MÃ¶ssbauer Spectroscopy and Its Chemical Applications - American

13 Novel Iron-Sulfur Clusters ECKARD

MÜNCK

Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: July 1, 1981 | doi: 10.1021/ba-1981-0194.ch013

Gray Freshwater Biological Institute, Department of Biochemistry, University of Minnesota, P.O. Box 100, Navarre, M N 55392

Three novel iron-sulfur clusters have emerged during the past few years from the study of various biological systems. Two of the new centers are components of the molybdenumand iron-containing protein (nitrogenase) of the biological nitrogen fixation system.

During the past few months

Möss

bauer data and x-ray diffraction studies have given evidence for a cluster containing three spin-coupled iron atoms and (probably equimolar amounts of) sulfide. W e characterized this center in detail for two ferredoxins from A . vinelandii and D . gigas. The new group also was present in aconitase from beef-heart

mitochondria and in glutamate

synthase

from E. coli. This chapter focuses mainly on the spectro scopic evidence for this new three-iron center.

i p v u r i n g the past 15 years a f a s c i n a t i n g s u b f i e l d of m o d e r n b i o c h e m i s t r y , the f a m i l y o f i r o n - s u l f u r p r o t e i n s , has d e v e l o p e d .

D u r i n g the past

d e c a d e this d i s c i p l i n e has m a t u r e d into a s o p h i s t i c a t e d b r a n c h of m o d e r n research, a n d i t has s p a w n e d t h e v i g o r o u s p u r s u i t of d e s i g n i n g s t r u c t u r a l m o d e l s f o r t h e m e t a l centers

discovered

i n the proteins.

This work

c u l m i n a t e d w i t h t h e syntheses of [ 2 F e - 2 S ] a n d [ 4 F e - 4 S ] centers b y R. H . H o l m ' s group

( I ) . A b o u t five years a g o i t a p p e a r e d t h a t t h e

fundamental units h a d been discovered. however,

were

characterized.

s i g n a l i n g that

S o m e strange E P R s p e c t r a ,

a d d i t i o n a l structures

remained

to b e

I n d e e d , three n o v e l i r o n - s u l f u r centers w e r e f o u n d since

1975. T w o of t h e n e w structures reside o n t h e m o l y b d e n u m - a n d i r o n containing protein

(MoFe

protein)

of nitrogenase; t h e y seem t o b e

specific i n g r e d i e n t s t o c a r r y o u t t h e b i o l o g i c a l n i t r o g e n fixation process. T h e t h i r d n o v e l cluster, a s p i n - c o u p l e d center c o n t a i n i n g t h r e e i r o n atoms a n d i n o r g a n i c s u l f u r , w a s d i s c o v e r e d v e r y r e c e n t l y ; d u r i n g t h e past f o u r m o n t h s w e f o u n d this s t r u c t u r e to b e present i n f o u r different p r o t e i n s 0065-2393/81/0194-0305$05.25/0 © 1981 American Chemical Society Stevens and Shenoy; Mössbauer Spectroscopy and Its Chemical Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1981.

306

M O S S B A U E R SPECTROSCOPY A N D ITS C H E M I C A L A P P L I C A T I O N S

a n d e n z y m e s , a n d at p r e s e n t i t appears t h a t this n e w c e n t e r m a y

be

d i s t r i b u t e d w i d e l y i n n a t u r e . S i n c e M o s s b a u e r s p e c t r o s c o p y has b e e n a n i n d i s p e n s a b l e t o o l i n c h a r a c t e r i z i n g this center a n d since t h e d e c i s i v e clues w e r e f u r n i s h e d b y this t e c h n i q u e , w e w i l l focus i n this c h a p t e r o n this n o v e l s t r u c t u r e . M o r e o v e r , w e w i l l h a v e the o p p o r t u n i t y to e m p l o y a n d discuss some n e w

Mossbauer methodologies

t h a t p r o m i s e to

be

f r u i t f u l i n u n r a v e l i n g the s p e c t r a l i n t r i c a c i e s of m u l t i m e t a l systems. I n the f o l l o w i n g w e

b r i e f l y s u m m a r i z e n o m e n c l a t u r e a n d salient

s p e c t r a l features of the f a m i l i a r i r o n - s u l f u r centers, m e n t i o n t h e n o v e l centers of nitrogenase, a n d p r o c e e d , i n t h e t h i r d section, w i t h a b r i e f h i s t o r y of t w o p r o t e i n s that c o n t a i n the n e w t h r e e - i r o n center.

I n the


f o u r t h section w e discuss recent M o s s b a u e r a n d E P R d a t a , a n d i n t h e fifth

section w e present a d i s c u s s i o n of t h e p r i n c i p a l results a n d a n

o u t l i n e of s o m e i n t e r e s t i n g f u t u r e w o r k . It s h o u l d b e e m p h a s i z e d that t h e w o r k p r e s e n t e d h e r e is the r e s u l t of a f r u i t f u l c o o p e r a t i o n

between

the M o s s b a u e r g r o u p

at the G r a y

F r e s h w a t e r B i o l o g i c a l I n s t i t u t e w i t h o t h e r g r o u p s , i n p a r t i c u l a r those of W . H . O r m e - J o h n s o n , M . I . T . , a n d A . V . X a v i e r , U n i v e r s i t y of L i s b o n , Portugal.

Familiar Centers and the Clusters of Nitrogenase I n this section w e w i l l b r i e f l y r e v i e w t h e p e r t i n e n t features of

[2Fe-

2S] a n d [ 4 F e - 4 S ] centers a n d of the c o f a c t o r centers a n d P-clusters of nitrogenase.

We

(geometrical)

w i l l not discuss r u b r e d o x i n , a l t h o u g h a n i n t e r e s t i n g

t w i s t r e g a r d i n g the s i m p l e s t i r o n - s u l f u r c e n t e r ( o n e

a t o m , no i n o r g a n i c s u l f u r ) w a s r e p o r t e d r e c e n t l y [2Fe-2S] Clusters. of the

[2Fe-2S]

centers

iron

(2).

T h e h i s t o r y of t h e e l u c i d a t i o n of t h e s t r u c t u r e by

means

of

spectroscopic

techniques

is

a

b e a u t i f u l e x a m p l e of h o w t e c h n i q u e s s u c h as the M o s s b a u e r effect, E P R , ENDOR,

N M R , magnetic susceptibility, a n d various optical

methods

c a n b e c o m b i n e d i n a v e r y p o w e r f u l w a y . A l u c i d r e v i e w has b e e n g i v e n by Palmer (3).

T h e most i m p o r t a n t b i o p h y s i c a l p r o p e r t y of the [ 2 F e - 2 S ]

centers is t h e i r a b i l i t y to m e d i a t e r e v e r s i b l e o n e - e l e c t r o n u t i l i z i n g t w o stable o x i d a t i o n states.

transfer

by

I n the o x i d i z e d f o r m the centers

h a v e a d i a m a g n e t i c ( S = 0 ) g r o u n d state; u p o n a one-electron r e d u c t i o n the famous E P R f e a t u r e at g = an S =

1.94 is o b s e r v e d , a s i g n a l r e s u l t i n g f r o m

1 / 2 g r o u n d state. I n 1966 G i b s o n a n d c o - w o r k e r s ( 4 )

proposed

a v e r y elegant m o d e l i n v o l v i n g a n t i f e r r o m a g n e t i c c o u p l i n g ( v i a s u l f u r bridges)

of the t w o i r o n atoms f o r the o b s e r v e d E P R features.

m o d e l w a s c o n f i r m e d e x p e r i m e n t a l l y b y D u n h a m et a l . ( 5 ) et a l . (6);

This

and Miinck

the d e c i s i v e c l u e w a s the o b s e r v a t i o n of p o s i t i v e a n d n e g a t i v e

m a g n e t i c h y p e r f i n e fields w i t h M o s s b a u e r spectroscopy.

Recently, the

salient features of the p r o p o s e d s t r u c t u r e f o r the [ 2 F e - 2 S ] centers w e r e

Stevens and Shenoy; Mössbauer Spectroscopy and Its Chemical Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1981.

13.

Iron-Sulfur

MUNCK

307

Clusters

c o n f i r m e d b y x - r a y d i f f r a c t i o n studies of t h e f e r r e d o x i n f r o m ( 7 ) ; this w o r k also establishes t h a t t h e c o m p o u n d s

phtensis

Spirulina

synthesized

b y H o l m ' s g r o u p are close s t r u c t u r a l m o d e l s of the a c t i v e centers f o u n d i n the e l e c t r o n transfer p r o t e i n s . [ 4 F e - 4 S ] Clusters.

