i l i t y of a n M o ( I V ) / M o ( V I ) c o u p l e b e i n g present i n n i t r o g e n a s e w i t h a different set of d o n o r atoms. A n o t h e r aspect of the d e p r o t o n a t e d ( a c i d i c ) l i g a n d effect manifests itself w h e n the oxo g r o u p s are r e m o v e d . tetradentate l i g a n d ( 7 )

F o r e x a m p l e , r e a c t i o n of the

with M o 0 ( C H 0 ) 2

5

7

2

2

gives a M o ( V I )

complex

of the f o r m M 0 O 0 L ( 8 9 ) , analogous to the d i t h i o c a r b a m a t e c o m p l e x of Mo (VI).

I n contrast, the r e a c t i o n of M o 0

4

2

" w i t h o-aminobenzenethiol

372


M0O4- + 3

|+2H

2

II

+

EtOH-H 0 2

HS

H N+


Mo|

(10)

4H 0 2

p r o c e e d s (67, 68, 89) a c c o r d i n g to R e a c t i o n 10. T h e p r o d u c t is f o r m a l l y a Mo (VI) amine.

c o m p l e x w h e r e i n the c o o r d i n a t e d l i g a n d is a

deprotonated

T h e f o r m a t i o n of this p r o d u c t is u n d e r s t o o d i n t h e l i g h t of t h e

p r e s e n c e of M o ( V I ) a n d its a c i d i t y - e n h a n c i n g p r o p e r t i e s (67, 68).

Thus,

w h e n the m o r e a c i d i c a q u o l i g a n d s ( oxos ) are r e m o v e d f r o m t h e c o o r d i n a t i o n sphere, the a c i d i t y manifests itself i n t h e i o n i z a t i o n of a c o o r d i n a t e d a m i n e l i g a n d w h i c h o r d i n a r i l y w o u l d n o t b e c o n s i d e r e d as a p o t e n t i a l l y i o n i z a b l e g r o u p i n g . T h e r e are n u m e r o u s examples i n c o o r d i n a t i o n c h e m i s t r y w h i c h s h o w t h e effect of o x i d a t i o n n u m b e r o n l i g a n d a c i d i t y . C o n s i d e r a t i o n of a l a r g e n u m b e r of examples ( 9 0 )

reveals that the p K

of a

a

c o o r d i n a t e d l i g a n d a t o m decreases b y a b o u t 6 - 1 0 u n i t s p e r u n i t c h a n g e i n the o x i d a t i o n n u m b e r of the m e t a l a t o m . T h i s effect is i l l u s t r a t e d i n m o l y b d e n u m c h e m i s t r y b y the a q u o ions (74).

In M o ( V I )

chemistry

i n s t r o n g a c i d solutions, t h e p r i n c i p a l species is t h o u g h t to b e (H 0) ] 2

4

present.

2 +

[Mo0 2

w h i l e for M o ( I I I ) i n a c i d s o l u t i o n , the i o n [ M o ( H 0 ) ] 2

S i m i l a r l y , for M o ( I I I ) , the species

6

3 +

[Mo( ( N H ) C H ) ] 2

2

6

4

3

3 +

is is

f o r m e d i n contrast to the r e s u l t f o r M o ( V I ) d i s c u s s e d a b o v e w h e r e t h e c o m p a r a b l e species M o ( N H S C H ) 6

4

3

w a s f o u n d . T h e results f r o m c o o r d i -

n a t i o n c h e m i s t r y i l l u s t r a t e t h a t l i g a n d s c o o r d i n a t e d to m o l y b d e n u m c a n e n g a g e i n p r o t o n transfer reactions w h i c h , t h r o u g h t h e effect of o x i d a t i o n state o n p K , c a n b e c o u p l e d to e l e c t r o n transfer reactions. a

HS

20.

STiEFEL

ET AL.

Molybdoenzymes

373

R e c e n t e l e c t r o n s p i n resonance studies i n o u r l a b o r a t o r y a d d w e i g h t to the n o t i o n that protons o n c o o r d i n a t e d n i t r o g e n p a r t i c i p a t e i n c a t a l y t i c steps.

R e a c t i o n 11 w a s d i s c o v e r e d

Mo(S CNEt2)(NHSC6H )2. 2

4

(91), l e a d i n g t o t h e i s o l a t i o n o f

The monomeric

Mo(V)

complex

formed

d i s p l a y s s u p e r h y p e r f i n e s p l i t t i n g ( 9 2 ) f r o m t w o e q u i v a l e n t n i t r o g e n atoms as w e l l as t w o e q u i v a l e n t h y d r o g e n atoms as i l l u s t r a t e d i n F i g u r e 4. T h e c o u p l i n g constants w h i c h h a v e b e e n c o n f i r m e d b y p r e p a r a t i o n o f t h e N-CH

3

a n d N - D complexes are A

N

= 2.4 a n d A


p r e p a r a t i o n o f the N - d e u t e r o c o m p l e x

H

= 7.4 gauss. T h e f a c i l e

f r o m its N - p r o t e o

analog and

C H O D attests t o t h e e x c h a n g e a b i l i t y o f t h e p r o t o n i n q u e s t i o n . 3

s i g n a l , w i t h its r e l a t i v e l y l a r g e v a l u e o f A the possibility of N - H groups enzymes.

W h i l e these

H

N

being coordinated

compounds

This

a n d l o w v a l u e for A , reaffirms to molybdenum i n

d o n o t represent m o d e l s

for the

m o l y b d e n u m site o f e n z y m e s , t h e y nevertheless i l l u s t r a t e that t h e k e y p r o t o n ( s ) i n v o l v e d i n t h e c a t a l y t i c step m a y b e associated w i t h l i g a n d a t o m ( s ) b o u n d d i r e c t l y to m o l y b d e n u m .

Journal of the American Chemical Society

Figure 4. EPR signal for Mo(S CN(C H ) )(SNHC H\) -displaying proton and nitrogen superhyperfine splitting (92) 2

2

5

2

6

2

Mechanistic

Considerations

M e c h a n i s t i c speculations about the molybdoenzymes

must b e con-

s i d e r e d t o b e i n t h e i r i n f a n c y w i t h the p o s s i b l e e x c e p t i o n of those f o r x a n t h i n e oxidase.

A l t h o u g h the d e t a i l e d s t r u c t u r a l n a t u r e o f the m o l y b -

d e n u m site i s u n k n o w n , t h e r e is sufficient i n f o r m a t i o n f r o m b i o c h e m i c a l a n d c o o r d i n a t i o n c h e m i s t r y studies t o a l l o w i n f o r m e d a r g u m e n t s t o b e drawn.

