Insights into the Role of Nickel in Hydrogenase - Advances in

2 Insights into the Role of Nickel in Hydrogenase Michael J. Maroney, Michelle A . Pressler, Shaukat A. Mirza, Joyce P. Whitehead, Ryzard J. Gurbiel, and Brian M . Hoffman 1

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Department of Chemistry, University of Massachusetts, Amherst, M A 01003 Downloaded by PURDUE UNIV on April 9, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/ba-1995-0246.ch002

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Department of Chemistry, Northwestern University, Evanston, IL 60280

The nickel site in Thiocapsa roseopersicina hydrogenase was ex amined in the five redox forms defined by electron paramagnetic resonance spectroscopy at 77 Κ by use of X-ray absorption spec troscopy. These studies show that the nickel site is remarkably in sensitive to changes in the redox state of the enzyme, a result that is inconsistent with nickel-centered redox chemistry. Model studies of a series of nickel complexes with ligands of the type RN(CH CH S) show that the products of one-, two-, and four -electronoxidations all reflect sulfur-centered chemistry. The role of the nickel site in binding hydrogen was explored by using a combination of electron nuclear double resonance and X-ray ab sorption spectroscopic techniques. These studies do not completely rule out a role for the nickel site, but they point to the possibility that nickel is not the hydrogen-binding site. These results are dis cussed within the context of biological and inorganic chemical lit erature pertaining to nickel thiolate complexes. 2

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Η YDROGENASES ( H a s e s ) a r e a w i d e l y d i s t r i b u t e d class o f e n z y m e s f o u n d i n b o t h prokaryotes a n d eukaryotes that catalyze t h e reversible t w o - e l e c t r o n o x i d a t i o n o f m o l e c u l a r h y d r o g e n ( e q 1) ( 1 - 3 ) . T h u s , H a s e s may function to provide r e d u c i n g equivalents for energy p r o d u c t i o n v i a the uptake a n d oxidation of H or m a y reduce H i n the production of 2

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H . H y d r o g e n o x i d a t i o n ( u p t a k e ) is g e n e r a l l y c o u p l e d w i t h p h o s p h o r y l a t i o n a n d u l t i m a t e l y w i t h t h e r e d u c t i o n o f i n o r g a n i c substrates s u c h as S 0 " (Desulfovibrio species), C O (Methanobacterium s p e c i e s ) , N 0 " (e.g., Paracoccus denitrificans), o r 0 (Alcaligenes a n d Nocardia). H a s e s 2

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0065-2393/95/0246-0021/$ 11.60/0 © 1995 American Chemical Society

Thorp and Pecoraro; Mechanistic Bioinorganic Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1996.

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M E C H A N I S T I C BIOINORGANIC CHEMISTRY

m a y also p l a y a r o l e i n c y c l i n g h y d r o g e n p r o d u c e d i n o t h e r

systems

(e.g., n i t r o g e n a s e ) o r i n g e n e r a t i n g a p r o t o n g r a d i e n t (1-4). H

^ 2H

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+ 2e"

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H y d r o g e n a s e s h a v e b e e n g r o u p e d i n t o t h r e e classes (J) that are b a s e d on the inorganic content of the enzymes and are immunologically a n d b i o c h e m i c a l l y d i s t i n c t (5). W i t h t h e p o s s i b l e e x c e p t i o n o f a r e c e n t l y purified enzyme, N ,N -methylenetetrahydromethanopterin dehyd r o g e n a s e f r o m Methanobacterium thermoautotrophicum, t h a t possesses H a s e a c t i v i t y b u t d o e s n o t c o n t a i n F e (6, 7), a l l H a s e s c o n t a i n F e , S clusters. F o u r enzymes have b e e n rigorously shown to contain only F e a n d S " (1). W i t h t h e e x c e p t i o n o f a H a s e f r o m D . vulgaris, t h e s e F e o n l y e n z y m e s are m o n o m e r i c p r o t e i n s o f 6 0 - k D a m o l e c u l a r w e i g h t that are e x t r e m e l y 0 sensitive ( f « a f e w m i n u t e s i n air) a n d i r r e v e r s i b l y deactivated. Because they catalyze both H oxidation and H production at h i g h rates i n v i t r o ( V ( H e v o l u t i o n ) « 6 0 0 0 umo\ m i n " m g " ) ; V ( H o x i d a t i o n ) « 2 0 , 0 0 0 umo\ m i n " m g " ) , t h e y a r e f r e q u e n t l y r e f e r r e d t o as b i d i r e c t i o n a l H a s e s . 5

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Downloaded by PURDUE UNIV on April 9, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/ba-1995-0246.ch002

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S i n c e t h e d i s c o v e r y o f N i as a b i o l o g i c a l c o m p o n e n t o f Methanobacterium bryantii i n 1 9 8 0 (8) a n d t h e s u b s e q u e n t i d e n t i f i c a t i o n o f t h e N i c o n t a i n i n g c o m p o n e n t as a H a s e (9), d o z e n s o f e x a m p l e s o f H a s e s t h a t r e q u i r e a s i n g l e N i a t o m as w e l l as F e , S c l u s t e r s h a v e b e e n c h a r a c t e r i z e d . H a s e s b e l o n g i n g t o t h e N i , F e class a r e g e n e r a l l y a s s o c i a t e d w i t h h y d r o g e n o x i d a t i o n i n v i v o a n d a r e w i d e l y d i s t r i b u t e d ; e x a m p l e s are k n o w n f r o m f e r m e n t a t i v e (JO), S 0 - r e d u c i n g (5, 11), m e t h a n o g e n i c (12), p h o t o s y n t h e t i c (4, 13), f a c u l t a t i v e (14), a n d a e r o b i c b a c t e r i a (3, 15). T h e N i , F e H a s e s a r e o f t e n a/3 d i m e r s w i t h s u b u n i t m o l e c u l a r w e i g h t s o f 2

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approximately 60 and 30 k D a . I n enzymes containing more than t w o s u b u n i t s (e.g., i n Alcaligenes eutrophus, a n affyd t e t r a m e r (16)), t w o s u b units w i t h the characteristic molecular weights are usually associated w i t h t h e H a s e activity. T h e N i , F e H a s e s have activities that are o n l y 1 - 1 0 % as l a r g e as t h o s e t y p i c a l o f t h e F e - o n l y e n z y m e s ( V ( H e v o l u t i o n ) « 4 5 0 umol m i n m g " ; V ( H o x i d a t i o n ) « 1 5 0 0 umo\ m i n " m g " ) (5) a n d a r e m o r e o x y g e n t o l e r a n t ( f i n air varies from several h o u r s t o s e v e r a l w e e k s (3)). F u r t h e r m o r e , t h e d e a c t i v a t e d e n z y m e s m a y be reductively reactivated. 2

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A m o n g t h e H a s e s c o n t a i n i n g N i a r e a class o f e n z y m e s t h a t also c o n t a i n S e : t h e N i , F e , S e H a s e s (5). T h e S e is g e n e r a l l y p r e s e n t as a s i n g l e s e l e n o c y s t e i n e r e s i d u e , m o s t n o t a b l y i n D. baculatus. T h e s e l e n o c y s t e i n e r e s i d u e has b e e n s h o w n t o b e e n c o d e d b y a n i n t e r n a l T G A c o d o n (17), t o c o n s t i t u t e a c o n s e r v a t i v e r e p l a c e m e n t f o r a c y s t e i n e r e s i d u e i n e n z y m e s l a c k i n g selenocysteine (17), a n d to b e o n e o f t h e N i l i g a n d s (18, 19). H o w e v e r , e x a m p l e s o f e n z y m e s t h a t c o n t a i n l a b i l e S e a r e also k n o w n i n w h i c h t h e H a s e g e n e d o e s n o t c o n t a i n t h e T G A c o d o n 2

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Insights into the Role of Nickel in Hydrogenase

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(20). A l t h o u g h s e l e n o c y s t e i n e - c o n t a i n i n g e n z y m e s a r e c l e a r l y r e l a t e d t o t h e N i , F e e n z y m e s (21), t h e y a r e e v e n m o r e 0 t o l e r a n t t h a n a r e t h e N i , F e e n z y m e s a n d are t y p i c a l l y isolated i n air i n a f o r m that does not r e q u i r e r e d u c t i v e a c t i v a t i o n (5). T h e c a t a l y t i c a c t i v i t i e s o f t h e N i , F e , S e e n z y m e s are e v e n l o w e r t h a n those f o u n d i n the N i , F e e n z y m e s ( V ( H e v o l u t i o n ) « 4 5 0 umo\ m i n " m g " ; V ( H o x i d a t i o n ) « 1 0 0 umo\ m i n " m g " ) (5). 2