C l u s t e r s of t h e c u b a n e [ 4 F e - 4 S ] t y p e are sta

b i l i z e d b y p r o t e i n s i n three o x i d i z e d states, a l t h o u g h a g i v e n p r o t e i n a p p a r e n t l y u t i l i z e s o n l y a single r e d o x p a i r . I n the Chromatium

vinosum

h i g h - p o t e n t i a l i r o n p r o t e i n ( H i P I P ) the cluster operates at a m i d p o i n t p o t e n t i a l of + 4 2 0 m V . o b s e r v e d at g

z

(8));

=

I n the o x i d i z e d f o r m a n a x i a l E P R s i g n a l is

2.12 a n d gj_ =

2.04

(see O r m e - J o h n s o n a n d Sands

u p o n a one-electron r e d u c t i o n a d i a m a g n e t i c c l u s t e r g r o u n d state


results. I n contrast, most other p r o t e i n s s t a b i l i z e the [ 4 F e - 4 S ] center at a m i d p o i n t p o t e n t i a l of a b o u t —400 m V . I n the l a t t e r case ( e x a m p l e s are the ferredoxins f r o m Bacillus

a n d Bacillus

polymyxa

stearoihermophilus)

t h e o x i d i z e d state is d i a m a g n e t i c w h i l e t h e r e d u c e d state ( S =

1/2)

y i e l d s a s i g n a l at g =

1.92,

and g

x

=

1.94 ( t y p i c a l g-values are g~ — 2.06, g

y

1.88 as o b s e r v e d for t h e B. polymyxa

=

p r o t e i n ; see R e f .

T h e o c c u r r e n c e of three o x i d a t i o n states for t h e [ 4 F e - 4 S ]

8).

clusters has

b e e n r a t i o n a l i z e d b y C a r t e r et a l . ( 9 ) b y t h e three-state h y p o t h e s i s w h i c h proposes t h a t the clusters of r e d u c e d H i P I P a n d the o x i d i z e d ferredoxins are i n e q u i v a l e n t d i a m a g n e t i c states. i n t o the E P R - a c t i v e ( + 1 )

T h e ferredoxins c a n b e

n o m e n c l a t u r e of i r o n - s u l f u r centers (10)). for r e d u c e d f e r r e d o x i n is g

reduced

state ( h e r e w e use t h e n e w rules for

a v

2 is for o x i d i z e d H i P I P .

T h e r e are m a n y examples i n the l i t e r a t u r e w h e r e signals a r o u n d g =

2.01

have been observed i n i r o n - s u l f u r proteins u n d e r o x i d i z i n g conditions. T h e s e features o f t e n h a v e b e e n r e f e r r e d to as " H i P I P - t y p e " signals, a n d f r e q u e n t l y i t w a s c o n c l u d e d ( o r t a c i t l y a s s u m e d ) t h a t a [ 4 F e - 4 S ] cluster i n the ( + 3 )

o x i d a t i o n l e v e l is present. I n the next section w e discuss a

t h r e e - i r o n center as t h e source of m a n y of the r e p o r t e d signals. The P-Cluster of Nitrogenase.

T h e P - c l u s t e r s of n i t r o g e n a s e are

i r o n - s u l f u r centers w i t h u n i q u e spectroscopic p r o p e r t i e s ( t h e s y m b o l P i n d i c a t e s that these centers ( f o u r p e r m o l e c u l e ) component cussed

of

r e s i d e o n the p r o t e i n

t h e M o F e p r o t e i n , i n contrast to t h e M - c e n t e r s , d i s

later, w h i c h b e l o n g

cofactor ( F e M o - c o )

to

the

iron-

of n i t r o g e n a s e ) .

and molybdenum-containing

T h e e l u c i d a t i o n of t h e i r s t r u c t u r a l

features so far has b e e n e n t i r e l y i n the r e a l m of M o s s b a u e r Z i m m e r m a n n et a l . (11)

a n d H u y n h et a l . (12)

spectroscopy.

have published detailed

analyses of M o s s b a u e r spectra o f nitrogenase f r o m Azotobacter (11)

a n d Clostridium

pasteurianum

vinelandii

A l l e v i d e n c e favors the P -

(12).

clusters to b e v a r i a n t s of f a m i l i a r [ 4 F e - 4 S ]

centers.

however,

I n the native protein

the P-clusters are q u i t e u n i q u e .

i s o l a t e d ) t h e P-clusters are d i a m a g n e t i c .

Spectroscopically, (as

A c c o r d i n g to the M o s s b a u e r

d a t a one of the i r o n sites ( l a b e l e d F e * ) is c l e a r l y r e c o g n i z a b l e as h i g h 2


308

M O S S B A U E R S P E C T R O S C O P Y A N D ITS C H E M I C A L A P P L I C A T I O N S

s p i n ferrous i n c h a r a c t e r ( t h i s r e m i n d s one of t h e t r a p p e d ferrous v a l e n c e state o b s e r v e d f o r r e d u c e d

[2Fe~2S]

c e n t e r s ) ; n o s u c h site has

been

o b s e r v e d f o r s t a n d a r d [ 4 F e - 4 S ] clusters. A o n e - e l e c t r o n o x i d a t i o n of t h e P-clusters y i e l d s a n e l e c t r o n i c g r o u n d state w i t h h a l f - i n t e g r a l e l e c t r o n i c spin, S >

3 / 2 ; c u r i o u s l y , this state is E P R - s i l e n t ( Z i m m e r m a n n et a l .

( 1 1 ) h a v e r a t i o n a l i z e d this o b s e r v a t i o n ) .

A t present i t is not clear w h a t

c h e m i c a l features d i s t i n g u i s h t h e P-clusters f r o m other [ 4 F e - 4 S ]

centers.

I t appears t h a t these s p e c t r o s c o p i c a l l y u n i q u e structures h a v e a [ 4 F e - 4 S ] core, as i n d i c a t e d b y core e x t r u s i o n experiments (11,13)

which yielded

t h r e e to f o u r [ 4 F e - 4 S ] clusters, i n a c c o r d a n c e w i t h c o n c l u s i o n s r e a c h e d f r o m M o s s b a u e r spectroscopy Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: July 1, 1981 | doi: 10.1021/ba-1981-0194.ch013

titrations

(II).

( f o u r clusters p e r m o l e c u l e )

T h i s suggests

s i g n a l different l i g a t i o n s of the P - c l u s t e r i r o n atoms. c o u l d i n c l u d e e x p a n s i o n to

and redox

that the u n i q u e s p e c t r o s c o p i c

five-coordination

Such

features

differences

or r e p l a c e m e n t of t h i o l a t e

l i g a n d s b y other n u c l e o p h i l e s . I f w e t e n t a t i v e l y a c c e p t the i d e a t h a t the P - c l u s t e r s are v a r i a n t s of [ 4 F e - 4 S ] centers, a n i n t r i g u i n g q u e s t i o n arises: T o w h a t o x i d a t i o n l e v e l i n C a r t e r s s c h e m e does t h e n a t i v e d i a m a g n e t i c state, P , b e l o n g ?

It

N

seems o b v i o u s to associate the o x i d a t i o n state P (+2)

state. H o w e v e r , the average i s o m e r shift S

ay

w i t h the diamagnetic

N

=

a n o x i d a t i o n l e v e l e v e n m o r e r e d u c e d t h a n the ( + 1 )

0.65 m m / s

0.58 m m / s ) , t h a t i s , t h e o x i d a t i o n state of s u p e r - r e d u c e d the ( + 0 )

suggests

state w h i c h has 8

«

ferredoxin,

state, is i m p l i c a t e d . T h i s is i n a c c o r d a n c e w i t h the o b s e r v a t i o n

of transient g =

1.94 signals u n d e r o x i d i z i n g c o n d i t i o n s .

(So far w e have

not b e e n a b l e to p r o v e u n a m b i g u o u s l y t h a t these signals r e s u l t i n d e e d f r o m the P - c l u s t e r s ; t h e l o w c o n c e n t r a t i o n of the g =

1.94 centers a n d

the presence of a b o u t 30 i r o n atoms i m p o s e f o r m i d a b l e difficulties.) P-clusters c a n b e r e v e r s i b l y o x i d i z e d i n t o the state P

o

x

The

(see F i g u r e 1 ) ;

recent Mossbauer experiments, i n collaboration w i t h W . H . O r m e - J o h n s o n a n d M . H . E m p t a g e , h a v e y i e l d e d a m i d p o i n t p o t e n t i a l of a b o u t m V for t h e P ° / P X

N

C 0 U

p l e ( f o r the A . vinelandii

the p o t e n t i a l seems to b e c o n s i d e r a b l y h i g h f o r a ( + l ) / ( - f - 0 ) O n the other h a n d , —250 m V is v e r y l o w f o r a ( + 3 ) / ( + 2 ) o n l y other p o s s i b l e assignment for t h e P

0

X

/P

N

—250

p r o t e i n ) . T h i s v a l u e for couple.

c o u p l e , the

p a i r . I t is c l e a r t h a t m u c h

i n t e r e s t i n g r e s e a r c h needs to b e d o n e . T h e Cofactor Center of Nitrogenase (M-Centers). s h o w n (14)

I n 1975 i t w a s

b y c o m b i n e d E P R a n d M o s s b a u e r studies t h a t t h e p r o m i n e n t

E P R features at g =

4.32, 3.67, a n d 2.01 o b s e r v e d f o r t h e A .

M o F e p r o t e i n r e s u l t f r o m a n S «= 3 / 2 s p i n - c o u p l e d cluster

vinelandii (M-center)

t h a t contains at least f o u r i r o n sites. ( T h e r e are t w o a p p a r e n t l y i d e n t i c a l M-centers per protein molecule.)

S u b s e q u e n t l y , R a w l i n g s et a l .

(15)

p r o v e d that the M - c e n t e r s c o n t a i n those m e t a l atoms t h a t c o m p r i s e t h e E P R - a c t i v e c o m p o n e n t of the i r o n - a n d m o l y b d e n u m - c o n t a i n i n g c o f a c t o r (FeMo-co)

of nitrogenase. X - r a y a b s o r p t i o n fine s t r u c t u r e studies


(16)

13.