H e r e w e first discuss e v i d e n c e for the n u c l e a r i t y o f t h e m o l y b -

d e n u m site a n d t h e n discuss b o t h oxo-transfer a n d p r o t o n - e l e c t r o n t r a n s f e r mechanisms for m o l y b d e n u m enzymes.

A final d i s c u s s i o n considers t h e

u n i q u e aspects o f nitrogenase a n d the p o s s i b l e reasons f o r t h e use o f m o l y b d e n u m i n enzymes. M o n o n u c l e a r v s . D i n u c l e a r Sites. A l l m o l y b d e n u m e n z y m e s c o n t a i n t w o m o l y b d e n u m atoms.

Dinuclear molybdenum

complexes

are

well

374

BIOINORGANIC

CHEMISTRY

k n o w n i n the c h e m i s t r y of M o ( V I ) , M o ( I V ) , a n d M o ( I I I ) d o m i n a n t r o l e i n t h e c h e m i s t r y of M o ( V ) .

II

and play a

T h e j u x t a p o s i t i o n of

the

b i o c h e m i c a l a n d i n o r g a n i c c h e m i c a l n u m e r o l o g y has l e d to the suggestion that this m a y b e m o r e t h a n m e r e c o i n c i d e n c e a n d t h a t t h e use of d i n u c l e a r m o l y b d e n u m i n t h e c a t a l y t i c sequences r e q u i r e s that t h e content of the enzymes b e d o u b l e d .

molybdenum

F u r t h e r m o r e , t h e r e are a t t r a c t i v e

c a t a l y t i c schemes w h i c h m a k e use of M o ( V )

complexes.

In particular,

the oxo transfer r e a c t i o n u s e f u l i n the o x i d a t i o n of t e r t i a r y p h o s p h i n e s


( 9 3 ) l e d to the d i s c o v e r y (78, 79, 93) of R e a c t i o n 12 i n w h i c h a d i n u c l e a r Mo 0 (R2dtc) 2

Mo(V)

3

complex

*± M o 0 ( R d t c ) 2

4

disproportionates

nuclear M o ( I V )

and M o ( V I ) .

2

2

+

MoO(R dtc) 2

(12)

2

as i t dissociates to p r o d u c e

As M o (IV)

and M o (VI)

mono-

are d i r e c t l y

i n t e r c o n v e r t i b l e b y a n oxo transfer r e a c t i o n , t h e y are v i a b l e p a r t i c i p a n t s i n c a t a l y t i c cycles.

A dinuclear M o ( V )

species of this n a t u r e c a n t h u s

s u p p l y either t h e o x i d i z i n g o r r e d u c i n g m e m b e r presents

a mechanism

by

which molybdenum

r e d u c i n g or o x i d i z i n g p o w e r .

of

this c o u p l e

enzymes

can

and

channel

S e v e r a l i n o r g a n i c reactions h a v e r e c e n t l y

b e e n e x p l a i n e d u s i n g this s c h e m e (80, 81).

T o date, h o w e v e r , R e a c t i o n

12 o n l y a p p l i e s w h e n the l i g a n d is a d i t h i o c a r b a m a t e or d i t h i o p h o s p h a t e . N e v e r t h e l e s s , w e r e there k n o w n d i n u c l e a r a c t i v e sites i n e n z y m e s , this w o u l d b e a n i m p o r t a n t m e c h a n i s m to c o n s i d e r . It appears, h o w e v e r , that i n w e l l s t u d i e d systems, the e v i d e n c e

for

d i n u c l e a r sites is o u t w e i g h e d b y that f o r m o n o n u c l e a r sites. T h e case for x a n t h i n e oxidase seems most e x p l i c i t . H e r e , the t w o

molybdenum

atoms are a c c o m p a n i e d b y t w o F A D groups as w e l l as t w o e a c h of t w o different types

of

Fe S 2

2

cluster. A l l c o m p o n e n t s ,

i n c l u d i n g subunits,

a p p e a r to b e present i n p a i r s a n d most m o d e r n treatments i n v o k e t w o separate c a t a l y t i c u n i t s , e a c h i n v o l v i n g one m o l y b d e n u m , one F A D , a n d one of e a c h of t h e F e S 2

2

systems.

T h e e x p e r i m e n t a l s u p p o r t for this is

i m p r e s s i v e . F i r s t , i t is clear that e a c h m o l y b d e n u m a t o m c a n a c c e p t t w o electrons

from

substrate.

T h i s i m p l i e s t h a t i f a d i n u c l e a r site

present, i t w o u l d b e r e q u i r e d to a c c e p t f o u r electrons.

were

It is n o t clear

w h y f o u r electrons s h o u l d b e a d d e d to a site w h i c h catalyzes a t w o e l e c t r o n substrate r e a c t i o n . S e c o n d , as d i s c u s s e d p r e v i o u s l y , t h e i n h i b i t o r a l l o x a n t h i n e b i n d s to a r e d u c e d f o r m of the e n z y m e c o n t a i n i n g M o ( I V ) w i t h o n l y one v e r y t i g h t b i n d i n g constant a n d a s t o i c h i o m e t r y of p r e c i s e l y one a l l o x a n t h i n e p e r one m o l y b d e n u m . accommodate i n a dinuclear model. amount

of

Mo(V)

sites has b e e n o b s e r v e d .

E P R work, no

T h e s e d a t a are v e r y difficult to F i n a l l y , despite a n e x t r a o r d i n a r y

spin-spin broadening

interaction

T h e r e f o r e , either the M o ( V )

always accompanied b y a diamagnetic m o l y b d e n u m partner

between state is [Mo(IV)

20.

STiEFEL

375

Molybdoenzymes

ET AL.

or M o ( V I ) ] , or t h e i n d i v i d u a l m o l y b d e n u m atoms are f a r apart.

The

l a t t e r i n t e r p r e t a t i o n seems f a r m o r e l i k e l y . T h e q u e s t i o n n o w arises as to w h y x a n t h i n e oxidase has a m o l e c u l a r w e i g h t of 300,000 w h e n the size of a c a t a l y t i c s u b u n i t is o n l y

150,000.

P e r h a p s the a n s w e r lies i n its p a r t i c u l a r i n t e g r a t i o n i n t o c e l l u l a r p h y s i o l o g y or is s i m p l y c a u s e d b y the p r o c l i v i t y of the e n z y m e to m a x i m i z e the n u m b e r of active sites p e r u n i t surface area; i.e., w h e n t w o

active

m o n o m e r s j o i n together, the n u m b e r of a c t i v e sites goes f r o m one to t w o ,


b u t the e x p o s e d surface area increases b y a f a c t o r less t h a n t w o .