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In general, the trends i n decreasing H a s e uptake and p r o d u c t i o n a c t i v i t y a r e p a r a l l e l e d b y i n c r e a s i n g affinities f o r H a n d h i g h e r H / H D r a t i o s i n p r o t o n - d e u t e r i u m e x c h a n g e assays (5). C l e a r l y , t h e p r e s e n c e o f N i a n d t h e a d d i t i o n o f s e l e n o c y s t e i n e h a v e a n effect o n t h e c a t a l y t i c activities and oxygen sensitivities of the H ases, although the physiol o g i c a l s i g n i f i c a n c e o f t h e s e d i f f e r e n c e s is n o t k n o w n . It a p p e a r s t h a t t h e only feature characteristic of all H a s e s may be m e t a l - s u l f u r bonds. O n e v i e w o f t h e r o l e p l a y e d b y N i is t h a t n a t u r e v a r i e s t h e m e t a l c o m p o s i t i o n o f the active site i n the e n z y m e i n o r d e r to i n f l u e n c e the r e a c t i v i t y of t h e m e t a l s t o w a r d H o r 0 . A n o t h e r v i e w is t h a t t h e r o l e o f N i is t o m o d i f y t h e a c t i v e s i t e , w h i c h m i g h t i n v o l v e c h e m i s t r y t h a t o c c u r s at F e o r at m e t a l l i g a n d s . W e u s e d s p e c t r o s c o p i c t e c h n i q u e s i n c o m b i n a t i o n w i t h a synthetic m o d e l a p p r o a c h to investigate the r o l e o f the N i c e n t e r i n hydrogenase. T h e s e studies are s u m m a r i z e d h e r e w i t h i n the context of the b i o l o g i c a l a n d i n o r g a n i c l i t e r a t u r e p e r t a i n i n g to N i thiolate complexes. 2

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O n e o f t h e m o s t i n t e r e s t i n g a s p e c t s o f t h e H a s e N i site is its assoc i a t i o n w i t h r e d o x c h e m i s t r y i n v o l v i n g u n u s u a l f o r m a l o x i d a t i o n states o f N i . I n c o n t r a s t w i t h N i ( I I ) , w h i c h has a n e v e n n u m b e r o f e l e c t r o n s , t h e p r e s e n c e o f N i i n H a s e s is o f t e n d e t e c t e d b y t h e a p p e a r a n c e o f characteristic r h o m b i c e l e c t r o n p a r a m a g n e t i c resonance ( E P R ) signals ( g = 2 . 4 - 2 . 0 ) i n o x i d i z e d a n d r e d u c e d s a m p l e s o f t h e e n z y m e (2, 3). T h e s e signals h a v e b e e n a s s o c i a t e d w i t h a n S = V2 N i s p e c i e s , f r o m t h e o b s e r v a t i o n o f h y p e r f i n e s p l i t t i n g a r i s i n g f r o m N i - l a b e l e d s a m p l e s (22) a n d have p r o v i d e d the p r i n c i p a l b i o p h y s i c a l p r o b e o f the N i site. T h e " N i E P R signals" i n the e n z y m e can be distinguished f r o m those arising f r o m F e , S c l u s t e r s b e c a u s e t h e y c a n b e o b s e r v e d at 7 7 K , w h e r e a s t h o s e f r o m the F e , S clusters r e q u i r e t e m p e r a t u r e s b e l o w 3 0 K to b e c o m e o b s e r v a b l e . T h e E P R signals o b s e r v e d at 7 7 K h a v e b e e n u s e d t o m o n i t o r t h e r e d o x state o f t h e e n z y m e (2, 3, 23-25) a n d t o i n f e r a n i n t e r a c t i o n o f t h e N i site w i t h i n h i b i t o r s (e.g., C O ) (26), H (27), a n d a n o t h e r p a r a m a g n e t (e.g., a n F e , S c l u s t e r ) (3). 2

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A m o d e l f o r t h e r e d o x c h e m i s t r y o f t h e N i site b a s e d o n r e d o x t i t r a t i o n s o f H a s e is s u m m a r i z e d i n F i g u r e 1. N i , F e H a s e s a r e g e n e r a l l y i s o l a t e d i n a i r as a c o m b i n a t i o n o f t w o f u l l y o x i d i z e d a n d i n a c t i v e f o r m s that can be d i s t i n g u i s h e d b y t h e i r N i E P R spectra a n d t h e i r k i n e t i c s of activation. F o r m A (g = 2 . 3 1 , 2 . 2 3 , a n d 2.02) r e q u i r e s extensive i n c u 2

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Form A g = 231,2.23,2.02 oxidized, inactive +e

it"--

no redox ?

FormB g = 233,2.16,2.01 oxidized, inactive +e

150 mV

Form Bnerf epr silent inactive

FomiArcrf


epr silent inactive

ca.-150mV)

(H

no redox

Em = -270 m V 2H"

FormC g = 2.19,2.14,2.02 active

+ 2e"

•390 mV Fully reduced epr silent inactive Figure 1. A model for the interconversion ofNi,Fe hydrogenase, based on the work of Cammack et al. (3) and Albracht et al (28). The example potentials and pH dependencies are those determined for D. gigas hydrogenase at pH 7 vs. NHE (3). b a t i o n u n d e r H o r t r e a t m e n t w i t h s t r o n g r e d u c i n g agents to b e a c t i v a t e d , w h e r e a s f o r m B ( g = 2 . 3 3 , 2 . 1 6 , a n d 2 . 0 1 ) is i n s t a n t a n e o u s l y a c t i v a t e d b y exposure to H . B o t h forms are i n i t i a l l y r e d u c e d b y H to f o r m an E P R s i l e n t state o f t h e N i c e n t e r . R e c e n t l y o b t a i n e d e v i d e n c e i n d i c a t e s t h a t f o r m B m a y b e c o n v e r t e d to f o r m A , b u t f o r m A is n o t c o n v e r t e d t o f o r m B p r i o r t o r e d u c t i o n . T h i s d a t a i m p l i e s t h a t t h e E P R s i l e n t state m a y b e a n e q u i l i b r i u m m i x t u r e o f t w o f o r m s , o n e t h a t is o x i d i z e d t o f o r m A , t h e o t h e r t o f o r m B (28). I n a n y e v e n t , s t u d i e s o f t h e m a g n e t i c properties of the N i center i n the E P R silent intermediate form of H a s e f r o m D. baculatus (an e n z y m e t h a t c o n t a i n s a s e l e n o c y s t e i n e N i l i g a n d ) i n d i c a t e t h a t t h e N i is d i a m a g n e t i c a n d m u s t t h e r e f o r e b e N i ( I I ) (29). 2

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F u r t h e r e x p o s u r e to H r e s u l t s i n t h e f o r m a t i o n o f a t h i r d E P R a c t i v e f o r m , form C (g = 2.19, 2.14, and 2.02). R e d o x titrations show that the 2


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i n t e n s i t y o f t h i s s i g n a l r e a c h e s a m a x i m u m at t h e s a m e p o t e n t i a l s assoc i a t e d w i t h c a t a l y t i c a c t i v i t y ; t h u s , f o r m C has b e e n a t t r i b u t e d t o a c a t a l y t i c a l l y a c t i v e f o r m o f t h e e n z y m e (3). F o r m C m a y b e f u r t h e r r e d u c e d t o a f u l l y r e d u c e d f o r m t h a t is E P R s i l e n t at 7 7 K . R e d o x t i t r a t i o n s o f s e v e r a l H a s e s h a v e b e e n p e r f o r m e d t o d e t e r m i n e t h e p o t e n t i a l s assoc i a t e d w i t h t h e t r a n s f o r m a t i o n s o f t h e N i E P R s p e c t r a (2, 3 ) . T h e p o tentials associated w i t h t h e redox processes o f N i i n H a s e generally l i e b e t w e e n ~ 0 a n d - 4 1 4 m V (vs. N H E ) , t h e l a t t e r b e i n g t h e p o t e n t i a l o f t h e H / H c o u p l e at p H 7. T h e p o t e n t i a l s d e t e r m i n e d v a r y s o m e w h a t w i t h t h e s o u r c e o f t h e e n z y m e u s e d ; t h o s e d e t e r m i n e d f o r D. gigas a r e s h o w n i n F i g u r e 1. 2

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T h e nature o f t h e l i g a n d e n v i r o n m e n t o f t h e N i c e n t e r has b e e n addressed b y use o f a combination of spectroscopic techniques. X - r a y a b s o r p t i o n s p e c t r a l d a t a a r e n o w a v a i l a b l e f o r N i , F e H a s e s f r o m D. gigas (30-32), M . thermoautotrophicum (33), a n d Thiocapsa roseopersicina (34, 35) a n d f o r t h e N i , F e , S e e n z y m e f r o m D. baculatus (19). I n g e n e r a l , t h e s e s t u d i e s i n d i c a t e t h a t t h e N i site is 5 - 6 c o o r d i n a t e a n d c o n t a i n s at least t w o S - d o n o r l i g a n d s at a d i s t a n c e o f 2 . 2 A . B e c a u s e o f the lack of visible hyperfine interactions i n the enzyme spectra, E P R has n o t b e e n o f m u c h v a l u e i n p r o b i n g t h e l i g a n d e n v i r o n m e n t o f t h e N i . H o w e v e r , t h e u s e o f i s o t o p e s w i t h m a g n e t i c n u c l e i has p r o v e d u s e f u l . Studies u s i n g b a c t e r i a g r o w n o n S - e n r i c h e d m e d i a r e v e a l that t h e N i s i g n a l i n t e r a c t s w i t h 1 - 2 s u l f u r a t o m s (36). E P R h a s also s h o w n t h a t C O interacts strongly w i t h the u n p a i r e d spin, consistent w i t h b i n d i n g o f t h i s i n h i b i t o r t o t h e N i c e n t e r (26). O n t h e o t h e r h a n d , s t u d i e s u s i n g 0 i n the deactivation o f the e n z y m e (formation o f forms A a n d B) revealed only weak hyperfine interactions, leading to the conclusion t h a t 0 d o e s n o t i n t e r a c t d i r e c t l y w i t h N i b u t is b o u n d i n t h e v i c i n i t y o f t h e N i i n b o t h o x i d i z e d f o r m s (26). E l e c t r o n s p i n e c h o e n v e l o p e m o d ulation ( E S E E M ) studies reveal an i n t e r a c t i o n w i t h a N atom i n m a n y (37-39), b u t n o t a l l (39) cases; i t is n o t c l e a r , h o w e v e r , w h e t h e r t h i s interaction represents ligation b y a N - d o n o r ligand. 2