MUNCK

Iron-Sulfur

COFACTOR M

CENTER M

M

S = 0

S =

M

3/

S>1

2

(EPR active)

P - CLUSTER


309

Clusters

frfi

,D---jS S-—D S =0 (EPR s i l e n t )

Figure 1. Stable oxidation states of the cofactor centers and P-clusters of nitrogenase. The hexagon symbol was used to indicate a structure with (most probably) six iron atoms; no structural features are suggested. The cubane structure depicted for the P-clusters is very probable but not an established fact. The symbol D represents three iron atoms that give rise to a Mossbauer quadrupole doublet D; the Fe site yields a doublet labeled Fe (see Refs. 11, 12, 14). 2+

2+

o n t h e m o l y b d e n u m of i s o l a t e d F e M o - c o a n d of h o l o p r o t e i n s t r o n g l y suggest t h a t t h e M - c e n t e r s c o n t a i n i r o n , m o l y b d e n u m , a n d s u l f u r . T h e exact s t o i c h i o m e t r i c ratios of these three elements are at present t h e subject of i n t e n s i v e r e s e a r c h i n m a n y l a b o r a t o r i e s . B y c o m b i n i n g M o s s b a u e r a n d E P R spectroscopy (15),

o n h o l o p r o t e i n s (12,17,18)

and FeMo-co

i t w a s f o u n d t h a t e a c h M - c e n t e r most l i k e l y contains six i r o n atoms

a n d o n e m o l y b d e n u m . T h i s n u m b e r agrees q u i t e w e l l w i t h values ( a b o u t 7 i r o n a t o m s ) o b t a i n e d r e c e n t l y b y c h e m i c a l analysis o f F e M o - c o

(13).

T h e M - c e n t e r , i n t h e p r o t e i n , c a n b e s t a b i l i z e d i n three d i s t i n c t o x i d a t i o n states (see F i g u r e 1 ) .

So f a r t h e r e d u c e d state M

R

has b e e n

observed

o n l y w h e n nitrogenase is p r e p a r e d u n d e r n i t r o g e n fixing c o n d i t i o n s . T h e d i a m a g n e t i c state M

o

x

is a t t a i n e d b y o x i d a t i o n i n a i r or w i t h r e d o x dyes

s u c h as t h i o n i n e . M a n y laboratories h a v e m a d e c o n s i d e r a b l e efforts to assess w h e t h e r i s o l a t e d c o f a c t o r m a t e r i a l contains a m n i o acids s e r v i n g as e x t e r n a l l i g a n d s to t h e M - c e n t e r s a n d w h e t h e r a n y o r g a n i c c o m p o n e n t is present at a l l . C u r r e n t l y i t appears that n o s u c h c o m p o n e n t s

are present a n d t h a t t h e

c o f a c t o r is r e m o v e d f r o m t h e p r o t e i n as a n a n i o n . I t has b e e n

shown

r e c e n t l y that n o n e of t h e i n g r e d i e n t s of t h e o r i g i n a l e x t r a c t i o n p r o c e d u r e of S h a h a n d B r i l l is a b s o l u t e l y r e q u i r e d ( B . K . B u r g e s s , W . E . N e w t o n , a n d E . I . S t i e f e l , p r i v a t e c o m m u n i c a t i o n ) . A p p a r e n t l y , buffer a n d solvent m o l e c u l e s c o o r d i n a t e t o t h e M - c e n t e r s i n F e M o - c o to r e p l a c e l i g a n d s f u r n i s h e d b y t h e p r o t e i n . T h i s is q u i t e s u r p r i s i n g since M o s s b a u e r a n d E P R studies d e m o n s t r a t e t h e s t r u c t u r a l s i m i l a r i t y of t h e F e M o - c o cluster to t h e p r o t e i n - b o u n d M - c e n t e r s . R e p o r t s f r o m v a r i o u s laboratories s u g gest t h e presence of a b o u t f o u r s u l f u r atoms i n F e M o - c o .

Since precise


310

MOSSBAUER SPECTROSCOPY A N D ITS C H E M I C A L APPLICATIONS

s u l f u r analyses are e x t r e m e l y difficult to o b t a i n i n the p r e s e n c e of m o l y b d e n u m , these findings, as m o s t results for F e M o - c o , s h o u l d be c o n s i d e r e d as tentative. W e s h o u l d l i k e to p o i n t out t h a t t h e o b s e r v e d i s o m e r shifts of the M - c e n t e r s d o not d e m a n d a s u l f u r c o o r d i n a t i o n of t h e i r o n atoms ( n o r d o t h e y r u l e out a s u l f u r e n v i r o n m e n t , see R e f . 1 9 ) . T h i s s h o u l d n o t d i s c o u r a g e chemists f r o m c o n t i n u i n g to d e s i g n cofactor m o d e l s b y c l e v e r l y combining and modifying [4Fe-4S]

cubes; h o w e v e r , o t h e r i r o n c o o r d i

nations are w o r t h c o n s i d e r a t i o n . O n e m i g h t c o n t e m p l a t e the p o s s i b i l i t y that some i r o n atoms of F e M o - c o are l i n k e d b y oxo-bridges.

It should be

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

l s

O or

1 7

0.

( M a s s spectroscopy a n d s p i n - e c h o m o d u l a t i o n experiments c o u l d p r o v i d e Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: July 1, 1981 | doi: 10.1021/ba-1981-0194.ch013

useful information.)

Background on Two Ferredoxins I n 1971 L e G a l l ' s g r o u p i n M a r s e i l l e i s o l a t e d a v e r y i n t e r e s t i n g f e r r e d o x i n f r o m the s u l f a t e - r e d u c i n g b a c t e r i u m Desulfovibrio

T h e basic

gigas.

s u b u n i t of this p r o t e i n has 57 a m i n o acids ( 6 c y s ) of k n o w n s e q u e n c e T h e f e r r e d o x i n is i s o l a t e d i n t w o different o l i g o m e r i c forms. F d I I

(20).

is a t e t r a m e r f o r m e d f r o m i d e n t i c a l m o n o m e r s .

It has b e e n r e p o r t e d

that F d I I mediates e l e c t r o n transfer b e t w e e n c y t o c h r o m e

c

(21)

a n d the

3

sulfite reductase system, w h i l e another o l i g o m e r i c f o r m , t h e t r i m e r i c F d I , c a n serve as a c a r r i e r i n t h e p h o s p h o r o c l a s t i c r e a c t i o n .

The

magnetic

p r o p e r t i e s of the i r o n - s u l f u r centers i n t h e t w o o l i g o m e r i c forms

are

d r a s t i c a l l y different (22).

R e d u c e d F d I exhibits E P R features at g

1.92, g

2.07, signals i n d i c a t i v e of [ 4 F e - 4 S ] centers i n

y

1.94, a n d g

=

the ( + 1 )

z

=

x

=

state. I n sharp contrast, F d I I exhibits a n e a r l y i s o t r o p i c s i g n a l

around g =

2.01 i n the o x i d i z e d state; this s i g n a l vanishes u p o n a o n e -

electron reduction.

T h e m i d p o i n t p o t e n t i a l s of F d I a n d F d I I

d e t e r m i n e d to b e at —455 m V a n d —130 m V , r e s p e c t i v e l y .

were

Although

t h e r e are n o t a b l e differences b e t w e e n H i P I P a n d F d I I i n g-values a n d r e d o x p o t e n t i a l , the e n t i r e p a t t e r n of E P R observations w a s i n t e r p r e t e d b y C a m m a c k et a l . (22)

as i n d i c a t i n g [ 4 F e - 4 S ]

clusters s t a b i l i z e d i n

different r e d o x p a i r s i n F d I a n d F d I I , a c c o r d i n g to C a r t e r s three-state s c h e m e (see the s e c o n d s e c t i o n ) . T h e i d e a of a [ 4 F e - 4 S ] center s t a b i l i z e d i n different r e d o x p a i r s b y t h e same m o n o m e r i c u n i t has c a u s e d

con

s i d e r a b l e excitement. Shethna and subsequently Y o c h and A n i o n have isolated a ferredoxin ( M W 14400) f r o m A . vinelandii equal

amounts

of

labile sulfur.

c o n t a i n i n g 6 - 8 i r o n atoms a n d a b o u t Subsequently,

r e p o r t e d p o t e n t i o m e t r i c t i t r a t i o n s of

Sweeney

et a l .

t h e p r o t e i n , a n d suggested

(23) the

presence of t w o [ 4 F e - 4 S ] cores t h a t are E P R - a c t i v e i n t h e o x i d i z e d state a n d t h a t are e a c h r e d u c e d b y a o n e - e l e c t r o n step i n t o a n E P R - s i l e n t state.


13.