This

a r g u m e n t is a f a m i l i a r one i n heterogeneous catalysis w h e r e attempts are often m a d e to m a x i m i z e the n u m b e r of a c t i v e sites p e r u n i t surface area to p r o d u c e m o r e efficient catalysts. T h e other m o l y b d e n u m enzymes e a c h c o n t a i n d u p l i c a t e p r o s t h e t i c groups a n d p a i r e d s u b u n i t s i n a d d i t i o n to t w o m o l y b d e n u m atoms.

Many

of the experiments p e r f o r m e d for x a n t h i n e oxidase h a v e also b e e n c a r r i e d o u t w i t h a l d e h y d e oxidase a n d sulfite oxidase, a n d t h e r e is n o e v i d e n c e for c h e m i c a l M o - M o c o u p l i n g i n these e n z y m e s .

T h u s , i n oxidases, the

e v i d e n c e for m o n o n u c l e a r m o l y b d e n u m sites appears s t r o n g , a n d i n v i e w of the d u p l i c a t e s u b u n i t s a n d c o m p o s i t i o n f o u n d , i t is reasonable assume a s i m i l a r s i t u a t i o n i n reductases as w e l l .

to

H o w e v e r , at present,

insufficient i n f o r m a t i o n bars a f u l l g e n e r a l i z a t i o n . E x c e p t f o r nitrogenase,

a l l substrate

h a l f - r e a c t i o n s i n v o l v e the a d d i t i o n or r e m o v a l of o x y g e n .

O x o T r a n s f e r Mechanisms.

T h e simplest

m a n n e r of r e p r e s e n t i n g these reactions, i n v o l v e s the d i r e c t transfer of a n o x y g e n a t o m to or f r o m substrate, e.g., R e a c t i o n s 13 a n d 14. F u r t h e r m o r e , M V - * N 0 or

S0

3

2

- +

2

- +

[0]

(13)

[ 0 ] -> S 0 ~ 4

(14)

2

i t is k n o w n that, at least w i t h some reactants, v a r i o u s

molybdenum

c o m p l e x e s w i l l u n d e r g o s u c h a n a p p a r e n t l y s i m p l e oxo transfer ( 8 1 , 9 3 ) , e.g., R e a c t i o n 15. T h i s o b s e r v a t i o n suggests t h a t o x y g e n a t o m transfer Mo0 (R dtc) 2

2

+ P ( C H ) - * MoO(R dtc) 6

5

3

2

+

2

OP(C H ) 6

5

3

is a r e a c t i o n w o r t h c o n s i d e r i n g for the m o l y b d e n u m e n z y m e s ( 7 7 ) . a m e c h a n i s m f o r n i t r a t e reductase

could involve Reaction

ο ι

ο

χ 1

I

\

I —Mo (IV)-

* o

+ î

11

Such where

ο

N)

— M o (VI)—

16

(15)

(16)

376

BIOINORGANIC

cleavage

of t h e N - O b o n d

p r e s u m a b l y occurs

f o r m a t i o n of t h e m u l t i p l e M o - O b o n d .

CHEMISTRY

concertedly

II

with

the

O n e of the p r o b l e m s w i t h this

t y p e of m e c h a n i s m for n i t r a t e reductase i n v o l v e s the r e m o v a l of the oxo group

on molybdenum

to regenerate

the o p e n

site.

Heretofore,

oxo

r e m o v a l reactions g e n e r a l l y r e q u i r e d s t r o n g a c i d (74, 78, 79) or a n oxo r e m o v a l agent s u c h as a p h o s p h i n e

(74,

Recently, thiol

81, 93, 94).

l i g a n d s h a v e , u n d e r c e r t a i n c o n d i t i o n s , also r e m o v e d oxo groups (91, 95, 96, 9 7 ) .

I n most cases u n f o r t u n a t e l y , a s u l f u r - d o n o r l i g a n d r e p l a c e d the


oxo g r o u p . H o w e v e r , i n other instances (95, 9 6 ) , r e m o v a l of a n oxo g r o u p w i t h c o n c o m i t a n t r e d u c t i o n of M o ( V I ) to M o ( I V ) b y t w o electrons has b e e n affected b y thiols w h i c h are o x i d i z e d to the d i s u l f i d e i n the process M o (VI) 0

2

(R dtc) + 2 C H S H - »

Mo(IV)0(R dtc) 2

(Reaction 17).

2

2

6

5

+ CcH SSC6H 5

5

+

H 0 2

O x o r e m o v a l to leave a n o p e n site m i g h t b e effected i n

this m a n n e r i n enzymes p a r t i c u l a r l y u n d e r h y d r o p h o b i c c o n d i t i o n s .

The

r e l a t e d p r o b l e m i n nitrogenase m a y b e o v e r c o m e b y A T P , w h i c h m a y f u n c t i o n i n oxo r e m o v a l f r o m m o l y b d e n u m . F o r the m o l y b d e n u m oxidases, the reverse oxo transfer r e a c t i o n c a n b e p o s t u l a t e d w h e r e i n a n o x o m o l y b d e n u m ( V I ) species donates oxo substrate. F o r e x a m p l e , the o x i d a t i o n of a l d e h y d e s

/

H

R—C.

+

\

II

M o (VI)

M o (IV)

+

( R e a c t i o n 18)

R—C

/°

(18)

\

Ο

to can

O H

b e affected b y a M o ( V I ) species.

A l t h o u g h the r e a c t i o n is s t o i c h i o m e t -

r i c a l l y a c c e p t a b l e , i t is not clear h o w the a l d e h y d e C - H b o n d is a c t i v a t e d for cleavage.

A s i m i l a r p r o b l e m occurs for x a n t h i n e o x i d a t i o n . F o r this

reason, a n d to m a k e use of the e x p e r i m e n t a l e v i d e n c e for p r o t o n transfer, the schemes i n v o l v i n g c o u p l e d e l e c t r o n - p r o t o n transfer w e r e

proposed

(66, 67, 68) a n d are d i s c u s s e d b e l o w . Coupled Proton—Electron Transfer

Mechanisms.

The

suggestive

e v i d e n c e for p r o t o n transfer i n x a n t h i n e oxidase has b e e n discussed a b o v e . T h e k e y p i e c e of e x p e r i m e n t a l i n f o r m a t i o n is the p r o t o n s u p e r h y p e r f i n e s p l i t t i n g i n t h e M o ( V ) E P R s i g n a l of x a n t h i n e oxidase.