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M o r e i n f o r m a t i o n r e g a r d i n g possible N i - b i n d i n g ligands has b e e n obtained from an examination o f homologies i n 17 amino acid sequences, c o u p l e d w i t h s i t e - d i r e c t e d m u t a g e n e s i s o n Escherichia coli H a s e - 1 a n d studies o f the N i b i n d i n g capacity a n d catalytic activity o f the mutants (21). T h e s e s t u d i e s i n d i c a t e d t h a t t h e l a r g e s u b u n i t o f N i , F e H a s e s c o n tains t h e N i - b i n d i n g site a n d is h i g h l y c o n s e r v e d . W i t h i n t h e a m i n o a c i d sequence of the large subunits are t w o fully conserved sequences: R - X C - X - G - C near the amino terminus and D - P - C - X - X - C near the carboxyl t e r m i n u s . T h e o n l y e x c e p t i o n s a r e t h e case o f t w o N i , F e , S e e n z y m e s , i n w h i c h t h e first c y s t e i n e r e s i d u e i n t h e c a r b o x y l t e r m i n a l r e g i o n is s u b s t i t u t e d b y t h e k n o w n N i l i g a n d , s e l e n o c y s t e i n e , a n d i n t h e case o f M . thermoautotrophicum F - r e d u c i n g H a s e , i n w h i c h t h e c o n s e r v e d 2

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g l y c i n e is s u b s t i t u t e d b y c y s t e i n e . T h e c o n s e r v e d s e q u e n c e s c o n t a i n six possible N i ligands: four cysteines, an aspartate, a n d an arginine. T h e mutagenesis a n d b i o c h e m i c a l studies of the mutants indicate that these six a m i n o acids a r e p o t e n t i a l N i l i g a n d s . H i s t i d i n e s flanking t h e c o n s e r v e d r e g i o n s w e r e less w e l l c o n s e r v e d , a n d m u t a n t s l a c k i n g t h e s e r e s i d u e s were active H ases. 2


T h e structure that emerges f r o m the p h y s i c a l a n d b i o c h e m i c a l s t u d ies is o n e t h a t c o n t a i n s a 5 - 6 c o o r d i n a t e N i a t o m i n a m i x e d l i g a n d e n v i r o n m e n t , f e a t u r i n g at least t w o c y s t e i n a t e l i g a n d s a n d o n e site t h a t is a v a i l a b l e f o r b i n d i n g e x o g e n o u s l i g a n d s .

Ni Redox Chemistry V a r i o u s s c h e m e s u s i n g f o r m a l N i o x i d a t i o n states I V - 0 h a v e b e e n u s e d t o a c c o u n t f o r t h e a p p e a r a n c e a n d d i s a p p e a r a n c e o f E P R s i g n a l s assoc i a t e d w i t h t h e N i s i t e (3, 24, 40). T w o o f t h e s e s c h e m e s a r e s u m m a r i z e d i n T a b l e I. P r o p o s a l A s i m p l y assigns o x i d a t i o n states o f III—0, w i t h o d d o x i d a t i o n states c o r r e s p o n d i n g t o E P R a c t i v e s p e c i e s . S u c h r e d o x c h e m i s t r y w i t h i n a 4 0 0 - m V p o t e n t i a l r a n g e is u n p r e c e d e n t e d i n N i c h e m i s t r y . A l t e r n a t i v e l y , p r o p o s a l B uses o n l y o n e - e l e c t r o n r e d o x c h e m i s t r y f o r N i b u t suggests t h a t s o m e h o w t h e N i site b e c o m e s r e o x i d i z e d at a lower p o t e n t i a l , i m p l y i n g t h a t t h e s t r u c t u r e a n d t h e p r o t o n a t i o n state o f t h e N i site h a v e c h a n g e d . In the absence of data regarding the redox role of the N i ligands, the F e , S clusters, or other groups nearby, or data that have a direct bearing on changes i n the electron density and charge density of the N i , s c h e m e s a s s i g n i n g f o r m a l o x i d a t i o n states to t h e N i a t o m offer n o c h e m i c a l o r m e c h a n i s t i c i n s i g h t . T o assess t h e r o l e o f N i - c e n t e r e d r e d o x c h e m i s t r y i n v o l v i n g w i d e l y d i f f e r i n g o x i d a t i o n states f o r N i , w e h a v e e x a m i n e d t h e N i K - e d g e X - r a y a b s o r p t i o n s p e c t r u m o f s a m p l e s o f T. roseopersicina H a s e p o i s e d i n e a c h o f t h e five states d e f i n e d b y E P R s p e c t r a o b s e r v e d at 7 7 K (35). T h e E P R s p e c t r a o b t a i n e d f r o m s a m p l e s 2

Table I.

Redox Schemes E (mV vs. NHEat pH 7.0) m

Observed Phenomenon EPR of form A EPR of form B EPR silent intermediate Appearance of form C signal Disappearance of form C signal Reductive activation Oxidative deactivation

-150

-270 -390 -310 -133

Proposal A

Proposal B

Ni(III) Ni(III) Ni(ll) Ni(I) Ni(0)

Ni(III) Ni(III) Ni(II) Ni(III) Ni(II)


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27

frozen i n sample holders for the X - r a y absorption spectroscopy ( X A S ) e x p e r i m e n t are s h o w n i n F i g u r e 2. T h e s e s p e c t r a d e m o n s t r a t e t h a t > 8 0 % o f t h e N i p r e s e n t i n t h e e n z y m e is p o i s e d i n t h e d e s i r e d f o r m . T h e y also i l l u s t r a t e t h e s e q u e n t i a l c h a n g e i n t h e E P R s p e c t r u m t h a t is o b s e r v e d as t h e e n z y m e is r e d u c e d . T h e N i K - e d g e X - r a y a b s o r p t i o n s p e c t r a c o r r e s p o n d i n g to t h e E P R s p e c t r a are s h o w n i n F i g u r e 3 a n d s u m m a r i z e d i n T a b l e II. T h e most s t r i k i n g f e a t u r e o f t h e H a s e N i K - e d g e X A S s p e c t r a is t h e l a c k o f s e n s i t i v i t y t o t h e o x i d a t i o n state o f t h e e n z y m e as d e t e r m i n e d b y t h e E P R s p e c t r u m o f t h e N i c e n t e r . T h e e d g e s d o n o t e x h i b i t a s i g n i f i c a n t shift to l o w e r e n e r g y u p o n r e d u c t i o n of the e n z y m e . V a l u e s for the e d g e e n e r g y are w i t h i n 0.2 e V o f e a c h o t h e r w i t h the e x c e p t i o n o f F o r m A (the s a m e f o r m a l o x i d a t i o n state as f o r F o r m B ) , w h i c h has a n e d g e e n e r g y t h a t is < 1 e V h i g h e r i n e n e r g y .


2

T h e X - r a y a b s o r p t i o n e d g e e n e r g y is a s e n s i t i v e m e a s u r e o f t h e c h a r g e r e s i d i n g o n t h e m e t a l c e n t e r a n d is t h e r e f o r e a g o o d p r o b e o f m e t a l - c e n t e r e d r e d o x p r o c e s s e s . K - e d g e e n e r g y shifts h a v e b e e n w i d e l y u s e d to m o n i t o r r e d o x c h e m i s t r y i n m e t a l l o e n z y m e s . T h e results f r o m several m e t a l l o p r o t e i n studies are s h o w n i n T a b l e III. I n g e n e r a l , shifts o f < 1 e V i n d i c a t e t h a t t h e r e d o x c h e m i s t r y i n v o l v e d is n o t l o c a l i z e d o n t h e m e t a l c e n t e r . T h i s is c l e a r l y t h e c a s e f o r t h e N i s i t e i n T. roseopersicina H a s e . 2

T h i s a p p r o a c h was u s e d to e x a m i n e the r e d o x c h e m i s t r y o f the C u site i n g a l a c t o s e o x i d a s e (41), w h i c h h a d b e e n p r o p o s e d t o c o n t a i n a n u n u s u a l C u ( I I I ) c e n t e r (52). T h e l a c k o f a s i g n i f i c a n t C u K - e d g e e n e r g y shift b e t w e e n t h e o x i d i z e d a n d r e d u c e d f o r m s o f t h e p r o t e i n d e m o n strated that the r e d o x c h e m i s t r y was not m e t a l - c e n t e r e d a n d i m p l i c a t e d another redox active site. T h e crystal structure o f the p r o t e i n subsequently revealed a novel thioether composed of a cysteine and a tyros i n a t e l i g a n d o f t h e C u s i t e t h a t is l i k e l y t o b e i n v o l v e d i n t h e r e d o x p r o c e s s (53). T h e pre-edge region of the spectra obtained for redox-poised samp l e s o f T. roseopersicina s h o w n o e v i d e n c e o f a p e a k o r s h o u l d e r n e a r 8 3 3 8 e V that has b e e n a s s i g n e d t o a I s ^ 4 p t r a n s i t i o n ( w i t h s h a k e d o w n c o n t r i b u t i o n s ) (54-57) a n d is o b s e r v e d o n l y i n t h e X A S s p e c t r a o f p l a n a r f o u r - c o o r d i n a t e c o m p l e x e s a n d p y r a m i d a l five-coordinate c o m p l e x e s (32, 5 8 ) . I n s e v e r a l cases, it is p o s s i b l e to r e s o l v e a w e a k p e a k n e a r 8 3 3 2 e V t h a t has b e e n a s s i g n e d t o a I s -*> 3 d t r a n s i t i o n ( F i g u r e 3) (32, 5 9 , 60). T h e I s ^ 3 d t r a n s i t i o n is s y m m e t r y - f o r b i d d e n i n c e n t r o s y m m e t r i c p o i n t g r o u p s , b u t is e x p e c t e d t o g a i n i n t e n s i t y i n g e o m e t r i e s t h a t a l l o w p - d m i x i n g t o o c c u r (58, 61). I n g e n e r a l , t h e I s 3 d peaks range f r o m 0 to 0.015(5) e V i n the e n z y m e , i n ratios of 0 - 0 . 1 3 r e l a t i v e to the area of t h e ( E t N ) [ N i C l ] p r e - e d g e p e a k (58). T h e l o w i n t e n s i t y o f t h i s f e a t u r e i n t h e H a s e s p e c t r a is c o n s i s t e n t w i t h e i t h e r a p l a n a r f o u r - c o o r d i n a t e z