Iron-Sulfur

MUNCK

311

Clusters

T h e y p r o p o s e d that the t w o clusters o p e r a t e d i n the ( + 3 ) / ( + 2 )

states

as does H i P I P , a l t h o u g h the t w o centers h a v e d r a s t i c a l l y different m i d p o i n t p o t e n t i a l s ( + 3 2 0 m V a n d —450 m V ) . B o t h centers, i n the o x i d i z e d state, e x h i b i t n e a r l y i s o t r o p i c E P R signals a r o u n d g = (24)

2.01. H o w a r d et a l .

i n t e r p r e t e d t h e i r cluster d i s p l a c e m e n t experiments as i n d i c a t i v e of

t w o [ 4 F e - 4 S ] centers, w h i l e A v e r i l l et a l . ( 2 5 ) one s u c h cluster. I n 1979, Stout (26)

f o u n d e v i d e n c e for o n l y

r e p o r t e d the analysis of a n x - r a y

d i f f r a c t i o n s t u d y at 4 - A r e s o l u t i o n , s u g g e s t i n g t h e presence of one 2S] cluster a n d one [ 4 F e - 4 S ] center.

[2Fe-

W e w e r e q u i t e e x c i t e d a b o u t this

r e p o r t since it suggested t h a t s o m e t h i n g n e w w a s to b e d i s c o v e r e d : S i n c e f a m i l i a r [ 2 F e - 2 S ] centers are E P R - a c t i v e o n l y i n the r e d u c e d state, a n d Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: July 1, 1981 | doi: 10.1021/ba-1981-0194.ch013

since the p r e s u m e d [ 2 F e - 2 S ] center y i e l d e d a n E P R s i g n a l i n the o x i d i z e d state, the p u b l i s h e d d a t a suggested t h a t either a h i g h e r o x i d a t i o n state of a [2Fe-2S] According

center w a s a t t a i n e d or that a n e w s t r u c t u r e w a s to

present

spectroscopic

evidence

present.

and a

(27,28)

recent

r e i n t e r p r e t a t i o n of the e l e c t r o n d e n s i t y m a p at 2.5-A r e s o l u t i o n , a n o v e l t h r e e - i r o n center is present i n the t w o ferredoxins.

Spectroscopic Evidence for a New Three-Iron Center Together w i t h co-workers W . H . O r m e - J o h n s o n a n d M . H . E m p t a g e w e have studied a landii

(27).

5 7

F e - e n r i c h e d s a m p l e of the f e r r e d o x i n f r o m A .

S h o r t l y after w e s u b m i t t e d t h e Azotobacter

vine-

w o r k w e started

a s t u d y of F d I I , together w i t h A . V . X a v i e r , J . J . G . M o u r a , a n d I . M o u r a (28). Fd

I n the f o l l o w i n g w e treat b o t h p r o t e i n s b u t w e focus m a i n l y o n

I I since t h e presence

of o n l y one

cluster facilitates the analysis

c o n s i d e r a b l y . T h e r e a d e r m a y k e e p i n m i n d t h a t t h e results w e r e o b t a i n e d d u r i n g the past f e w m o n t h s a n d that m a n y details n e e d to b e w o r k e d out.

A l l c h e m i c a l , M o s s b a u e r , a n d E P R d a t a r e p o r t e d here o n F d I I

w e r e t a k e n o n t h e selfsame s a m p l e ; i r o n a n d s p i n concentrations quoted per monomer. 5 7

Fe

are

T h e Mossbauer data were taken on a sample w i t h

i n natural abundance.

(The

s u l f a t e - r e d u c i n g D . gigas

bacteria

p r o d u c e sulfide w h i c h p r e c i p i t a t e s i r o n i n t h e g r o w t h m e d i u m a n d t h u s p r e c i o u s dollars i f

5 7

F e were used.)

T h e F d I I s a m p l e w a s a n a l y z e d f o r e i g h t stable a m i n o a c i d s ; t h e i r d i s t r i b u t i o n w a s f o u n d to fit t h e k n o w n sequence. T h u s , t h e s a m p l e w a s p u r e i n p r o t e i n a n d t h e p r o t e i n c o n c e n t r a t i o n w a s therefore k n o w n . I r o n analyses

yielded repeatedly,

to

within

3%,

3 iron

atoms/monomer.

T h e M o s s b a u e r d a t a w i l l p r o v e t h e absence of a n y d i s c e r n a b l e i r o n impurity.

A n E P R s p e c t r u m of o x i d i z e d F d I I is s h o w n i n F i g u r e 2.

W e s t u d i e d the E P R s a t u r a t i o n b e h a v i o r o f the s a m p l e over a w i d e r a n g e of t e m p e r a t u r e s a n d f o u n d that o n l y one E P R - a c t i v e species is present. A g o o d r e p r e s e n t a t i o n of the o b s e r v e d s p e c t r u m w a s o b t a i n e d b y c h o o s i n g



312

M O S S B A U E R S P E C T R O S C O P Y A N D ITS C H E M I C A L A P P L I C A T I O N S

g = 2.02

g=2.00

Figure 2 . EPR spectrum of oxidized Fd II from D . gigas recorded at 6 K. The derivative of the absorption is plotted vs. the magnetic induction. gi =

2.02, g

2

=

2.00, a n d g

=

3

q u a n t i t a t e d the spectra at 6, 8,

1.97

for spectral simulations.

a n d 12 K

against a

s t a n d a r d a n d f o u n d a s p i n c o n c e n t r a t i o n of 0.93 atoms.

±

We

copper-EDTA

0.12 s p i n s / 3 i r o n

T h u s , i f one c a n e s t a b l i s h that a l l i r o n atoms i n the s a m p l e are

connected

w i t h the E P R - a c t i v e center, i t f o l l o w s t h a t a center w i t h 3

c o v a l e n t l y l i n k e d i r o n atoms is present. T h e M o s s b a u e r d a t a w i l l p r o v i d e t h e answer. F i g u r e 3 A shows a M o s s b a u e r s p e c t r u m of o x i d i z e d F d I I t a k e n at 7 7 K . T h e s o l i d l i n e is a least-squares fit g e n e r a t e d b y a s s u m i n g t h a t o n l y one species is present, t h a t is, b y a s s u m i n g t h a t a l l i r o n atoms i n the s a m p l e are e q u i v a l e n t .

T h e q u a l i t y of t h e fit, the s y m m e t r y of

s p e c t r u m , a n d the sharpness of t h e a b s o r p t i o n lines (0.27 m m / s )

the

support

this a s s u m p t i o n . A l s o , there is no e v i d e n c e for a n y i r o n i m p u r i t y . T h e Mossbauer parameters A E 0.03)

mm/s

Q

=

(0.54 ±

0.03)

m m / s and S =

a r e p r a c t i c a l l y the same as those o b s e r v e d

rubredoxin (29).

for

(0.27

=b

oxidized

I n p a r t i c u l a r , the i s o m e r shift suggests a t e t r a h e d r a l

c o o r d i n a t i o n of s u l f u r atoms.

M o r e o v e r , t h e d a t a s h o w t h a t the i r o n

atoms are h i g h - s p i n f e r r i c ( S =

5/2)

i n character. I s o l a t e d S =

5/2 iron

o b v i o u s l y is not present; o t h e r w i s e , a n E P R s i g n a l t y p i c a l of t h e f e r r i c i o n should be observed. F i g u r e 4 shows a s p e c t r u m of t h e s a m p l e t a k e n at 1.5 K . A b r o a d spectrum exhibiting paramagnetic

h y p e r f i n e s t r u c t u r e is o b s e r v e d ,

in

a c c o r d a n c e w i t h t h e i n f o r m a t i o n f r o m E P R w h i c h shows s l o w e l e c t r o n i c



13.

Iron-Sulfur

MUNCK

313

Clusters

i

i

VELOCITY

i

(mm/s)

Figure 3. Zero-field Mossbauer spectra of Fd II from D . gigas. The sample had 26mM iron with Fe in natural abundance: (A) spectrum of oxidized, EPR-active Fd II taken at 77 K; (B) spectrum of reduced Fdll taken at 4.2 K. The solid lines are the result of a least-squares fitting of the quadrupole doublets to the data. 57

i

(

i

i

T

1

1

T

1

•

LU

7\ \ : \/

V

1

1

.

0

2

/

^o.o

o SI

< i

- 4 - 2

1

.

VELOCITY

1

.

(mm/s)

Figure 4. Mossbauer spectrum of oxidized Fd II taken at 1.5 K in a parallel field of 600 G. The solid line plotted over the data is the result of adding three theoretical spectra computed from Equation 1 using the parameters quoted in Table I.


314

M O S S B A U E R SPECTROSCOPY A N D ITS C H E M I C A L

APPLICATIONS

s p i n r e l a x a t i o n b e l o w 10 K . N o t e t h a t t h e d o u b l e t - t y p e s p e c t r u m a r o u n d z e r o v e l o c i t y is q u i t e

different f r o m t h e d o u b l e t o b s e r v e d i n F i g u r e 3 A .

B e f o r e w e discuss the s p e c t r a l d e c o m p o s i t i o n i n d i c a t e d i n F i g u r e 4, w e present s o m e d a t a t a k e n o n t h e f e r r e d o x i n f r o m A . vinelandii

(the protein

w a s l a b e l e d i r o n - s u l f u r p r o t e i n I I I b y S h e t h n a , b u t here w e w i l l l a b e l i t A v I I I for b r e v i t y ) . A M o s s b a u e r s p e c t r u m of s e m i - r e d u c e d A v I I I is s h o w n i n F i g u r e 5. I n the s e m i - r e d u c e d state the h i g h - p o t e n t i a l center of A v I I I is r e d u c e d w h i l e the l o w - p o t e n t i a l center is i n t h e o x i d i z e d , E P R - a c t i v e state. spectrum

shows

essentially t w o

components:

a n intense

The

quadrupole

d o u b l e t ( i n d i c a t e d b y the b r a c k e t ) a n d a m a g n e t i c c o m p o n e n t

(traced


o u t b y the s o l i d l i n e ) . T h e q u a d r u p o l e d o u b l e t has to b e associated w i t h the h i g h - p o t e n t i a l center.