M o d e l studies

(89, 92) h a v e i n d i c a t e d that a c o o r d i n a t e d n i t r o g e n is the l i k e l y l o c a t i o n of the p r o t o n ( a l t h o u g h c o o r d i n a t e d o x y g e n is not e l i m i n a t e d ) . d i n a t i o n c h e m i s t r y f u r t h e r shows that p r o t o n transfer c a n b e

Coor coupled

to e l e c t r o n transfer t h r o u g h the effect of o x i d a t i o n state o n the p K c o o r d i n a t e d l i g a n d s (66, 67, 68).

a

of

The combined biochemical and inor-

20.

377

Molybdoenzymes

STIEFEL E T A L .

g a n i c i n f o r m a t i o n leads t o a m e c h a n i s t i c suggestion f o r x a n t h i n e oxidase w h i c h is d e p i c t e d i n F i g u r e 5. T h e e v i d e n c e f o r t h e presence o f v a r i o u s m o l y b d e n u m

oxidation

states has b e e n p r e s e n t e d p r e v i o u s l y . T h e r e s t i n g e n z y m e ( u p p e r r i g h t o f F i g u r e 5 ) is a s s u m e d t o c o n t a i n M o ( V I ) .

I n this h i g h o x i d a t i o n state, a t

least s o m e o f t h e l i g a n d s o n m o l y b d e n u m m u s t b e d e p r o t o n a t e d a n d a n i t r o g e n a t o m o f t h e p r o t e i n is so d e p i c t e d xanthine c a n then coordinate

to M o ( V I )

i n the

figure.

Substrate

t h r o u g h its 9-nitrogen. T h e


C = N o f t h e 8- a n d 9 - p u r i n e positions is t h e n p o l a r i z e d b y t h e M o ( V I ) c a u s i n g t h e 8-carbon t o b e c o m e s u s c e p t i b l e t o n u c l e o p h i l i c attack. A l t h o u g h there is e v i d e n c e f o r a p r o t e i n b o u n d persulfide b e i n g t h e n u c l e o p h i l i c agent ( 6 5 ) , f o r s i m p l i c i t y i n this s c h e m e , O H " assumes that r o l e . ( W e r e t h e persulfide i n v o l v e d , i t w o u l d h a v e t o b e s u b s e q u e n t l y lyzed b y O H " or H

2

hydro

0 anyway. )

I n c o n j u n c t i o n w i t h t h e n u c l e o p h i l i c attack at t h e 8-carbon, t w o electrons c o u l d flow f r o m x a n t h i n e to p r o d u c e M o ( I V )

w h i c h requires

a c o n c o m i t a n t decrease i n p K o f the p r o t e i n n i t r o g e n , r e s u l t i n g i n transfer a

ο

Xanthine

Mo(IV)-N-

1st International Conference on Chemistry and Uses of Molybdenum Figure 5. Proposed coupled proton-electron transfer scheme for xanthine oxidase activity (66, 67, 68)

378

BIOINORGANIC

CHEMISTRY

II

of t h e p r o t o n t o the m o l y b d e n u m site. T h i s process c o u p l e s t h e t r a n s f e r of a p r o t o n f r o m substrate w i t h the t w o - e l e c t r o n transfer. U r i c a c i d ( o r its p e r s u l f i d o p r e c u r s o r ) is n o w c o o r d i n a t e d t o the M o ( I V ) , a n d r e a c t i v a t i o n o f the site m u s t i n v o l v e d i s s o c i a t i o n o f p r o d u c t ,

two-electron

o x i d a t i o n o f m o l y b d e n u m , a n d loss o f a p r o t o n f r o m c o o r d i n a t e d n i t r o g e n . T h e o r d e r o f these events is n o t c l e a r a n d , i n fact, m a y not b e s u s c e p t i b l e to t e m p o r a l d e s i g n a t i o n . I t is clear, h o w e v e r , that this t w o - e l e c t r o n r e a c t i v a t i o n process o c c u r s


i n t w o s e q u e n t i a l o n e - e l e c t r o n steps w h i c h cause t h e a p p e a r a n c e o f the M o ( V ) E P R s i g n a l . F u r t h e r m o r e , the p r o t o n ( o r i g i n a l l y f r o m substrate) m a y r e m a i n i n p l a c e o n the p r o t e i n n i t r o g e n d u r i n g o x i d a t i o n f r o m M o ( I V ) to M o ( V ) a n d m a y thus b e r e s p o n s i b l e f o r the s u p e r h y p e r f i n e s p l i t t i n g . I n s u p p o r t of this m e c h a n i s m , the use of 8 - d e u t e r o x a n t h i n e causes this s i g n a l t o a p p e a r i n i t i a l l y i n its deutero f o r m . A k e y aspect of this s i g n a l is t h e a p p a r e n t p K

a

o f 8 for this p r o t o n i n the M o ( V )

of o x i d a t i o n state o n p K

a

state. T h e effect

r e q u i r e s that i n t h e M o ( I V ) state, t h e p K o f a

this p r o t o n w o u l d b e v e r y h i g h ( p e r h a p s 14 o r g r e a t e r ) w h e r e a s i n t h e M o ( V I ) state this p K w o u l d b e q u i t e l o w ( p e r h a p s 2 o r l o w e r ) . a

Thus,

t h e ( I V ) state w o u l d c o n t a i n a s t r o n g l y b a s i c p r o t e i n n i t r o g e n i n agree m e n t w i t h its p o s t u l a t e d r o l e i n c l e a v i n g t h e C - H b o n d . f o r the M o ( V I ) state, the l o w p K

a

Furthermore,

w o u l d i n d i c a t e t h a t the c o o r d i n a t e d

n i t r o g e n w o u l d b e d e p r o t o n a t e d thus p r e p a r i n g t h e site t o re-enter t h e catalytic cycle ( F i g u r e 5 ) . T h e i n h i b i t o r a l l o x a n t h i n e b i n d s v e r y s t r o n g l y t o x a n t h i n e oxidase b u t o n l y w h e n the m o l y b d e n u m is i n the f u l l y r e d u c e d [ M o ( I V ) ] state. I n F i g u r e 6, this extra s t r o n g b i n d i n g is i n t e r p r e t e d as r e s u l t i n g f r o m t h e possession b y a l l o x a n t h i n e o f t h e f u l l r e c o g n i t i o n c a p a b i l i t y f o r the a c t i v e

ι xanthine

Figure 6. alloxanthine

alloxanthine

Suggested binding mode for to the Mo(IV) site in the xan thine oxidase

20.