4

2

4

2


28



g=2.32

1

2800

1

1

1

3000 3200 3400 Magnetic Field (Gauss)

1

3600

Figure 2. EPR spectra from Thiocapsa roseopersicina hydrogenase obtained on samples used in XAS experiments at 77 K. The spectra are arranged in order of decreasing redox potential top to bottom. Forms A and B correspond to oxidized enzyme, SI is an EPR silent intermediate, form C is an active form of the enzyme that is also EPR-active, and R is the fully reduced enzyme. (Reproduced from reference 35. Copyright 1993 American Chemical Society.)



2.

MARONEY ET AL.

^


1

8300

1

8320

1

1

8340 8360 8380 X-ray Energy (eV)

29

1—

8400

Figure 3. Nickel K-edge X-ray absorption spectra obtained on samples of Thiocapsa roseopersicina hydrogenase poised in thefiveforms defined by the EPR spectra at 77 K. (Reproduced from reference 35. Copyright 1993 American Chemical Society.)

g e o m e t r y [peak areas o f 0 - 0 . 0 2 9 ( 5 ) e V (58)] o r a s i x - c o o r d i n a t e g e o m e t r y [peak areas o f 0 . 0 0 6 ( 5 ) - 0 . 0 4 0 ( 5 ) e V (58)]. G i v e n t h e a b s e n c e o f a I s 4 p t r a n s i t i o n t h a t is e x p e c t e d f o r t h e p l a n a r a n d p y r a m i d a l g e o m e t r i e s 2

(shoulder), the spectra obtained from all o f the H a s e samples are most 2

consistent w i t h a six-coordinate or a

five-coordinate

trigonal-bipyramidal

N i site. T h e post-edge X - r a y absorption near-edge structure ( X A N E S ) obs e r v e d f o r t h e five r e d o x states r e m a i n s n e a r l y c o n s t a n t , also s u g g e s t i n g t h a t t h e s t r u c t u r e o f t h e N i site d o e s n o t c h a n g e d u r i n g r e d u c t i o n o f the e n z y m e . T h e lack of a significant change i n the structure o f the N i site is also s e e n f r o m a n analysis o f t h e first c o o r d i n a t i o n s p h e r e e x t e n d e d X - r a y a b s o r p t i o n fine s t r u c t u r e ( E X A F S ) d a t a f o r t h e N i site ( F i g u r e 4 ) , w h i c h c a n b e fit w i t h 2 - 3 S,C1 d o n o r s at 2 . 2 3 ± 0 . 0 3 A a n d 3 ± 1 N , 0 d o n o r s at 2 . 0 0 ± 0 . 0 6 A r e g a r d l e s s o f o x i d a t i o n state. T h i s r e s u l t c a n b e contrasted w i t h expectations for metal-centered redox chemistry based on the structures of [ N i ( p d t c ) ] ~ a n d [ N i ( p d t c ) ] " , w h i c h show a dem

2

n

2

2

crease o f 0 . 1 4 A i n the average N i - S b o n d l e n g t h u p o n o x i d a t i o n o f N i ( I I ) t o N i ( I I I ) (62). A l o g i c a l c o n c l u s i o n is t h a t t h e r e d o x c h e m i s t r y associated w i t h the processes that give rise to the E P R spectra characteristic of H a s e are largely not centered i n N i orbitals. 2


30


Table II.

Enzyme Redox State


Form A Form B SI Form C

Ni K-Edge Energy ±0.2 (eV)

R

8340.4 8339.4 8339.8 8339.6 8339.5

Form C + light

8339.4

E P R and Ni K-Edge Data

ls-+3d Peak Area ±0.005 eV (relative to NiCl ~)

% Ni EPR Detectable

%of EPR Active Ni Poised

(0.132) (0.351) (0.123) (0) (0.061)

91 90 0 80 0

78 85 (100) 100 (100)

0.012 (0.140)

65

100

2

0.015 0.040 0.014 Mo(lV)

2.2 2.0 2.2 3.1

a3

41 42 43

0.5 0.5

Cu (II)^Cu (I) Ni(III)—Ni(II) a3

ligand Fe,S cluster

? metal metal metal metal


46

49 49 50 49 51

MARONEY E T AL.


31


2.

"h

1

1

1

1

2

4

6

8

10

1

12

k(A" ) 1

Figure 4. First coordination sphere (backtransform window = 1.1-2.7 A) Fourier-filtered Ni K-edge EXAFS spectra from redox-poised Thiocapsa roseopersicina hydrogenase samples (O) andfits( ):formA, (3 ± 1)N,0 at 1.97(2) A + (2 ± 1)S at 2.23(2) A; form B, (3 ± 1)N,0 at 1.95(2) A + (2 ± 1)S at 2.24(2) A, S7, (2 ± 1)N,0 at 2.03(2) A + (3 ± 1)S at 2.24(2) A; form C, (2 ± 1)N,0 at 2.01(2) A+ (2±l)Sat 2.25(2) A; R, (2 ± 1)N,0 at 2.03(2)A+(2±1)S at 2.24(2) A. (Reproduced from reference 35. Copyright 1993 American Chemical Society.)


32


T h e m o s t a p p r o p r i a t e d e s c r i p t i o n o f t h e N i o x i d a t i o n state i n a l l forms o f the e n z y m e appears to b e N i ( I I ) , b a s e d o n a v a l e n c e b o n d s u m analysis of the best E X A F S m o d e l ( T h o r p , H . H . , U n i v e r s i t y o f N o r t h C a r o l i n a , u n p u b l i s h e d data). T h i s m o d e l is i n a g r e e m e n t w i t h s t u d i e s o f the magnetic properties of the E P R silent intermediate of the H a s e f r o m D. baculatus t h a t d e m o n s t r a t e d t h a t t h e N i site w a s d i a m a g n e t i c , a l b e i t i n a n e n z y m e t h a t has a s e l e n o c y s t e i n a t e N i l i g a n d (29).


2

A l t h o u g h N i - c e n t e r e d r e d o x c h e m i s t r y does not appear to b e i n volved, redox chemistry clearly occurs i n the e n z y m e and cannot be a t t r i b u t e d solely to e i t h e r the F e , S clusters present or the N i c e n t e r . D e t a i l e d studies of the redox c h e m i s t r y associated w i t h the F e , S clusters h a v e b e e n p e r f o r m e d o n t h e N i , F e e n z y m e f r o m D . gigas (63, 64) a n d o n t h e F e , N i , S e e n z y m e f r o m D. baculatus (40). T h e s e s t u d i e s u s e d r e d o x titrations a n d E P R spectroscopy to v a r y a n d m o n i t o r the r e d o x state o f t h e e n z y m e s a n d m a g n e t i c field-dependent Mossbauer experim e n t s t o e x a m i n e t h e c l u s t e r t y p e s , s p i n states, a n d r e d o x states o f t h e F e , S c l u s t e r s p r e s e n t i n e a c h s a m p l e . I n t h e o x i d i z e d D. gigas e n z y m e , t h e s e s t u d i e s i d e n t i f i e d t h r e e c l u s t e r s : a n o x i d i z e d , p a r a m a g n e t i c (S = V2, g = 2.02) [ 3 F e , 4 S ] cluster a n d t w o d i a m a g n e t i c [ 4 F e , 4 S ] clusters. T h e [ 3 F e , 4 S ] c l u s t e r is r e d u c e d at —70 m V t o a n i n t e g e r s p i n c l u s t e r (S = 2, g = 12) t h a t is n o t r e d u c e d f u r t h e r , b u t its E P R s p e c t r u m is s e n s i t i v e to t h e r e d o x states o f t h e o t h e r c l u s t e r s . T h e t w o [ 4 F e , 4 S ] c l u s t e r s a r e r e d u c e d at d i f f e r e n t p o t e n t i a l s (—290 a n d — 3 4 0 m V ) a n d display distinct and unusual spectral properties i n their r e d u c e d forms. O n l y b r o a d , i l l - d e f i n e d E P R signals f r o m one [ 4 F e , 4 S ] cluster are observed, suggestive of s p i n - s p i n interactions w i t h other paramagnets. I n c o n t r a s t , t h e D. baculatus e n z y m e w a s s h o w n n o t t o c o n t a i n a [ 3 F e , 4 S ] c l u s t e r i n e i t h e r a n o x i d i z e d o r r e d u c e d state. T h i s e n z y m e has t w o [4Fe,4S] clusters that e x h i b i t M o s s b a u e r p r o p e r t i e s similar to those c h a r a c t e r i z e d f o r t h e D. gigas e n z y m e a n d h a v e s i m i l a r m i d p o i n t r e d o x potentials o f - 3 1 5 m V . H o w e v e r , these clusters e x h i b i t E P R signals (g = 1.94) t h a t a r e t y p i c a l o f r e d u c e d [ 4 F e , 4 S ] c l u s t e r s , i n d i c a t i n g t h a t i n t e r a c t i o n w i t h t h e [ 3 F e , 4 S ] c l u s t e r i n D. gigas H a s e is r e s p o n s i b l e f o r the u n u s u a l E P R properties of the [4Fe,4S] clusters i n that e n z y m e . T h e p o t e n t i a l s d e t e r m i n e d for t h e F e , S c l u s t e r s i n t h e s e t w o e n z y m e s i n d i c a t e t h a t t h e o x i d i z e d f o r m s o f H a s e (forms A a n d B ) w i l l c o n t a i n a n o x i d i z e d [ 3 F e , 4 S ] c l u s t e r (if p r e s e n t ) a n d t h a t it w i l l b e r e d u c e d i n t h e E P R s i l e n t forms. T h e potentials d e t e r m i n e d for [4Fe,4S] clusters i n d i c a t e that the a c t i v e f o r m ( f o r m C ) has at least o n e r e d u c e d [ 4 F e , 4 S ] c l u s t e r a n d t h a t t h e f u l l y r e d u c e d state c o n t a i n s o n l y r e d u c e d c l u s t e r s . +