I n m o s t respects this center behaves

indeed

l i k e a n H i P I P - t y p e [ 4 F e - 4 S ] cluster. W e w i l l n o t discuss t h i s c o m p o n e n t here a n y f u r t h e r , b u t w i l l c o n c e n t r a t e o n the m a g n e t i c m a t e r i a l . T o evaluate the magnetic spectrum the f o l l o w i n g remarks m i g h t be h e l p f u l . F o r a system w i t h S =

1/2 exhibiting an isotropic Zeeman term

a n d i s o t r o p i c m a g n e t i c h y p e r f i n e i n t e r a c t i o n , the i n t e r n a l m a g n e t i c p r o d u c e d b y the d o u b l e t is J ?

i n t

=

—A>/g /? . n

n

system the e x p e c t a t i o n v a l u e o f the e l e c t r o n i c s p i n < S > ( o r a n t i p a r a l l e l ) to t h e a p p l i e d m a g n e t i c

field

w i l l be parallel

Thus r7

H.

field

F o r an isotropic

i n t

will

be

p a r a l l e l to it also, regardless of the o r i e n t a t i o n of t h e m o l e c u l e r e l a t i v e to the a p p l i e d field. S i n c e 7 ?

int

is ( e s s e n t i a l l y ) the q u a n t i z a t i o n axis of the

n u c l e u s , the n u c l e a r A m — 0 fines w i l l b e q u e n c h e d w h e n a s a m p l e is s t u d i e d i n a field a p p l i e d p a r a l l e l to the o b s e r v e d g a m m a r a d i a t i o n . I n transverse field the A m =

0 lines w i l l h a v e m a x i m u m i n t e n s i t y . I f

we

r e c o r d a M o s s b a u e r s p e c t r u m i n p a r a l l e l a n d transverse field a n d s u b t r a c t the " t r a n s v e r s e " s p e c t r u m f o r t h a t o b t a i n e d i n p a r a l l e l

field,

we

will

2 oF o

i

5-

r—

CL

cr O

_

10-

-4

-2

0

VELOCITY

2 (mm/s)

4 The Journal of Biological Chemistry

Figure 5. Mossbauer spectrum of Fe-enriched ferredoxin from A . vine landii taken at 4.2 Kin a 500-G parallel field (27). The bracket indicates a doublet resulting from the high-potential [4Fe-4S] center. The solid lines in Figures 5 and 6 are theoretical spectra for Sites 1 and 2, computed with the parameters of Table I. 57



13.

MUNCK

Iron-Sulfur

315

Clusters

-4

-2

0

4

2

VELOCITY

(mm/s) The Journal of Biological Chemistry

Figure

6.

Mossbauer

spectra

of the oxidized Av III (21).

low-potential

center of

Spectrum c was obtained by removing the contribution of the high-potential center quadrupole doublet, using methods as described in Ref. 11. Spectra a and b are theoretical spectra belonging to Sites 1 and 2. Spectrum d is a difference spectrum, obtained by subtracting a 4.2-K spectrum taken in transverse field from that of Figure 5. The solid line in d is a theoretical difference spectrum computed with the parameters of Table I.

generate a difference s p e c t r u m w i t h t h e positions o f t h e t w o n u c l e a r A m =

0 lines c l e a r l y i d e n t i f i e d . F o r A v I I I this p r o c e d u r e has a n o b v i o u s

benefit: t h e h i g h - p o t e n t i a l center that y i e l d s t h e intense d o u b l e t i n F i g u r e 5 is d i a m a g n e t i c , a n d therefore, its s p e c t r u m is p r a c t i c a l l y i n s e n s i t i v e t o t h e d i r e c t i o n of a w e a k

(500 G ) applied

field.

cancels w h e n t h e difference s p e c t r u m is f o r m e d .

T h u s , its contribution A difference

spectrum

obtained for A v III, shown i n F i g u r e 6 d , should be attributable entirely to t h e E P R - a c t i v e , l o w - p o t e n t i a l center. t w o sets of A m =

N o t e that t h e s p e c t r u m e x h i b i t s

0 lines ( i n d i c a t e d b y t h e b r a c k e t s ) , p r o v i n g t h e

presence o f at least t w o m a g n e t i c a l l y u n e q u i v a l e n t subsites. difference s p e c t r u m t h e g e n e r a l features of t h e t w o m a g n e t i c

F r o m the components

are o b t a i n e d q u i t e r e a d i l y . W e c o m p u t e d t h e o r e t i c a l spectra f o r t h e t w o subcomponents from the spin H a m i l t o n i a n ( S = cH = pH-g-S

+ S-A-I

+

eQV 12

Zi

- gp n

n

1/2).

H-I

[ 3 / / - 7 ( 7 + 1) + * ( J „

- V ) ]

a

T h e g-values f o r A v I I I w e r e t a k e n f r o m t h e E P R d a t a , g

x

2.01.

(1)

= g

y

= g

z

=

A E Q is i n d e p e n d e n t of t e m p e r a t u r e a n d is k n o w n f r o m t h e h i g h -


316


t e m p e r a t u r e d a t a . T h e m a g n e t i c h y p e r f i n e c o u p l i n g constants of the t w o i d e n t i f i e d sites are f o u n d f r o m the t o t a l s p l i t t i n g s ( o r t h e s p l i t t i n g s of t h e Am =

0 p a i r s ) . T o account for some details of the l i n e shape w e f o u n d i t

necessary

to i n t r o d u c e some m a g n e t i c

anisotropy.

The

s o l i d lines i n

F i g u r e s 6a a n d b are t h e o r e t i c a l s p e c t r a of Sites 1 a n d 2; the c u r v e i n F i g u r e 6 d is a t h e o r e t i c a l difference s p e c t r a is d i s p l a y e d i n F i g u r e 5.

s p e c t r u m , w h i l e the s u m of

both

T h e s p e c t r a l p a r a m e t e r s are l i s t e d i n

T a b l e I. I n s t r o n g a p p l i e d fields the s i g n of r 7

i n t

c a n be d e t e r m i n e d . W e h a v e

s t u d i e d samples of F d I I a n d A v I I I i n fields u p to 60 k G a n d f o u n d t h a t A < 0 f o r Site 1 w h i l e A > 0 f o r Site 2 (see T a b l e I ) . S i n c e the m a g n e t i c Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: July 1, 1981 | doi: 10.1021/ba-1981-0194.ch013

h y p e r f i n e c o u p l i n g constants of s i n g l e h i g h - s p i n f e r r i c irons are a l w a y s n e g a t i v e , the o b s e r v a t i o n of a p o s i t i v e c o u p l i n g constant gives evidence for spin c o u p l i n g

strong

(5,6).

A t this stage of t h e analysis w e h a v e c l e a r l y i d e n t i f i e d t w o i r o n sites that are associated w i t h the S = s i g n a l present a n d since t w o

1 / 2 center. S i n c e there is o n l y one E P R

field-dependent

m a g n e t i c M o s s b a u e r spectra

are o b s e r v e d , b o t h i r o n sites m u s t p a r t i c i p a t e i n a s p i n - c o u p l e d H o w e v e r , there is one p r o b l e m : t w o S =

complex.

5 / 2 irons c a n n o t b e

to y i e l d the o b s e r v e d h a l f - i n t e g r a l system s p i n S =

1/2;

coupled

a third half-

i n t e g r a l s p i n is r e q u i r e d . T h e F d I I s p e c t r u m of F i g u r e 4 is v e r y s i m i l a r to the

magnetic

s p e c t r u m of t h e A v I I I l o w - p o t e n t i a l center except t h a t the

magnetic

splittings of Sites 1 a n d 2 differ less for F d I I , y i e l d i n g a less-resolved spectrum.

H o w e v e r , the m i s s i n g s p i n shows u p q u i t e n i c e l y i n F d I I :

T h e t h i r d i r o n site, t r a c e d b y the d o t t e d l i n e i n F i g u r e 4, is c h a r a c t e r i z e d b y a very small magnetic hyperfine interaction, A ^

3.5 M H z . W e h a v e

just c o m p l e t e d a series of h i g h - f i e l d studies of o x i d i z e d F d I I . T h e d a t a s h o w t h a t A < 0 for Site 1 w h i l e A > Table I.

Hyperfine Parameters

0 for Site 2, b u t the s i g n of A f o r

for

Oxidized Three-Iron

A . V i n e l a n d i i Ferredoxin Site No. A»(MHz) A j, ( M H z ) il.(MHs) A# (mm/s) 8 (mm/s) Q

1 -37 -42 -45 +0.63 0.27

2

3 +17 +17 +17 +0.63 0.27

4 4 4 0.6 0.3-0.4

Centers

0

D . gigas Fd II 1 -27 -44 -44 +0.54 0.27

2

3 +29 +16 +16 +0.54 0.27

(+)3.5 (+)3.5 ( + )3.5 +0.54 0.27

° Isomer shifts are quoted at 77 K relative to iron metal at room temperature. T o account for the details of line shape some magnetic anisotropy seems to be required. However, within the limited resolution microheterogeneities could have similar effects. The sign of A for Site 3 is extremely difficult to determine and should be considered as tentative. For the simulations of the A v III spectra we used v — 0.7 and a linewidth of 0.3 m m / s ; for F d II we used v = 1 and 0.4 m m / s for the linewidth. For further details see Refs. 27 and 28.