STiEFEL

379

Molybdoenzymes

ET AL.

site c o u p l e d w i t h the f o r m a t i o n of a n a d d i t i o n a l h y d r o g e n b o n d w i t h t h e e n z y m e t h r o u g h t h e k e y p r o t o n . F u r t h e r m o r e , this t i g h t e n z y m e - i n h i b i t o r c o m p l e x c l e a r l y resembles t h e p r o p o s e d t r a n s i t i o n state ( i n t h e c o u p l e d p r o t o n - e l e c t r o n transfer m e c h a n i s m ) f o r the c a t a l y z e d r e a c t i o n , as m a n y s u c h complexes do. M a n y e x p e r i m e n t a l observations

o n x a n t h i n e oxidase a c t i v i t y are

c o r r e l a t e d b y this scheme, a n d at present, there a p p e a r to b e n o m a j o r inconsistencies.

T h e c o u p l e d p r o t o n - e l e c t r o n transfer s c h e m e (66,

67,


68 ) has b e e n successfully i n c o r p o r a t e d i n t o a n o v e r a l l m e c h a n i s t i c s c h e m e (69)

w h i c h e x p l a i n s , w i t h great e c o n o m y , a large a m o u n t of r a t h e r d e

m a n d i n g data, from both kinetic a n d electron uptake experiments. N o n e of the other m o l y b d e n u m e n z y m e s h a v e b e e n s t u d i e d as t h o r o u g h l y as x a n t h i n e oxidase, b u t b o t h a l d e h y d e oxidase a n d sulfite oxidase d i s p l a y d a t a consistent w i t h t h e i r use of t h e M o ( I V ) - M o ( V ) - M o ( V I ) t r i a d of o x i d a t i o n states d u r i n g catalysis. T h e i r M o ( V ) E P R signals are s i m i l a r to those f r o m x a n t h i n e oxidase, i n c l u d i n g the l a r g e n e a r - i s o t r o p i c s u p e r h y p e r f i n e s p l i t t i n g for a s i n g l e p r o t o n w i t h a p f C close to 8 a

(20).

T h e s e observations r e q u i r e s t r u c t u r a l s i m i l a r i t i e s of the m o l y b d e n u m sites and

suggest m e c h a n i s t i c s i m i l a r i t i e s as w e l l .

Thus, coupled

electron-

p r o t o n transfer processes h a v e b e e n suggested for b o t h a l d e h y d e oxidase a n d sulfite o x i d a t e (66, 67, 68).

F o r the f o r m e r , a m e c h a n i s m closely a k i n

to x a n t h i n e oxidase a c t i o n is suggested ( R e a c t i o n 19 ) i n w h i c h C — H b o n d R

O —Mo

R

•OIT ^

-

A,

° "

(VI)—Ν

^

0

(19)

H

Η \

— M o (IV)—Ν

b r e a k i n g is assisted b y n u c l e o p h i l i c attack ( a g a i n p o s s i b l y b y p e r s u l f i d e (97))

w i t h t h e p r o t o n b e i n g t r a n s f e r r e d to the m o l y b d e n u m

c o n j u n c t i o n w i t h the e l e c t r o n transfer process.

site i n

I n fact, x a n t h i n e oxidase

w i l l o x i d i z e a l d e h y d e s (98), a n d a l d e h y d e oxidase w i l l h a n d l e a v a r i e t y of purines (25).

T h e k e y feature of the m o l y b d e n u m site is its a b i l i t y to

abstract i n a c o n c e r t e d m a n n e r t w o electrons a n d a p r o t o n f r o m substrate c o u p l e d w i t h a n u c l e o p h i l i c attack o n the c a r b o n b e a r i n g the p r o t o n to be transferred. T h e c o u p l e d p r o t o n - e l e c t r o n transfer m e c h a n i s m c a n also b e a p p l i e d to t h e m o l y b d e n u m reductases. F o r n i t r a t e reductase, a s c h e m e s u c h as R e a c t i o n 20 is p o s s i b l e . A M o ( I V ) - M o ( V I ) c o u p l e is u s e d to i l l u s t r a t e this, a n d w h i l e s u c h a c o u p l e is v i a b l e for some n i t r a t e reductases, t h e M o ( I I ) - M o ( I V ) or the M o ( I I I ) - M o ( V ) c o u p l e c o u l d also b e

accommodated

380


0

0

"

Ν

+

OH-

(20)

Ο'

— M o (VI)

•Mo ( I V ) — Ν .

w i t h i n the p r o t o n - e l e c t r o n transfer scheme.

II

Ν.

T h i s s c h e m e is s i m p l y the

reverse of t h a t for the oxidases. T h e l o w e r o x i d a t i o n state w o u l d h a v e a Bioinorganic Chemistry—II Downloaded from pubs.acs.org by UNIV LAVAL on 04/09/16. For personal use only.

p r o t o n a t e d l i n g a n d ( h e r e s h o w n as n i t r o g e n ) a n d d u r i n g t w o - e l e c t r o n r e d u c t i o n of substrate, this l i g a n d w o u l d increase i n a c i d i t y a n d t r a n s f e r its p r o t o n to a n o x y g e n a t o m o n n i t r a t e , m a k i n g the o x y g e n a b e t t e r leaving group a n d facilitating nitrite production. T h i s process c a n b e c o n t r a s t e d d i r e c t l y w i t h the oxo transfer s c h e m e ( R e a c t i o n 16) d i s c u s s e d a b o v e . I n either case, the c l e a v a g e of t h e N - O b o n d is assisted b y the b i n d i n g of o x y g e n to a n e l e c t r o p h i l e ( t o m o l y b d e n u m itself i n the oxo transfer m e c h a n i s m or to p r o t o n ( s ) i n the c o u p l e d p r o t o n - e l e c t r o n transfer s c h e m e ) . A l t h o u g h t h e c o u p l e d p r o t o n - e l e c t r o n transfer m e c h a n i s m w o u l d p o s s i b l y h a v e the a d v a n t a g e of l e a v i n g a n o p e n site o n m o l y b d e n u m to restart the c y c l e , there is no s t r o n g d a t a to s u p p o r t e i t h e r of these m e c h a n i s m s at present. F o r nitrogenase, the s i t u a t i o n is less c e r t a i n .