2 +

+

2 +

+

2

2

T h e potentials d e t e r m i n e d for r e d u c t i o n of the F e , S clusters indicate that t h e y are not d i r e c t l y r e s p o n s i b l e for a l l o f the changes i n the E P R s p e c t r u m that h a v e b e e n a s s o c i a t e d w i t h N i . F u r t h e r m o r e , b e c a u s e t h r e e electrons appear to b e i n v o l v e d i n the r e d o x c h e m i s t r y associated w i t h


2.

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33


N i , m a n y e n z y m e s (e.g., D. baculatus a n d T. roseopersicina) d o n o t c o n tain the n u m b e r o f clusters that w o u l d b e r e q u i r e d to account for t h r e e one-electron redox processes. C l e a r l y , other redox centers must be p r e s e n t . F r e q u e n t l y , h i g h f o r m a l o x i d a t i o n state i n t e r m e d i a t e s i n b i o l o g y i n v o l v e o x i d a t i o n o f s o m e o t h e r g r o u p r a t h e r t h a n t h e m e t a l c e n t e r (e.g., galactose oxidase (41), M n 0 - e v o l v i n g c o m p l e x ( O E C ) (65), h o r s e r a d i s h p e r o x i d a s e c o m p o u n d I (66, 67)). I n t h i s r e g a r d , it is w o r t h n o t i n g t h a t the only N i - c o n t a i n i n g functional m o d e l r e p o r t e d that features a m i x e d d o n o r l i g a n d e n v i r o n m e n t c o m p o s e d o f N - , O - , a n d S - d o n o r s uses o n l y t h e N i ( I I / I ) c o u p l e t o c a t a l y z e t h e H - D e x c h a n g e r e a c t i o n (68). It is p o s s i b l e t h a t p r o t e i n c h e m i s t r y , s u c h as t h a t o b s e r v e d f o r g a l a c t o s e o x idase, might e x p l a i n some of the redox c h e m i s t r y that occurs i n H a s e . A n o t h e r p o s s i b i l i t y is t h a t t h e c y s t e i n a t e s b o u n d t o t h e N i site m i g h t account for some of the redox c h e m i s t r y o f H a s e , a n d i f the changes w e r e l o c a l i z e d to a large extent o n the S - d o n o r ligands, t h e y m i g h t not affect t h e N i s i t e . W e h a v e e x p l o r e d t h i s p o s s i b i l i t y u s i n g N i c o m p l e x e s of alkyl thiolate ligands. 2


2

2

Redox Chemistry of Nickel Thiolates T h e s i m i l a r i t y o f the o x i d i z e d e n z y m e E P R spectra (forms A a n d B) to t h o s e o b t a i n e d f r o m N i ( I I I ) c o o r d i n a t i o n c o m p l e x e s (69-73) l e d t o t h e a s s i g n m e n t o f t h e E P R signals t o l o w - s p i n t e t r a g o n a l N i ( I I I ) c e n t e r s w i t h t h e u n p a i r e d s p i n i n t h e d 2 o r b i t a l (g < g ). T h i s a s s i g n m e n t , c o u p l e d w i t h the identification of S-donor ligands i n the coordination sphere of N i a n d t h e l o w o x i d a t i o n p o t e n t i a l o f t h e b i o l o g i c a l N i s i t e , has m o t i v a t e d a n u m b e r o f s t u d i e s a i m e d at p r o d u c i n g N i ( I I I ) c o m p l e x e s w i t h t h i o l a t e l i g a n d s , a n d s e v e r a l e x a m p l e s a r e n o w k n o w n (74). A l t e r n a t i v e l y , t h e S = Vi E P R signals c o u l d b e a s s i g n e d to N i ( I ) s p e c i e s ( p a r t i c u l a r l y a p p r o p r i a t e for t h e r e d u c e d e n z y m e ) , a n d N i ( I ) m o d e l c o m p o u n d s w i t h t h i o late l i g a t i o n a n d r h o m b i c E P R s p e c t r a h a v e b e e n r e p o r t e d (75, 76). M a n y of these models w e r e d e v e l o p e d w i t h the goal of u n d e r s t a n d i n g the factors that l e a d to s t a b i l i z a t i o n o f Ni(III) or N i ( I ) , r a t h e r t h a n to e x p l o r e t h e r e d o x c h e m i s t r y o f N i t h i o l a t e s l i k e t h o s e t h a t m i g h t exist i n H a s e . z

z

x>y

2

Nickel(II) coordination compounds w i t h simple N - or O - d o n o r l i g a n d s g e n e r a l l y l e a d to r e d o x p o t e n t i a l s o f > + l V f o r t h e o x i d a t i o n o f N i ( I I ) t o N i ( I I I ) , a n d o f < - 1 V for t h e r e d u c t i o n o f N i ( I I ) t o N i ( I ) , p o t e n t i a l s that are w e l l b e y o n d t h e r e l e v a n t r a n g e (69, 70, 73). H o w e v e r , ligands featuring deprotonated amides, thiocarboxylates, and oxime l i g a n d s , w h i c h a r e k n o w n t o s t a b i l i z e h i g h e r o x i d a t i o n states o f m e t a l s b u t have little b i o l o g i c a l r e l e v a n c e , have b e e n s h o w n to l o w e r the o x i d a t i o n p o t e n t i a l o f N i ( I I ) (74). T h e o x i d a t i o n o f o n e a l k y l t h i o l a t e c o m p l e x points to the s t a b i l i z a t i o n o f f o r m a l l y Ni(III) centers b y thiolate ligands, a l t h o u g h it is n o t y e t c l e a r w h i c h c e n t e r s a r e p r i m a r i l y i n v o l v e d i n t h e


34


oxidation. T h e sterically encumbered complex N i ( n b d t ) " undergoes a r e v e r s i b l e o n e - e l e c t r o n o x i d a t i o n to a f o r m a l l y N i ( I I I ) s p e c i e s t h a t e x h i b i t s a n a x i a l E P R s p e c t r u m (g|| = 2 . 1 4 , g = 2 . 0 5 ) at p o t e n t i a l s as n e g a t i v e as - 0 . 7 6 V ( 7 7 ) . 2