13.

Iron-Sulfur

MUNCK

317

Clusters

Site 3 is e x t r e m e l y difficult to d e t e r m i n e . F i r s t , i n o r d e r to assess the shape of the h i g h - f i e l d spectra of Site 3 one has to u n d e r s t a n d the features of the h i g h - f i e l d spectra of Sites 1 a n d 2 i n d e t a i l ; u n f o r t u n a t e l y , t h e b r o a d spectra i n d i c a t e the p r e s e n c e of m a g n e t i c anisotropics as w e l l as micro-heterogeneities

( t h e latter also seem to b e i n d i c a t e d b y the h i g h -

field t a i l of the E P R s p e c t r u m of F i g u r e 2 ) .

S e c o n d , since the m a g n e t i c

s p l i t t i n g is v e r y s m a l l for Site 3, m a g n e t i c anisotropics c o u l d r e s u l t i n p o s i t i v e a n d n e g a t i v e c o m p o n e n t s of the A tensor.

I f w e assume

an

i s o t r o p i c A tensor for Site 3, t h e h i g h - f i e l d d a t a o n F d I I suggest A >

0.

( B e c a u s e of the s m a l l h y p e r f i n e field the n u c l e a r A m = Site 3 s p e c t r u m o v e r l a p w i t h the A m =

0 lines of t h e

db 1 lines. T h u s , this site is not


d i s c e r n i b l e i n the difference s p e c t r u m i n F i g u r e 6d.) T h e t h e o r e t i c a l spectra s h o w n i n F i g u r e s 4 a n d 6 fit t h e d a t a q u i t e n i c e l y . T h e p a r a m e t e r s q u o t e d i n T a b l e I are tentative a n d c e r t a i n l y n o t u n i q u e . W e b e l i e v e , h o w e v e r , t h a t t h e y represent t h e essential features of the o b s e r v e d spectra. A t this stage of the analysis w e h a v e o b t a i n e d , i n o u r v i e w ,

com

p e l l i n g e v i d e n c e for the existence of a n o v e l t h r e e - i r o n center. T h e case c a n b e s t r e n g t h e n e d f u r t h e r b y s t u d y i n g the one-electron r e d u c e d centers. A

M o s s b a u e r s p e c t r u m of r e d u c e d F d I I , t a k e n at 4.2 K i n z e r o

applied

field,

is s h o w n i n F i g u r e 3 B .

T h e one-electron

r e d u c t i o n has

t r a n s f o r m e d the spectra of a l l irons of F i g u r e 4 i n t o t w o sharp q u a d r u p o l e doublets.

D o u b l e t I has A E

Q

=

(1.47 ±

0.02) m m / s w h i l e D o u b l e t I I has A E (0.30 ±

Q

=

0.03) m m / s a n d 8 = (0.47 ±

(0.46

±

0.03) m m / s a n d 8

=

0 . 0 2 ) m m / s ( i s o m e r shifts are q u o t e d r e l a t i v e to i r o n m e t a l at

298 K ) . A least-squares fit to t h e d a t a ( F i g u r e 3 B ) s h o w e d ,

somewhat

f o r t u i t o u s l y , t h a t the t w o sites are i n the r a t i o of 2.0:1. T h e spectra s h o w n i n F i g u r e 7 s h o w that t h e t h r e e - i r o n centers h a v e a v e r y d i s t i n c t i v e M o s s b a u e r signature. F i g u r e 7 A shows a g a i n t h e z e r o field s p e c t r u m of F i g u r e 3 B ; a s p e c t r u m t a k e n i n a p a r a l l e l field of 600 G is s h o w n i n F i g u r e 7 B . N o t e t h a t b o t h q u a d r u p o l e d o u b l e t s h a v e v i r t u a l l y disappeared

a n d t h a t the

weak

m a g n e t i c h y p e r f i n e interactions.

external

field

has

induced

sizeable

T h i s observation proves immediately

that the e l e c t r o n i c g r o u n d state of the r e d u c e d cluster is p a r a m a g n e t i c , that is, S >

1.

( T h e s p i n m u s t b e a n i n t e g e r because t h e cluster is

r e d u c e d b y a one-electron step.)

F u r t h e r m o r e , it is v e r y clear t h a t the

centers u n d e r c o n s i d e r a t i o n h a v e no s p e c t r a l r e s e m b l a n c e to H i P I P as suggested

i n the l i t e r a t u r e ; r e d u c e d H i P I P has S =

0 ( a n d four i r o n

sites). F i g u r e 8 shows a s p e c t r u m of r e d u c e d F d I I t a k e n at 4.2 K i n a p a r a l l e l field of 10 k G . A b e a u t i f u l l y r e s o l v e d m a g n e t i c h y p e r f i n e p a t t e r n is o b s e r v e d .

W e f o u n d that the h y p e r f i n e fields are essentially s a t u r a t e d

i n a p p l i e d fields of about 2 k G . F o r H >

10 k G w e o b s e r v e d a p a t t e r n


318

MOSSBAUER SPECTROSCOPY A N D ITS C H E M I C A L

CL 0.5

or

O

APPLICATIONS

°/o


CO CD

correct;

0 a n d thus p a r t i c i p a t e s i n s p i n - c o u p l i n g . A t present t h e

system s p i n of the r e d u c e d t h r e e - i r o n centers is not k n o w n . J . J . G . M o u r a has p e r f o r m e d r o o m - t e m p e r a t u r e s u s c e p t i b i l i t y studies of r e d u c e d F d I I u s i n g the N M R m e t h o d at 270 M H z . T h e d i a m a g n e t i c corrections

are

n o w b e i n g assessed so the r o o m - t e m p e r a t u r e s u s c e p t i b i l i t y of the r e d u c e d t h r e e - i r o n center s h o u l d b e a v a i l a b l e soon. T o explore the p h y s i c s of the spectra i n F i g u r e s 7 a n d 8 a b i t m o r e , l e t us assume t h a t S =

2, the v a l u e p r e s e n t l y i n d i c a t e d . T h e fact t h a t a

s i z a b l e i n t e r n a l m a g n e t i c field is o b s e r v e d i n a p p l i e d fields of o n l y 100 G shows t h a t the e l e c t r o n i c r e l a x a t i o n rate is s l o w at 4.2 K . F u r t h e r m o r e , since the m a g n e t i c h y p e r f i n e fields saturate a l r e a d y i n w e a k a p p l i e d the lowest electronic s p i n levels are t w o closely s p a c e d states of separation A (for F d I I w e f o u n d A =

fields, energy

0.35 c m " ) ; for t h e h i g h - s p i n 1

ferrous i r o n the p h y s i c s of s u c h situations has b e e n discussed i n d e t a i l (2,29,31,32).

T h e d i s t i n c t i v e feature of t h e r e d u c e d t h r e e - i r o n centers

is the o b s e r v a t i o n that the values for A are m u c h s m a l l e r t h a n those f o r other c o m p o u n d s .

T h i s c a n b e u s e d to i d e n t i f y the p r e s e n c e of r e d u c e d

t h r e e - i r o n clusters i n proteins w i t h m o r e t h a n one m e t a l center.

L e t us

discuss this for r e d u c e d A v I I I . I n r e d u c e d A v I I I b o t h i r o n centers are E P R - s i l e n t . T h e H i P I P - t y p e [ 4 F e - 4 S ] center is i n the same d i a m a g n e t i c state as o b s e r v e d ( i n F i g u r e 5)

f o r s e m i - r e d u c e d A v I I I ; i n a d d i t i o n , the l o w - p o t e n t i a l center

has

b e c o m e r e d u c e d . A M o s s b a u e r s p e c t r u m of a s a m p l e t a k e n i n zero

field

at 4.2 K is s h o w n i n F i g u r e 9 a ; the s p e c t r u m i n F i g u r e 9 b w a s t a k e n i n a p a r a l l e l field of 500 G .

N o t e t h a t the a p p l i c a t i o n of a 5 0 0 - G field has

e l i c i t e d a m a g n e t i c c o m p o n e n t , most c o n s p i c u o u s l y i n t h e v e l o c i t y r a n g e


320


0 2 O J* o


8go T

c

-4

-2

0

2

4

VELOCITY (mm/s) The Journal of Biological Chemistry

Figure 9. Mossbauer spectra of fully reduced Av III, taken at 4.2 K (a) in zero-field and (b) in a parallel field of 600 G (27). The spectrum in (c), obtained by subtracting the spectrum in (b) from that in (a), should be compared with that shown in Figure 7A. f r o m + 2 m m / s to + 4

mm/s.