F i r s t , the

metal(s)

present at the a c t i v e site, the o x i d a t i o n s t a t e ( s ) , a n d the s t a t e ( s )

of

a g g r e g a t i o n are v i r t u a l l y u n k n o w n at present. T h u s , a n y s u g g e s t i o n m a d e f o r nitrogenase m u s t b e v i e w e d as h i g h l y s p e c u l a t i v e a n d u s e f u l o n l y to t h e extent to w h i c h i t suggests f u r t h e r e x p e r i m e n t s o n e n z y m e s o r m o d e l systems.

W i t h this i n m i n d , c o u p l e d e l e c t r o n - p r o t o n transfer schemes

c a n b e suggested f o r nitrogenase.

Again, while a ( I V ) - ( V I )

c o u p l e is

u s e d to i l l u s t r a t e the process, other t w o - e l e c t r o n couples are also p o s s i b l e . T h e k e y step i n s u c h a process, as v i s u a l i z e d i n F i g u r e 7 a for a c e t y l e n e r e d u c t i o n , i n v o l v e s the c o u p l e d transfer of t w o p r o t o n s a n d t w o electrons to substrate. T h e k n o w n cis stereochemistry of the a d d i t i o n is consistent w i t h this p r o p o s a l . F o r d i n i t r o g e n r e d u c t i o n , a t w o - m e t a l - s i t e h y p o t h e s i s ( 52, 53, 66-68, 9 6 ) , as s h o w n i n F i g u r e 7 b , m i g h t b e i n v o k e d w i t h d i n i t r o g e n first b i n d i n g e n d - o n ( p e r h a p s to i r o n ) a n d t h e n a d d i t i o n a l l y b i n d i n g s i d e - o n to t h e same site at w h i c h a c e t y l e n e r e d u c t i o n occurs. T h e first r e d u c t i o n p r o d u c t of d i n i t r o g e n w o u l d t h e n b e a b o u n d c i s - d i i m i d e species i n a g r e e m e n t w i t h t h e i n t e r p r e t a t i o n of the d i h y d r o g e n i n h i b i t i o n a n d H D p r o d u c t i o n r e a c tions of nitrogenase d i s c u s s e d e a r l i e r . T h e m o l y b d e n u m site c o u l d t h e n be reactivated twice more, w i t h hydrazine and

finally

ammonia being

s e q u e n t i a l l y f o r m e d . F o r n i t r o g e n a s e there are c l e a r l y a d d i t i o n a l e x p e r i m e n t a l observations w h i c h r e m a i n to b e i n t e g r a t e d w i t h a n d m u s t reflect u p o n the eventual mechanistic conclusions.

20.

STiEFEL

381

Molybdoenzymes

ET AL.

N i t r o g e n a s e differs

Nitrogenase—Additional Considerations.

from

a l l other m o l y b d e n u m e n z y m e s i n s e v e r a l i m p o r t a n t w a y s . I t is t h e o n l y k n o w n m o l y b d e n u m e n z y m e to consist of t w o separately i s o l a b l e p r o t e i n s a n d to r e q u i r e A T P h y d r o l y s i s i n its c a t a l y t i c c y c l e .

Its substrate h a l f -

reactions are the o n l y ones w h i c h d o not ( i n a n y o b v i o u s w a y ) transfer of o x y g e n atoms.

T h e nitrogenase system evolves

involve

dihydrogen

w h e n s u p p l i e d w i t h A T P a n d r e d u c t a n t i n t h e absence of r e d u c i b l e s u b strate.

A l l substrate

reactions

(but

not

ATP-dependent


e v o l u t i o n ) are i n h i b i t e d b y c a r b o n m o n o x i d e .

dihydrogen

T h e presence of substrate

appears to c u r t a i l the d i h y d r o g e n e v o l u t i o n r e a c t i o n b u t , f o r some s u b strates at least ( i n c l u d i n g d i n i t r o g e n ) , i t seems i m p o s s i b l e to e l i m i n a t e d i h y d r o g e n e v o l u t i o n c o m p l e t e l y (46, 58, 5 9 ) . I n this section, w e address dihydrogen

evolution

first

a n d then the A T P utilization reaction

of

nitrogenase. α

Ην

,Ν—

^

Η

MoOv)— ÎSL

Η

Ην

^

Ν —Μο(νι) —

*N =

'

Ν

N>

N^

— M o —

Ν

1

J

/

Figure 7. Proposed proton-electron transfer step for nitrogenase. (a) C H reduction to C H ; (b) N re duction to bound N H (66, 67, 68). 2

2

2

2

U

2

2

Dihydrogen Evolution Reaction. T h e d i h y d r o g e n e v o l u t i o n r e a c t i o n of nitrogenase is c e r t a i n l y a t h e r m o d y n a m i c a l l y r e a s o n a b l e one, i.e., t h e site w h i c h reduces d i n i t r o g e n s h o u l d h a v e sufficient p o t e n t i a l to dihydrogen.

evolve

T h e m o l e c u l a r m e c h a n i s m b y w h i c h this arises is t o t a l l y

u n k n o w n a l t h o u g h this w i l l n o t stop us f r o m s p e c u l a t i n g . I n v i e w of t h e l a c k of i n h i b i t i o n b y c a r b o n m o n o x i d e

a n d its A T P d e p e n d e n c e , i t is

a s s u m e d t h a t d i h y d r o g e n e v o l u t i o n i n nitrogenase occurs b y a process different f r o m t h a t w h i c h occurs i n h y d r o g e n a s e

(56).

As

hydrogenase

contains o n l y i r o n - s u l f u r clusters a n d n o m o l y b d e n u m ( 5 6 ) , t h e m o l y b d e n u m site ( t h e p r e s u m e d substrate r e d u c t i o n site) m a y b e the l o c a t i o n of t h e d i h y d r o g e n e v o l u t i o n r e a c t i o n i n nitrogenase. proton-electron

transfer scheme,

the fully

I n the

coupled

a c t i v a t e d site h a v i n g

the

382


p o t e n t i a l to donate t w o protons a n d t w o electrons c a n react to d i h y d r o g e n e i t h e r w i t h or w i t h o u t the a i d of w a t e r .

evolve

If water were i n -

v o l v e d , a n M o - O l i n k a g e m i g h t b e f o r m e d w i t h A T P h e l p i n g to t h a t oxo l i g a n d f r o m m o l y b d e n u m .

II

remove

A l t e r n a t i v e l y , i t is possible t h a t a

m e t a l h y d r i d e or d i h y d r i d e is i n v o l v e d w h i c h , u p o n r e d u c t i v e e l i m i n a t i o n or r e a c t i o n w i t h protons, p r o d u c e s

dihydrogen.