L

R e d u c t i v e c h e m i s t r y is n o t r e a d i l y a c c e s s i b l e i n N i ( n b d t ) " , p r e s u m ably because of the negative charge on the complex. O n l y one system has b e e n r e p o r t e d t h a t p r o d u c e s s t a b l e N i ( I I I ) a n d N i ( I ) c o m p l e x e s (76), and no system w i t h redox potentials for the Ni(III/II) a n d Ni(II/I) couples d i f f e r i n g b y 4 0 0 m V has b e e n c h a r a c t e r i z e d . N i ( t e r p y ) ( S A r ) c a n b e r e d u c e d b y N a S 0 to give a Ni(I) c o m p l e x w i t h an axial E P R s p e c t r u m (gn = 2 . 2 5 , g = 2 . 1 3 ) t h a t is c a p a b l e o f r e v e r s i b l y b i n d i n g C O a n d H " . T h e C O adduct and the H ~ adduct b o t h give r h o m b i c E P R spectra ( C O adduct: g = 2.24, g = 2.14, g = 2.05; H " adduct: g = 2.24, g = 2.19, g = 2 . 0 5 ) . C h e m i c a l o x i d a t i o n o f t h e s e c o m p l e x e s is d i f f i c u l t a n d l e a d s t o d e c o m p o s i t i o n o f t h e c o m p l e x (76). H o w e v e r , i f t h e t e r p y l i g a n d is c h a n g e d to 2 , 6 - b i s [ ( l - p h e n y l i m i n o ) e t h y l ] p y r i d i n e ( D A P A ) , b o t h o x i d i z e d a n d r e d u c e d c o m p l e x e s m a y b e p r e p a r e d (76). T h i s c h a n g e is a t t r i b u t e d t o t h e p r e s e n c e o f a less e x t e n s i v e 7r-system i n t h e D A P A l i g a n d . R e d u c t i o n of the Ni(II) c o m p l e x N i ( D A P A ) ( S P h ) b y N a S 0 leads to c h e m i s t r y similar to that o b s e r v e d for the t e r p y c o m p l e x e s . T h e Ni(I) c o m p l e x t h a t f o r m s has a r h o m b i c E P R s p e c t r u m ( g = 2 . 2 6 , g = 2 . 1 4 , g = 2 . 0 9 ) a n d r e a c t s w i t h C O t o f o r m a a d d u c t t h a t also has a r h o m b i c E P R spectrum (g = 2.20, g = 2.15, g = 2.02). O x i d a t i o n of N i ( D A P A ) ( S P h ) w i t h F e ( C N ) " leads to the f o r m a t i o n of a f o r m a l l y N i ( I I I ) s p e c i e s w i t h a n a x i a l E P R s p e c t r u m (g„ = 2 . 0 3 ; g = 2 . 2 1 ) . T h e o x i d a t i o n p r o d u c t does not b i n d C O , b u t does b i n d C N ~ to give a c o m p l e x w i t h a r h o m b i c E P R spectrum (g = 2.26, g = 2.21, g = 2.04). T h e r e d u c e d c o m p l e x also b i n d s C N " , b u t C O is c a p a b l e o f d i s p l a c i n g t h e C N " l i g a n d . B e c a u s e C O b i n d s to active H a s e (form C ) to f o r m a N i C O a d d u c t (26), t h i s m o d e l c h e m i s t r y p o i n t s t o a m o r e r e d u c e d N i c e n t e r in the active protein. 2

2

2

2

4


±

x

2

Y

3

2

3

2

2

2

x

4

2

3

x

2

2

6

3

3

±

x

2

3

2

W e h a v e f o c u s e d o u r m o d e l i n g efforts o n a s y s t e m t h a t c o n t a i n s alkyl thiolate ligands, i n analogy w i t h cysteinate coordination i n the e n z y m e . R e a c t i o n o f N i ( O A c ) w i t h a series o f t r i d e n t a t e ligands [ R N ( C H C H S H ) ] leads to the f o r m a t i o n o f a series o f d i m e r i c c o m p l e x e s , { N i [ R N ( C H C H S ) ] } [R = C H C H S C H (1), C H C H S C H P h (2), C H (3), C H P h (4), C H C H C H ( P h ) (5)], c o n t a i n i n g d i s t o r t e d planar N i centers coordinated b y three thiolate donors and a tertiary a m i n e (78, 79) ( P r e s s l e r , M . A . a n d M a r o n e y , M . J . , U n i v e r s i t y o f M a s s a c h u s e t t s , u n p u b l i s h e d r e s u l t s ) . T h e s t r u c t u r e o f t h e s e d i m e r s is r e p r e s e n t e d b y 1 i n F i g u r e 5. T w o o f t h e t h i o l a t e l i g a n d s b r i d g e b e t w e e n the N i centers giving a butterfly-shaped cluster, w i t h the two planar N i complexes j o i n e d along an edge w i t h an angle of 105° b e t w e e n the N i ( S ) p l a n e s i n t h e e x a m p l e s h o w n . T h i s t y p e o f s t r u c t u r e is c h a r a c 2

2

2

2

2

3

b r

2

2

2

2

2

2

2

2

3

2

2

2

2


2

MARONEY ET AL.


35


2.

Figure 5. The oxidative chemistry of a series of Ni thiolate complexes. Crystallographically characterized structures are shown as ORTEP diagrams.


36


t e r i s t i c o f this class o f l i g a n d s (80) a n d has b e e n o b s e r v e d for t h e formally Ni(III,II) mixed-valent one-electron oxidation product of the Ni(II) complex of a tertiary phosphine ligand w i t h three pendant arylthiolates ( P ( o - C H S ) ) (81). 6

4

3

T h e o x i d a t i v e c h e m i s t r y t h a t is c h a r a c t e r i s t i c o f t h e d i m e r s is s u m m a r i z e d i n F i g u r e 5. A s d i s c u s s e d b e l o w , t h e m o s t s t r i k i n g f e a t u r e is that t h e p r o d u c t s o f o x i d a t i o n r e v e a l S - c e n t e r e d c h e m i s t r y i n e v e r y case. D e s p i t e t h e o b v i o u s s h o r t c o m i n g s o f s u c h c o m p l e x e s as m o d e l s f o r a m o n o n u c l e a r five- o r s i x - c o o r d i n a t e b i o l o g i c a l N i s i t e , t h e r e d o x c h e m istry o f this series o f c o m p o u n d s p r o v i d e s insight into the possible roles of the N i thiolate ligands i n the redox chemistry exhibited b y H a s e . Downloaded by PURDUE UNIV on April 9, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/ba-1995-0246.ch002

2

O n e o f t h e s e c o m p l e x e s (1) p r o v i d e d t h e first e x a m p l e o f a N i ( I I ) thiolate c o m p l e x that u n d e r g o e s a r e v e r s i b l e o n e - e l e c t r o n o x i d a t i o n to a f o r m a l l y N i ( I I I ) - c o n t a i n i n g c o m p l e x (78). D a t a f r o m c y c l i c v o l t a m m e t r i c s t u d i e s o f t h e r e d o x c h e m i s t r y o f 1 a r e s h o w n i n F i g u r e 6. S c a n s t h a t p r o c e e d first i n t h e c a t h o d i c d i r e c t i o n (a) s h o w n o r e d u c t i o n s o f the Ni(II) d i m e r w i t h i n the limits p r o v i d e d b y the solvent or electrolyte u s e d (0.1 M n - B u 4 N ( C l 0 ) / C H C l 2 ) . S c a n n i n g to i n c r e a s i n g a n o d i c p o t e n t i a l s r e v e a l s t w o d i s t i n c t o x i d a t i o n s , t h e first o f w h i c h is q u a s i r e v e r s i b l e ( F i g u r e 6b) a n d has a n £ - 0 V v e r s u s N H E ( T a b l e I V ) . T h e fact that t h e p r o c e s s is r e v e r s i b l e suggests t h a t t h e p r o d u c t is s t i l l d i m e r i c , a n e x p e c t a t i o n t h a t is g i v e n c r e d e n c e b y t h e i s o l a t i o n a n d s t r u c t u r a l characterization of a mixed-valent Ni(II/III) dimer w i t h a core structure l i k e t h a t o b s e r v e d i n 1 (81). C o u l o m e t r i c m e a s u r e m e n t s s h o w t h a t t h e e l e c t r o c h e m i c a l p r o c e s s is a o n e - e l e c t r o n o x i d a t i o n , a n d t h e o x i d a t i o n p r o d u c t y i e l d s a r h o m b i c E P R s p e c t r u m ( g = 2 . 1 7 , g = 2 . 1 1 , g = 2.08) a p p r o p r i a t e for a n S = V2 s p e c i e s ( F i g u r e 7). T h i s s p e c t r u m is r e m i n i s c e n t o f t h o s e o b t a i n e d f r o m H a s e s i n t h a t i t is a r h o m b i c s p e c t r u m w i t h g values b e t w e e n g = 2.3 a n d 2.0 a n d displays no obvious l i g a n d h y p e r f i n e s p l i t t i n g s d e s p i t e t h e p r e s e n c e o f a n N - d o n o r l i g a n d . T h e first o x i d a t i o n has a n Z p / / p r a t i o n e a r 1 o n l y at h i g h s c a n r a t e s . T h e fact t h a t t h i s r a t i o b e c o m e s > 1 at s l o w e r s c a n r a t e s suggests t h a t t h e o x i d a t i o n p r o d u c t is u n s t a b l e , a n o b s e r v a t i o n t h a t is c o n f i r m e d b y E P R s p e c t r o s c o p y . 4

2

1 / 2

x

2

3

2

a

c

I f t h e v o l t a m m e t r i c s c a n is n o t r e v e r s e d a f t e r t h e first o x i d a t i o n , a s e c o n d o x i d a t i o n ( E p = 0 . 2 - 0 . 3 V ) is o b s e r v e d ( F i g u r e 6 c ) . T h i s o x i dation renders the lower potential oxidation irreversible ( E p = 0 - 0 . 1 V ) a n d also l e a d s t o t h e o b s e r v a t i o n o f a n i r r e v e r s i b l e r e d u c t i o n at n e g a t i v e p o t e n t i a l s ( E p = - 0 . 8 t o - 0 . 9 V ) . It has n o t b e e n p o s s i b l e t o measure accurately the n u m b e r of electrons involved i n either the second o x i d a t i o n or i n the c o u p l e d r e d u c t i o n process b y c o u l o m e t r y d u e to the f o r m a t i o n o f films o n t h e e l e c t r o d e s u r f a c e s . H o w e v e r , i n c o m p a r i s o n w i t h that o b s e r v e d for t h e first o x i d a t i o n , t h e p e a k c u r r e n t s are c o n s i s t e n t w i t h one- and t w o - e l e c t r o n processes, respectively. In spite of the i r reversible nature of the cyclic v o l t a m m o g r a m , continuous c y c l i n g bea

a

c


2.

MARONEY E T AL.