W e k n o w from the semi-reduced sample

t h a t the h i g h - p o t e n t i a l c e n t e r is i n a d i a m a g n e t i c state. T h u s , b y s u b t r a c t i n g the s p e c t r u m o f F i g u r e 9 b f r o m t h a t s h o w n i n F i g u r e 9 a its c o n t r i b u t i o n w i l l c a n c e l . T h e r e s u l t i n g difference s p e c t r u m , d i s p l a y e d i n F i g u r e 9c, is p r a c t i c a l l y i d e n t i c a l to t h a t o b t a i n e d w h e n t h e same p r o c e d u r e is u s e d for t h e F d I I d a t a of F i g u r e 7. T h e r e f o r e , t h e s p e c t r u m of r e d u c e d F d I I a n d the s p e c t r u m of t h e r e d u c e d l o w - p o t e n t i a l center of A v I I I are a l m o s t i d e n t i c a l . ( T h e o b s e r v a t i o n that t h e t i n y a b s o r p t i o n f e a t u r e at -f- 0.5 m m / s i n F i g u r e 9 a b r o a d e n s i n a w e a k m a g n e t i c

field

p r o v i d e d us w i t h t h e first c l u e of the existence of t h r e e - i r o n centers.) T h e theoretical spectra i n F i g u r e 8 were simulated using a spin Hamiltonian with S =

2.

I n a p p l i e d fields of m o d e r a t e strengths t h e

e l e c t r o n i c system has a n easy axis of m a g n e t i z a t i o n , a n d Sites I a n d I I a r e c h a r a c t e r i z e d b y i n t e r n a l m a g n e t i c fields of —237 k G a n d + 2 5 0 k G , respectively.

W e r e f r a i n at present f r o m p u b l i s h i n g a c o m p l e t e set of

h y p e r f i n e p a r a m e t e r s ; the a m b i g u i t y p r o b l e m c o m m o n l y associated w i t h m u l t i p a r a m e t e r fits r e q u i r e s m o r e d e t a i l e d studies. ( A set of p a r a m e t e r s u s e d to s i m u l a t e the 1 0 - k G s p e c t r u m m a y b e o b t a i n e d f r o m t h e a u t h o r . ) Discussion I n the p r e c e d i n g section w e d i s c u s s e d i n some d e t a i l t h e M o s s b a u e r results o b t a i n e d for F d I I a n d t h e l o w - p o t e n t i a l c e n t e r of the f e r r e d o x i n


13.

MUNCK

Iron-Sulfur

f r o m A . vinelandii.

321

Clusters

I n t h e f o l l o w i n g w e s u m m a r i z e a n d elaborate o n s o m e

of the salient features of the n e w t h r e e - i r o n centers. W e w i l l focus m a i n l y o n F d I I ; e x c e p t for m i n o r details the same conclusions a p p l y for A v I I I . A t temperatures a b o v e 15 K , w h e r e t h e e l e c t r o n i c s p i n r e l a x a t i o n is fast, a l l three i r o n sites of o x i d i z e d F d I I e x h i b i t the same doublet.

T h e o b s e r v e d i s o m e r shifts, 8 =

quadrupole

0.27 m m / s , s t r o n g l y

suggest

h i g h - s p i n f e r r i c - t y p e i r o n i n a t e t r a h e d r a l e n v i r o n m e n t of s u l f u r atoms. A l t h o u g h such an environment, b y analogy w i t h well-characterized com p o u n d s , is s t r o n g l y i m p l i c a t e d , w e cannot

r u l e out a site w i t h

three

t h i o l a t e l i g a n d s a n d , f o r e x a m p l e , one o x y g e n i c or nitrogeneous l i g a n d ; n o s u i t a b l e reference

compounds

are a v a i l a b l e for c o m p a r i s o n .


t h r e e i r o n atoms seem to b e e q u i v a l e n t w h e n o n l y A E

Q

While all

a n d 8 are c o n

s i d e r e d , t h e l o w - t e m p e r a t u r e d a t a r e v e a l d r a s t i c differences b e t w e e n t h e i r o n sites w h e n the m a g n e t i c p r o p e r t i e s are t a k e n i n t o account.

N o t only

d o the A values h a v e different signs, a s t r o n g i n d i c a t i o n of s p i n - c o u p l i n g , b u t t h e i r m a g n i t u d e s also differ a p p r e c i a b l y .

T h e r e are essentially t w o

p o s s i b i l i t i e s for r a t i o n a l i z i n g t h e v a s t l y different s p l i t t i n g s . F i r s t ,

the

i n t r i n s i c A values m i g h t b e different f o r the t h r e e sites, t h a t is, l i g a n d g e o m e t r y a n d c o v a l e n c y effects are at the root of the o b s e r v e d

differences.

H o w e v e r , this is u n l i k e l y because these w o u l d affect the values of 8 a n d A E , at least to some o b s e r v a b l e

extent.

q

Since 8 indicates tetrahedral

e n v i r o n m e n t s of s u l f u r atoms, one w o u l d expect the i n t r i n s i c A values to b e close to those o b s e r v e d for r u b r e d o x i n ^ 2 9 ) t h a t is, A ^

a n d desulforedoxin

- 2 2 M H z (defined b y A l f i • I w i t h S — 5 / 2 ) . x

(1),

I n our v i e w ,

the o b s e r v e d difference i n the m a g n e t i c s p l i t t i n g s reflects the m e c h a n i s m of s p i n - c o u p l i n g . W h a t i s j n e a s u r e d are A values r e f e r r i n g to the c o u p l e d system s p i n ; n a m e l y , S • A • I w h e r e S =

1/2.

I t is w e l l k n o w n f r o m the

analysis of the s p i n - c o u p l i n g of the [ 2 F e - 2 S ] centers (4,5,6) t i a l l y one measures the c o m p o n e n t s

t h a t essen

of the i n t r i n s i c i n t e r n a l fields a l o n g

the d i r e c t i o n of t h e system s p i n , w h i c h defines the f r a m e of

reference.

T . A . K e n t a n d B . H . H u y n h , i n o u r l a b o r a t o r y , are. p r e s e n t l y s t u d y i n g t h e p r o b l e m of c o u p l i n g three S = T o produce an S =

1/2

constants are r e q u i r e d .

5 / 2 spins to a r e s u l t a n t s p i n S =

1/2.

g r o u n d state, at least three different c o u p l i n g I t appears t h a t the e x p e r i m e n t a l results, b o t h

t h e signs a n d m a g n i t u d e s of the A values, are n i c e l y e x p l a i n e d i n s u c h a model. U p o n r e d u c t i o n b y a one-electron experience p r o f o u n d changes. is essentially a c c o m m o d a t e d

step, a l l three irons of F d I I

E v i d e n t l y the e l e c t r o n e n t e r i n g the cluster b y those t w o i r o n atoms that g i v e rise to

D o u b l e t I. T h e o b s e r v e d i s o m e r shift for the t w o Site I i r o n s , 8 = m m / s , is just b e t w e e n the values o b s e r v e d f o r f e r r i c (8 = a n d ferrous (8 =

0.7 m m / s )

rubredoxin (29).

r o u g h l y at the o x i d a t i o n l e v e l F e

2 < 5 +

0.3

0.47

mm/s)

B o t h irons of Site I are

w h i l e the i r o n of Site I I r e m a i n s


322 Fe

3 +


i n character.

T h e o b s e r v a t i o n t h a t t h e t w o S i t e I i r o n atoms are

i n d i s t i n g u i s h a b l e e v e n i n the w e l l - r e s o l v e d h i g h - f i e l d spectra t h a t t h e y reflect t w o sites i n e q u i v a l e n t

(chemical and

suggests

geometrical)

environments. N u m e r o u s H i P I P - t y p e E P R signals a r o u n d g

—

2.01 h a v e

been

r e p o r t e d i n the l i t e r a t u r e . I n p a r t i c u l a r , a n E P R s i g n a l s i m i l a r to those o b s e r v e d i n F d I I a n d A v I I I has b e e n r e p o r t e d f o r o x i d i z e d aconitase f r o m beef-heart m i t o c h o n d r i a ( 3 3 ) , the K r e b s cycle.

an important enzyme operating i n

H . B e i n e r t a n d J . - L . D r e y e r of

the

U n i v e r s i t y of

W i s c o n s i n h a v e p r o v i d e d us w i t h a s a m p l e of the r e d u c e d e n z y m e . l o w c o n c e n t r a t i o n of Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: July 1, 1981 | doi: 10.1021/ba-1981-0194.ch013

investigation. 5 7

5 7

F e has p u t some restrictions o n t h e

( W e used a 2 - m M sample w i t h

5 7

The

Mossbauer

F e i n natural abundance;

F e - e n r i c h e d beef hearts are m u c h m o r e expensive t h a n beef W e l l i n g t o n . )

T h e d i s t i n c t i v e signature of the t h r e e - i r o n centers, h o w e v e r , w a s a p p a r e n t : T w o spectra o b t a i n e d at 4.2 K i n z e r o field a n d 600 G are p r a c t i c a l l y i d e n t i c a l to those s h o w n f o r F d I I i n F i g u r e 7. T o g e t h e r w i t h t h e E P R information, the Mossbauer data strongly inplicate a three-iron center i n aconitase.

We

particular, we

f o u n d no evidence

f o r a n y other i r o n c o m p o n e n t .

found

of

no

evidence

a h i g h - s p i n ferrous

In

component

a l l u d e d to i n m a n y b i o c h e m i s t r y text books. Recently we

s t u d i e d a n o t h e r p r o t e i n t h a t exhibits a n H i P I P - t y p e

E P R s i g n a l i n t h e o x i d i z e d state. T h e e n z y m e g l u t a m a t e synthase f r o m E . coli has a c o m p l e x groups

(34).

s u b u n i t s t r u c t u r e a n d at least f o u r

U s i n g s i m i l a r difference spectroscopy

prosthetic

as d i s c u s s e d i n t h e

p r e v i o u s section, w e f o u n d that the e n z y m e contains a c l u s t e r w i t h t h e spectroscopic p r o p e r t i e s of t h r e e - i r o n centers.