I t is not p o s s i b l e

to

d i s t i n g u i s h b e t w e e n these p o s s i b i l i t i e s at present. T h e q u e s t i o n n o w arises as to w h y some substrates ( l i k e

acetylene)


c o m p l e t e l y c u r t a i l d i h y d r o g e n e v o l u t i o n w h i l e others ( s u c h as d i n i t r o g e n ) d o not.

M o s t d a t a i n d i c a t e t h a t the e v o l t u i o n of one m o l e of d i h y d r o -

gen per mole

of

dinitrogen reduced

is a p p r o a c h e d

at h i g h levels

of

d i n i t r o g e n ( 5 2 , 5 8 ) . A r e l a t e d p r o b l e m i n v o l v e s t h e fact that a c e t y l e n e is a n o n c o m p e t i t i v e i n h i b i t o r of n i t r o g e n fixation w h i l e d i n i t r o g e n is a c o m p e t i t i v e i n h i b i t o r of acetylene

reduction

(51, 57).

Several

hypotheses

h a v e b e e n a d v a n c e d to e x p l a i n these facts. THE

sions

L E A K Y S I T E H Y P O T H E S I S ( 5 1 , 9 9 , 100).

This

hypothesis

envi-

nitrogenase as a n e l e c t r o n s i n k w h i c h m u s t b e f u l l ( a t least six

electrons ) to r e d u c e d i n i t r o g e n . H o w e v e r , a f u l l s i n k m a y not a l w a y s b e m a i n t a i n e d b e c a u s e of l i m i t a t i o n s i n e l e c t r o n flow, a n d the sink m a y t h e n leak t w o electrons to f o r m d i h y d r o g e n .

If a c e t y l e n e is present, i t c a n

a c c e p t these t w o electrons f r o m the sink. T h e a c e t y l e n e - d i n i t r o g e n

inhi-

b i t i o n studies c a n t h e n b e r a t i o n a l i z e d as f o l l o w s . A c e t y l e n e c a n o v e r c o m e the p r e s e n c e of d i n i t r o g e n b y c o n t i n u a l l y r e m o v i n g e l e c t r o n pairs f r o m t h e s i n k a n d k e e p i n g it e m p t y . H o w e v e r , d i n i t r o g e n r e q u i r e s six electrons f o r r e d u c t i o n , a n d therefore a p a r t i a l l y filled e n z y m e w o u l d r e m a i n accessible to a c e t y l e n e .

T h u s , h i g h dinitrogen concentrations

cannot

effectively

e l i m i n a t e a c e t y l e n e r e d u c t i o n . I n this m o d e l , the i n a b i l i t y of h i g h d i n i t r o g e n t o t a l l y to e l i m i n a t e either d i h y d r o g e n or a c e t y l e n e r e d u c t i o n is a t t r i b u t e d to the i n a b i l i t y of the e n z y m e to k e e p t h e s i n k f u l l . commonly

Using

the

a c c e p t e d n o t i o n t h a t the i r o n p r o t e i n s u p p l i e s electrons, this

m o d e l w o u l d p r e d i c t that as t h e c o m p o n e n t r a t i o ( [ F e ] / [ M o - F e ] ) is i n c r e a s e d a n d the s i n k is k e p t m o r e n e a r l y f u l l , t h e d i h y d r o g e n e v o l u t i o n and

a c e t y l e n e r e d u c t i o n reactions c o u l d b e m o r e n e a r l y q u e n c h e d

by

d i n i t r o g e n . H o w e v e r , as m o s t d i h y d r o g e n e v o l u t i o n a n d a c e t y l e n e r e d u c t i o n e x p e r i m e n t s h a v e b e e n c a r r i e d out u s i n g n i t r o g e n a s e c o m p l e x or a fixed

[Fe]/[Mo-Fe]

ratio, this question

still awaits an

experimental

answer. THE

HYPOTHESIS.

FOUR-ELECTRON

This

hypothesis,

apparently

f a v o r e d b y S h i l o v (101,102), postulates that nitrogenase w o r k s b y a series of t w o f o u r - e l e c t r o n processes.

I n t h e r e d u c t i o n of d i n i t r o g e n , t h e

step w o u l d b e t h e p r o d u c t i o n of N H 2

p r o d u c t i o n of 2 N H

3

and H

2

4

w h i l e the second w o u l d be

first the

w h i c h n i c e l y explains t h e 1:1 s t o i c h i o m e t r y

f o r d i n i t r o g e n a n d d i h y d r o g e n d i s c u s s e d a b o v e . T h e r e s i d u a l r e d u c t i o n of

20.

STIEFEL E T A L .

383

Molybdoenzymes

acetylene i n the p r e s e n c e of d i n i t r o g e n is e x p l a i n e d b y a s e q u e n t i a l disass o c i a t i o n of a m m o n i a a n d d i h y d r o g e n f r o m the e n z y m e w i t h the a c t i v a t e d h y d r o g e n ( or protons a n d electrons ) o n t h e e n z y m e b e i n g a b l e to r e d u c e acetylene ( b u t o n l y to e t h y l e n e ). T h i s m o d e l does n o t f u l l y e x p l a i n w h y acetylene is o n l y r e d u c e d t o e t h y l e n e b y nitrogenase i n t h e absence of d i n i t r o g e n i f t h e f o u r - e l e c t r o n process is r e a l l y t h e b a s i c step. THE

EQUILIBRATING H Y D R O G E N / N I T R O G E NM O D E L .

This

model

(52,

103) postulates, i n close a n a l o g y to i n o r g a n i c systems, t h a t a d i h y d r i d e


site o n t h e e n z y m e is r e a c t i v e t o w a r d s d i n i t r o g e n b i n d i n g i n a m a n n e r exactly

analogous

t o t h e reactions

of k n o w n

molybdenum

d i h y d r i d e s (e.g., R e a c t i o n s 21 a n d 2 2 ) . T h e 1:1 N : H 2

FeH (PEtPh ) 4

2

MoH (dppe) 4

2

+ N

2

+ 2N

2

3

-> F e H N ( P E t P h )

d i r e c t l y e x p l a i n a b l e i n this m o d e l .