37


a) 8-1

ι 1000

1 500

Ί

0

mV

1 -500

1 -1000

Γ -1500

Figure 6. Voltammetric studies of {Ni[RN(CH CH S) ]} , R = CH CH SMe. A, initial scan in the cathodic direction. B, initial scan in the anodic direction, reversing the direction of the scan following the first oxidation. C, initial scan in the anodic direction, reversing the direction of the scan following the second oxidation. 2

2

2

2

2

2

t w e e n +1.5 a n d —1.5 V d o e s n o t l e a d t o a n y c h a n g e s (i.e., n o n e w p r o d u c t s a r e f o r m e d , a n d t h e d i m e r is n o t c o n s u m e d o n t h e c y c l i c v o l t a m m e t r i c t i m e scale). C y c l i c v o l t a m m o g r a m s o b t a i n e d o n t h e o n e e l e c t r o n oxidation p r o d u c t w e r e i d e n t i c a l to those o b t a i n e d f r o m the starting material. Ligands lacking a pendant thioether donor show very similar oxi d a t i v e c h e m i s t r y ( F i g u r e 8). T h e l a r g e s t d i f f e r e n c e is i n t h e first o x i d a t i o n , w h i c h is i r r e v e r s i b l e a n d o c c u r s at a m o r e p o s i t i v e p o t e n t i a l ( E p = 0 . 4 - 0 . 5 V ) . T h e oxidation remains a o n e - e l e c t r o n process that leads a


38

MECHANISTIC BIOINORGANIC CHEMISTRY

Table IV.

Electrochemical and E P R Data for the Oxidation of Complexes 1-5 Ep (mV vs. NHE)

Ep (mV vs. NHE)

2.17, 2.11, 2.08

260

-755

2.16, 2.11, 2.07

220

-900

2.21, 2.14, 2.03 2.20, 2.14, 2.02 2.21, 2.14, 2.02

880

-960

700

-950

800

-1200


a

Complex

(mV vs. NHE)

1

100

2

10

3

400

4

470

5

430

Characteristics quasireversible Ei/2 = +30 η = 1.0 quasireversible Ei/2 = +30 η = 1.1 irreversible η = 0.8 irreversible η = 0.9 irreversible η = 0.8

EPR g-values

a

c

N O T E : Potentials are taken from 250 mV/s cyclic voltammetric scans at a Pt button working electrode on 0.5 mM solutions of the complexes in 0.1 M n-Bu N(Cl0 ) at - 3 0 °C, and converted to N H E using the F c / F c + couple (= +440 mV vs. N H E ) ; the value of η was determined by controlled potential electrolysis at a Pt gauze electrode under the same conditions. 4

a

4

Samples were generated by electrolysis and measured as frozen solutions at 77 K.

t o t h e f o r m a t i o n o f a n E P R - a c t i v e S = V2 s p e c i e s ( F i g u r e 7). A l t h o u g h t h e g-values c h a r a c t e r i s t i c o f l i g a n d s w i t h N - a l k y l s u b s t i t u e n t s ( g i = 2 . 2 0 , g

2

= 2.14, g

3

= 2.02)

are distinct f r o m those that feature

pendant

t h i o e t h e r s (Table I V ) , t h e y are v i r t u a l l y i d e n t i c a l to those o b s e r v e d for t h e a c t i v e f o r m ( f o r m C ) o f H a s e (g 2

x

= 2.19, g

2

= 2.14, g

3

= 2.02). O n e

p o s s i b i l i t y that c o u l d account for the differences b e t w e e n these

two

classes o f d i m e r s is t h a t t h e p e n d a n t t h i o e t h e r d o n o r b e c o m e s c o o r d i n a t e d i n s o l u t i o n , a p o s s i b i l i t y t h a t is s u g g e s t e d b y t h e s o l i d - s t a t e s t r u c t u r e s o f a N i ( I I ) c o m p l e x w i t h a s i m i l a r l i g a n d (82) a n d o f t h e m i x e d valence Ni(III/II) dimer

(81).

A s is t h e case f o r t h e c o m p l e x e s w i t h p e n d a n t t h i o e t h e r d o n o r s , t h e N - a l k y l complexes show a second oxidation ( E p

a

= 0 . 7 - 0 . 9 V ) t h a t is

c o u p l e d to the f o r m a t i o n o f a n e w species w i t h an i r r e v e r s i b l e r e d u c t i o n (Figure 8c); the potential can be continuously c y c l e d b e t w e e n +1.5 a n d — 1.5 V w i t h o u t c h a n g i n g t h e v o l t a m m o g r a m . T h e redox c h e m i s t r y o b s e r v e d for these c o m p o u n d s b y u s i n g e l e c t r o c h e m i c a l m e t h o d s is s i m i l a r t o t h a t o b s e r v e d f o r t h i o l a t e s i n t h e a b s e n c e o f m e t a l i o n s (83, 84) a n d s t r o n g l y suggests t h e f o r m a t i o n o f d i s u l f i d e s . T h i s b e h a v i o r has also b e e n n o t e d i n t h e e l e c t r o c h e m i c a l s t u d y o f t h e o x i d a t i o n o f [ N i ( p d m t ) S P h ] " (85). T h e l a r g e s t d i f f e r e n c e


observed

2.


MARONEY E T AL.

1

39

+


2*

r

* "

'Vy— ~

~

A

v

r

2.5 2.4 2.3 2.2 2.1 2.0 1.9 g value

~

1.8

Figure 7. X-band EPR spectra of the one-electron oxidation products of complexes 1-5.

for the d i m e r s w o u l d appear to b e that the o x i d a t i o n o f the t w o thiolates i n v o l v e d i n t h e f o r m a t i o n o f t h e d i s u l f i d e o c c u r at d i f f e r e n t p o t e n t i a l s . A l t h o u g h t h e r e is n o p r o o f t h a t t h e t w o - e l e c t r o n o x i d a t i o n p r o c e s s l e a d s to a p r o d u c t that is s t i l l d i m e r i c , t h e fact t h a t t h e c y c l i c v o l t a m m o g r a m does not reveal the formation of n e w species even d u r i n g r e p e a t e d c y c l i n g is c o n s i s t e n t w i t h e i t h e r a d i m e r i c p r o d u c t o r m o n o n u c l e a r c o m p l e x e s t h a t r a p i d l y r e f o r m t h e s t a r t i n g d i m e r (85). D i s u l f i d e f o r m a t i o n i n t h e t w o - e l e c t r o n o x i d a t i o n p r o d u c t s is also s u p p o r t e d b y c h e m i c a l o x i d a t i o n s o f 1. T h e t w o - e l e c t r o n o x i d a t i o n o f each N i c e n t e r i n the d i m e r b y I leads to the f o r m a t i o n of a five-coord i n a t e m o n o n u c l e a r N i c o m p l e x w i t h a d i s u l f i d e l i g a n d (78). T h e s t r u c 2


40



«H

1000

-1000

0 mV

Figure 8. Voltammetric studies of [Ni(RN(CH CH S) ) ],R = CH . A, initial scan in the cathodic direction. B, initial scan in the anodic direction, reversing the direction of the scan following the first oxidation. C, initial scan in the anodic direction, reversing the direction of the scan following the second oxidation. 2

2

2

3

t u r e o f t h i s c o m p l e x , s h o w n i n F i g u r e 5 , f e a t u r e s a h i g h - s p i n (S = 1) five-coordinate Ni(II) c e n t e r i n a d i s t o r t e d p y r a m i d a l e n v i r o n m e n t c o m posed of a thioether ligand, a tertiary amine donor, two iodide ligands, and one sulfur of a disulfide. T h u s , the two-electron oxidation of each Ni(II) c e n t e r i n t h e d i m e r r e s u l t s i n t h e o n e - e l e c t r o n o x i d a t i o n o f a l l o f the thiolates. T h e frozen solutions containing the one-electron oxidation products reported i n F i g u r e 7 may be thawed, w h e r e u p o n they give isotropic spectra with g = g . A t r o o m t e m p e r a t u r e , t h e E P R signals are r a p i d l y lost d u e t o t h e f o r m a t i o n o f a n E P R s i l e n t p r o d u c t . T h i s c h e m i s t r y , w h i c h o c c u r s at a s l o w e r r a t e f o l l o w i n g t h e o n e - e l e c t r o n o x i d a t i o n , l i k e l y a c counts for the increasing value o f Z p / i p w i t h decreasing scan rate. T h e r a t e o f t h e loss o f t h e E P R s i g n a l c a n b e f o l l o w e d b y i n c u b a t i n g t h e i s o

a v e

a

c



2.

MARONEY E T AL.


41

s a m p l e s at s u b a m b i e n t t e m p e r a t u r e s a n d m e a s u r i n g t h e E P R s p e c t r a as a f u n c t i o n o f t i m e . T h e loss o f t h e E P R s i g n a l is first-order i n [ d i m e r ] ( F i g u r e 9 ) , a n d t h e s l o w e r r a t e o f s i g n a l loss e x h i b i t e d b y t h e d e r i v a t i v e c o n t a i n i n g a p e n d a n t t h i o e t h e r is c o n s i s t e n t w i t h t h e s t a b i l i z a t i o n o f t h e oxidation p r o d u c t v i a coordination o f this l i g a n d . T w o mechanisms for t h e d e c o m p o s i t i o n o f formally Ni(III) t h i o l a t e s t o d i s u l f i d e s h a v e b e e n s u g g e s t e d (85). T h e first m e c h a n i s m i n v o l v e s d i m e r i z a t i o n o f t h e o x i d i z e d complexes; the second involves dissociation of thiyl radicals a n d the s u b s e q u e n t f o r m a t i o n o f a d i s u l f i d e . T h e first-order d e c a y o f t h e E P R s i g n a l suggests t h a t c o u p l i n g o f t w o c o m p l e x e s d o e s n o t o c c u r i n t h e r a t e - d e t e r m i n i n g step. T h e data are consistent w i t h a r a t e - l i m i t i n g dissociation of a thiyl radical, followed b y the r a p i d formation of a disulfide. H y p o t h e t i c a l mechanisms for t h e c h e m i s t r y associated w i t h t h e products of one- a n d two-electron electrochemical oxidations are s u m m a r i z e d i n F i g u r e 10.