(This work was performed

i n cooperation w i t h W . H . O r m e - J o h n s o n a n d A . Rendina.) So f a r w e h a v e c o n c e n t r a t e d

e n t i r e l y o n the spectroscopy

of

the

t h r e e - i r o n centers a n d h a v e n o t m e n t i o n e d t h e results of core e x t r u s i o n experiments. aconitase

T h i s t e c h n i q u e has b e e n a p p l i e d to b o t h A v I I I ( 2 7 )

(35)

w i t h the r e s u l t that [ 2 F e - 2 S ]

and

centers are r e c o v e r e d i n

h i g h y i e l d s . W e suspect t h a t the t h r e e - i r o n centers m a y d e c o m p o s e to y i e l d [ 2 F e - 2 S ] cores u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s . A v e r i l l et a l . (25) h a v e r e p o r t e d that the a b s o r p t i o n s p e c t r u m of t h e A v I I I p r o t e i n i n 8 0 % d i m e t h y l s u l f o x i d e w i t h 10 m M b e n z e n e t h i o l c o u l d n o t b e s i m u l a t e d as t h e s u m of [ 2 F e - 2 S ] a n d [ 4 F e - 4 S ] centers. F u r t h e r m o r e , t h e s p e c t r a c h a n g e d s l o w l y w i t h t i m e . I t w o u l d b e i n t e r e s t i n g to k n o w at w h a t stage of t h e e x t r u s i o n p r o c e d u r e the t h r e e - i r o n centers d e c o m p o s e . E x p e r i m e n t a l d a t a o n this q u e s t i o n m i g h t s h e d some l i g h t o n s t r u c t u r a l features of t h r e e - i r o n centers.

the

A l s o , i f the t h i r d i r o n a t o m r e m a i n s o n t h e p r o t e i n ,

interesting isotopic-labeling experiments w o u l d be useful i f

(at

partially) reversible conditions could be found.


least

13.

MUNCK

Iron-Sulfur

323

Clusters

W h a t is t h e s t r u c t u r e of t h e n e w t h r e e - i r o n centers?

Based on x-ray

d i f f r a c t i o n studies at 2 . 5 - A r e s o l u t i o n , Stout et a l . ( 3 6 ) r e i n t e r p r e t e d t h e d a t a o n A v I I I a n d f o u n d t h a t a t h r e e - i r o n center best fits the e l e c t r o n density map.

T h e y also r e p o r t t h a t t h e c l u s t e r is d i s t i n c t l y p l a n a r a n d

that i t cannot be modeled w i t h [2Fe-2S]

or [ 4 F e - 4 S ]

structures.

The

p r o p o s e d m o d e l has not b e e n refined c r y s t a l l o g r a p h i c a l l y , b u t w h a t seems to e m e r g e is a p l a n a r s t r u c t u r e of three i r o n atoms finked b y three sulfurs. Six contacts ( m o s t l y cysteines) w i t h the p r o t e i n are suggested.

F o r details

t h e r e a d e r is r e f e r r e d to t h e a r t i c l e b y Stout et a l . ( 3 6 ) . W e p o i n t e d o u t e a r l i e r t h a t C a m m a c k et a l . ( 2 2 ) t h a t the centers of F d I a n d F d I I are [ 4 F e - 4 S ]

have

suggested

clusters s t a b i l i z e d at


different o x i d a t i o n levels b y the p r o t e i n e n v i r o n m e n t s . W e are p r e s e n t l y s t u d y i n g a n F d I s a m p l e w i t h M o s s b a u e r a n d E P R spectroscopy.

Although

t h e studies are not c o m p l e t e d , w e c a n u n e q u i v o c a l l y state t h a t the center that gives rise to the E P R s i g n a l at g

x

=

1.92, g

y

=

1.94, a n d g

=

z

is i n d e e d a [ 4 F e - 4 S ] center. T h u s t h e m o n o m e r i c u n i t c a n

2.07

accommodate,

i n the p r o p e r o l i g o m e r i c f o r m , t h r e e - i r o n centers a n d [ 4 F e - 4 S ]

clusters.

I t m i g h t a p p e a r t h a t the t e t r a m e r i c f o r m of t h e p r o t e i n a c c o m m o d a t e s t h r e e - i r o n centers, w h i l e the t r i m e r i c o l i g o m e r folds i n t o a c o n f o r m a t i o n that can accept [4Fe-4S] s a m p l e has a b o u t 2 5 %

centers.

H o w e v e r , w e f o u n d t h a t the F d I

of its i r o n i n t h e f o r m of t h r e e - i r o n centers.

A c c o r d i n g to the p u r i f i c a t i o n standards the s a m p l e is h o m o g e n e o u s i n t r i m e r s , that is, there is n o e v i d e n c e f o r a ( t e t r a m e r i c ) F d I I c o n t a m i n a n t . S i n c e e v e n m o l e c u l e s s u c h as r u b r e d o x i n a n d i n s u l i n c a n a c c o m m o d a t e [4Fe-4S]

centers u n d e r the p r o p e r c o n d i t i o n s , m u c h w o r k needs to

be

d o n e to i d e n t i f y the p h y s i o l o g i c a l l y a c t i v e centers of F d I . I t is clear t h a t s o m e e x c i t i n g p r o t e i n c h e m i s t r y lies a h e a d . A t present, extreme c a u t i o n needs to be exercised i n assessing the p h y s i o l o g i c a l significance of t h e c h e m i c a l a n d spectroscopic observations for F d I a n d F d I I . F i n a l l y , w e h a v e r e p o r t e d h e r e the presence of a n o v e l t h r e e - i r o n center i n f o u r p r o t e i n s . T h e r e is q u i t e a v a r i e t y of p r o t e i n s w i t h m a g n e t i c p r o p e r t i e s t h a t also suggest the presence of the t h r e e - i r o n center. t h e m are the ferredoxins f r o m P . ovalis ( 3 9 ) , M . smegmatis

( 3 7 ) , R . rubrum

( 4 0 ) , a n d T . thermophilus

Among

(38), M .

favum

(41).

Acknowledgment T h e work on A v III was performed i n collaboration w i t h W . H . O r m e - J o h n s o n of t h e M a s s a c h u s e t t s I n s t i t u t e of

Technology

a n d his

p o s t d o c t o r a l associates M . H . E m p t a g e a n d J . R a w l i n g s . T h e studies o n the D. gigas ferredoxins are the r e s u l t of a c o o p e r a t i o n w i t h A . V . X a v i e r , J . J . G . M o u r a , a n d I . M o u r a , v i s i t i n g scientists at the G r a y F r e s h w a t e r


324


B i o l o g i c a l I n s t i t u t e f r o m t h e U n i v e r s i t y of L i s b o n , P o r t u g a l . T h e D . gigas bacteria were

grown i n J . LeGall's laboratory i n Marseilles, France.

F i n a l l y , I a m g r a t e f u l to m y p o s t d o c t o r a l associates B . H . H u y n h a n d T . A . K e n t w h o d i d most of t h e spectroscopic w o r k d e s c r i b e d here.

Addendum R e c e n t l y w e h a v e d e m o n s t r a t e d ( 4 2 ) t h a t t h e m a g n e t i c p r o p e r t i e s of the o x i d i z e d t h r e e - i r o n centers c a n b e e x p l a i n e d w i t h a s i m p l e m o d e l of t h r e e h i g h - s p i n f e r r i c ions ( S = 5 / 2 ) e x c h a n g e - c o u p l e d S =

interactions Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: July 1, 1981 | doi: 10.1021/ba-1981-0194.ch013

to a system s p i n

1 / 2 . T h e results s h o w t h a t t h e three sites h a v e i n t r i n s i c m a g n e t i c

observed

like

rubredoxin;

t h e differences

i n the hyperfine

f o r t h e three sites reflect t h e g e o m e t r i c a l features

coupling.

fields

of spin-

I n p a r t i c u l a r , t h e s m a l l h y p e r f i n e field o b s e r v e d f o r Site 3 is

a t t r i b u t a b l e t o a n almost p e r p e n d i c u l a r o r i e n t a t i o n of t h e l o c a l s p i n t o t h e system s p i n . F u r t h e r m o r e , t h e m o d e l suggests s t r o n g l y that t h e t h r e e - i r o n center is a single, c o v a l e n t l y l i n k e d s t r u c t u r e ; i t s h o u l d n o t b e c o n s i d e r e d as a [ 2 F e - 2 S ] cluster w e a k l y c o u p l e d to a t h i r d i r o n a t o m . Together w i t h H . Beinert a n d J . - L . Dreyer w e have continued the M o s s b a u e r studies o f aconitase. D a t a o b t a i n e d f o r o x i d i z e d a n d r e d u c e d aconitase c l e a r l y s h o w t h e presence of a t h r e e - i r o n center.

Literature

Cited

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R E C E I V E D June 27, 1980.


MÃ¶ssbauer Spectroscopy and Its Chemical Applications - American

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