2

2

2

Mo(N ) (dppe) 2

2

2

+ H

3

and iron

s t o i c h i o m e t r y is

2

+ 2H

(21)

2

(22)

2

A l t h o u g h the apparent

competitive

i n h i b i t i o n of n i t r o g e n fixation b y d i h y d r o g e n is also e x p l a i n a b l e i n s u c h a m o d e l , o u r recent results (60) i n d i c a t e t h e absence of t r u e c o m p e t i t i v e i n h i b i t i o n i n nitrogenase s u c h that this l a t t e r p o i n t is m o o t .

Further, if

s u c h a n e q u i l i b r i u m w e r e o p e r a t i v e , d i h y d r o g e n i n h i b i t i o n o f other s u b strate reactions w o u l d b e p r o b a b l e . H o w e v e r , i n p r a c t i c e , o n l y d i n i t r o g e n r e d u c t i o n is i n h i b i t e d , a n d t h e l i k e l y m e c h a n i s m f o r t h a t process is d e s c r i b e d e a r l i e r i n this p a p e r . THE

REDUCTIVE

ELIMINATION HYPOTHESIS.

The

1:1

stoichiometry

of d i h y d r o g e n e v o l v e d a n d d i n i t r o g e n r e d u c e d c a n also b e e x p l a i n e d i f f o r m a t i o n a n d d i s p l a c e m e n t of d i h y d r o g e n at t h e m o l y b d e n u m site w e r e a n i n t e g r a l p a r t o f d i n i t r o g e n r e d u c t i o n b u t w e r e unnecessary f o r acetyl e n e r e d u c t i o n , i.e., t w o - e l e c t r o n a n d s i x - e l e c t r o n substrates a r e r e d u c e d v i a related b u t somewhat

different c a t a l y t i c cycles

(96).

evolution might occur b y reductive elimination from

a

Dihydrogen molybdenum

d i h y d r i d e , thus m a k i n g a n e l e c t r o n p a i r o n m o l y b d e n u m a v a i l a b l e f o r substrate r e d u c t i o n . W i t h d i n i t r o g e n , this d i h y d r o g e n e h m i n a t i o n m i g h t b e r e q u i r e d to i n i t i a t e e a c h r e d u c t i o n c y c l e w h e r e a s f o r acetylene, t h e d i h y d r o g e n e v o l u t i o n is r e q u i r e d o n l y i n i t i a l l y t o p r i m e t h e site f o r m u l t i p l e acetylene

r e d u c t i o n s , i.e., t h e site r e m a i n s p r i m e d after

acetylene

r e d u c t i o n b u t r e q u i r e s r e p r i m i n g after t h e s i x - e l e c t r o n d i n i t r o g e n r e d u c t i o n . T h e reason f o r the difference m a y i n v o l v e t h e l a r g e r n u m b e r o f oxo groups p r o d u c e d o n m o l y b d e n u m d u r i n g n i t r o g e n fixation a n d the consequent requirement for their removal. cussed

below)

A T P m a y also f u n c t i o n (as d i s -

i n this site c l e a r i n g ( o x o r e m o v a l )

w h i c h results i n

d i h y d r o g e n e v o l u t i o n . T h e n o n r e c i p r o c a l n a t u r e of t h e m u t u a l i n h i b i t i o n of d i n i t r o g e n a n d acetylene c o u l d b e e x p l a i n e d b y d i n i t r o g e n r e d u c t i o n r e q u i r i n g a n i n i t i a l a c t i v a t i o n at a s e c o n d m e t a l ( i r o n ) a t t h e site, w h i l e

384

BIOINORGANIC

CHEMISTRY

II

a c e t y l e n e does not. F u r t h e r e x p e r i m e n t a l e l a b o r a t i o n is c l e a r l y n e e d e d to d i s t i n g u i s h these p o s s i b i l i t i e s . The A T P Utilization Reaction.

U n d e r o p t i m u m conditions, nitro

genase r e q u i r e s t h e h y d r o l y s i s of 4 - 5 moles of A T P p e r e l e c t r o n p a i r t r a n s f e r r e d (48, 58, 5 9 ) .

A s this represents the e x p e n d i t u r e of 100 k c a l /

m o l e p e r d i n i t r o g e n r e d u c e d , a c l e a r l y p e r t i n e n t q u e s t i o n arises as to t h e r e a s o n f o r the A T P r e q u i r e m e n t .

A m o n g the m a n y suggestions for t h e

r o l e of A T P are those i n v o l v i n g A T P i n a c o n f o r m a t i o n a l c h a n g e of either


t h e i r o n a n d / o r m o l y b d e n u m - i r o n p r o t e i n s . W h i l e this f u n c t i o n of A T P m a y w e l l b e i m p o r t a n t , here w e focus o n those suggestions i n v o l v i n g specific c h e m i c a l i n t e r a c t i o n s b e t w e e n A T P a n d the m o l y b d e n u m site. S u c h suggestions

( w h i c h are n o t necessarily p r e c l u s i v e of a c o n c u r r e n t

c o n f o r m a t i o n a l c h a n g e ) g e n e r a l l y i n v o l v e A T P i n the g e n e r a t i o n of a n o p e n c o o r d i n a t i o n site o n m o l y b d e n u m . W e h a v e n o t e d p r e v i o u s l y that M o - O l i n k a g e s p e r v a d e the c h e m i s t r y of m o l y b d e n u m i n h i g h o x i d a t i o n states a n d that the r e m o v a l of

Mo-O

is n o t a n easy task. A T P , b y its a b i l i t y to act as e i t h e r a p h o s p h o r y l a t i n g agent or a p r o t o n source, c o u l d f a c i l i t a t e the oxo r e m o v a l r e a c t i o n .

One

p o s s i b i l i t y is that A T P couples p h o s p h o r y l a t i o n of the M o - O g r o u p w i t h r e d u c t i o n of the site b y t w o electrons. electron-phosphoryl

T h e rationale for the

coupled

g r o u p transfer is s i m i l a r to t h a t for t h e

coupled

e l e c t r o n - p r o t o n transfer w i t h the e l e c t r o p h i l i c p h o s p h o r y l g r o u p s e r v i n g i n p l a c e of the p r o t o n . 0

Il

0

T h u s , as s h o w n i n R e a c t i o n 23, M o - O

0

I I

I I

I

I

R—0—Ρ—Ο—Ρ—Ο—Ρ—Ο" +

1

ο-

could,

Ο"

0=Mo(VI)

2e"

0"

(ATP) 0

0

0

R — 0 — Ρ — 0 — Ρ — Ο " + Ό — P — 0 — M o (IV)

1

o-

I

(23)

I

o-

o-

(ADP) Pi + M o (IV)

Molybdoenzymes: The Role of Electrons, Protons, and Dihydrogen

Recommend Documents