1000

2000 3000 Time (sec)

4000

Figure 9. First-order kinetic plots of the decay of the EPR signals of 1 * at 25° (a) and 4 * at —25° (b). Solid lines representfitsobtained for k = 4.7 X 10~ s' and k= 7.2 X J O " s' , respectively. +

+

3

1

4

1



+ #

Figure 10. Hypothetical mechanisms for the oxidative chemistry of 1 and the reaction of the one-electron oxidation product, 2 . Left from The process corresponding to the slow loss of the EPR signal and two paths for the formation of disulfides upon a second one-electron oxidation. Path A: Retaining a dimeric structure, the two-electron reduction of the disulfide regenerates 1. Path B: The second one-electron oxidation cleaves the dimer leading to the formation of a mononuclear disulfide complex, in analogy with that shown in Figure 5 and a mononuclear Ni (II) complex that rapidly dimerizes to form 1, in analogy with chemistry known for similar complexes (85). Reduction of the disulfide leads to production of the same mononuclear Ni(II) complex.


2.


MARONEY E T AL.

43

T h e e l e c t r o n i c s t r u c t u r e o f t h e o n e - e l e c t r o n o x i d a t i o n p r o d u c t s has also b e e n e x p l o r e d . A t first g l a n c e , t h e o b s e r v a t i o n o f g - v a l u e s > 2 . 0 suggests t h a t a N i ( I I I ) c e n t e r has b e e n f o r m e d a n d t h a t s p i n - o r b i t c o u p l i n g f r o m m e t a l - c e n t e r e d o r b i t a l s is r e s p o n s i b l e f o r t h e o b s e r v e d g v a l u e s . H o w e v e r , e l e c t r o n s l o c a l i z e d o n S also e x p e r i e n c e s p i n - o r b i t c o u p l i n g o f s u f f i c i e n t m a g n i t u d e to g i v e r i s e to t h e o b s e r v e d g - v a l u e s . E P R s p e c t r a o f t h i y l r a d i c a l s (e.g., c y s t e i n y l r a d i c a l ) t a k e n i n H - b o n d d o n o r s o l v e n t s (e.g., M e O H ) r e v e a l a x i a l s p e c t r a w i t h g = 2 . 3 a n d g = 2.0 ( g = 2.1) (86). T h e s p e c t r a r e p o r t e d f o r t h e d i m e r s ( a n d m a n y o t h e r formally N i ( I I I ) t h i o l a t e s ) h a v e g = g = 2.1. Presumably, the spectra of t h i y l radicals are t y p i c a l l y axial d u e to the d e g e n e r a c y o f the p-orbitals that are not i n v o l v e d i n the S - C s-bond. T h i s d e g e n e r a c y c o u l d b e l i f t e d i n a m e t a l c o m p l e x , g i v i n g rise to r h o m b i c s p e c t r a w i t h g e ~ 2.1. T h u s , analysis of g-values does not p r o v i d e u n e q u i v o c a l i n formation r e g a r d i n g the nature of the m o l e c u l a r o r b i t a l that contains the u n p a i r e d spin, particularly because the orbital likely contains contributions from b o t h the sulfur and the metal atom. l{

±

a v e


a v e

i s o

aV

Additional information regarding the metal character of the orbital containing the u n p a i r e d spin can be obtained from the observation of metal hyperfine interactions. W h e n N i (J = %) is u s e d t o p r e p a r e t h e d i m e r , l i n e b r o a d e n i n g w i t h n o r e s o l v e d h y p e r f i n e c o u p l i n g is o b s e r v e d . T h e fact t h a t o n l y l i n e b r o a d e n i n g is o b s e r v e d is c o n s i s t e n t w i t h o n l y a s m a l l a m o u n t o f s p i n d e n s i t y i n t e r a c t i n g w i t h t h e N i c e n t e r s . T h e effects o f N i s u b s t i t u t i o n a r e i l l u s t r a t e d i n F i g u r e 11 a n d a r e s i m i l a r i n d i m e r s w i t h a n d w i t h o u t p e n d a n t t h i o e t h e r s . A l t h o u g h t h e p r o b l e m is n o t a simple one, attempts w e r e m a d e to simulate the E P R spectra a n d estimate the m a x i m u m value of h y p e r f i n e c o u p l i n g constants. T h e simulations s h o w n i n F i g u r e 11 w e r e o b t a i n e d f o r t w o N i c e n t e r s w i t h o u t c o n s t r a i n i n g the m a g n i t u d e o f the h y p e r f i n e i n t e r a c t i o n to b e the same for b o t h c e n t e r s . I n fact, t h e v a l u e s o b t a i n e d w e r e e s s e n t i a l l y t h e s a m e a n d l e a d to average values for the t w o N i centers o f | A 1 , | A 1 , a n d | A 1 = 2 . 1 , 1 0 . 1 , a n d 1 0 . 0 G , a n d 1 0 . 5 , 0 . 0 , a n d 4.1 G f o r c o m p o u n d s 1 a n d 4, r e s p e c t i v e l y . T h e fact t h a t e q u a l p a r t i c i p a t i o n o f t w o N i c e n t e r s is r e q u i r e d to s i m u l a t e t h e E P R s p e c t r a suggests that t h e r a d i c a l c a t i o n d i m e r s a r e e x a m p l e s o f d e l o c a l i z e d m i x e d - v a l e n c e c o m p l e x e s . T h i s n o t i o n is consistent w i t h the structure obtained f r o m a similar Ni(II/III) complex, i n w h i c h t h e N i c e n t e r s a r e s t r u c t u r a l l y i n d i s t i n g u i s h a b l e (81). 6

1

6 1

2

2

3

W h e n the magnitudes of the dipolar hyperfine interactions i n the d i m e r s are c o m p a r e d w i t h a t h e o r e t i c a l v a l u e o f 6 7 . 5 G f o r a n u n p a i r e d s p i n l o c a l i z e d i n a N i 3 d o r b i t a l (87), i t c a n b e s e e n t h a t t h e m o l e c u l a r o r b i t a l c o n t a i n i n g t h e u n p a i r e d s p i n has a r e l a t i v e l y s m a l l c o n t r i b u t i o n f r o m N i ( < 3 0 % ) . A s i m i l a r s i t u a t i o n is o b s e r v e d i n H a s e s . W h e n b a c t e r i a a r e r a i s e d o n a s o u r c e o f N i , h y p e r f i n e is o b s e r v e d i n t h e E P R s i g n a l o r i g i n a t i n g f r o m H a s e (22). (In fact, o b s e r v a t i o n o f t h i s h y p e r f i n e w a s 2

6 1

2



44


2.5 2.4 2.3 2.2 2.1 2.0 1.9 g-value Figure 11. X-band EPR spectra of Ni derivatives of I (top) and 4 * (bottom) at 77 K. Simulated spectra assuming Lorentzian line shapes are shown as dashed lines. Simulations were obtained by using the program NEWSIM. Initial g-values and linewidths for the simulations were obtained from the complexes with natural abundance Ni and were used as a starting point for the refinement of the hyperfine components. Thefinalrefinement allowed the g-values, linewidths, and hyperfine values to vary, although this did not result in significant changes in the g-values or linewidths. The bestfitsshown above result from using two independently refined sets of hyperfine tensor elements for each of the Ni centers in the dimer, although the hyperfine parameters obtained for each Ni center are nearly the same and were subsequently averaged. (gi = 2.168, g = 2.111, g = 2.077; G = 8.83 G, G = 8.95 G, G = 9.14 G; |A,| = 2.1 G, \A \ = 10.0 G, \A \ = 9.7 G). 4+·: ( = 2.209, g = 2.141, g = 2.028; d = 6.21 G, G = 14.29 G, G = 12.06 G; I A , I = 10.8 G, \A \ = 0.0 G, \A \ =4.5G). 61

2

1

2

3

gl

3

2

2

3

+

+ #

3

2

3

2

3

t h e basis f o r a s s i g n i n g t h e E P R s i g n a l s t o N i - c o n t a i n i n g s p e c i e s (S).) I n t h e case o f a c t i v e H a s e ( f o r m C ) , t h e h y p e r f i n e is h i g h l y a n i s o t r o p i c , a n d r e s o l v e d h y p e r f i n e c o u p l i n g o f ~ 2 0 G is o b s e r v e d o n l y o n t h e h i g h est field f e a t u r e ; t h e h y p e r f i n e o b s e r v e d f o r t h e o t h e r t w o g - v a l u e s is v e r y s m a l l . I f t h e v a l u e o b s e r v e d at g is u s e d t o e s t i m a t e t h e s p i n d e n s i t y o n N i , a v a l u e o f ~ 3 0 % is o b t a i n e d . T h i s v a l u e is v e r y c l o s e t o t h a t o b s e r v e d f o r t h e d i m e r s a n d suggests t h a t t h e o r b i t a l c o n t a i n i n g t h e u n p a i r e d s p i n is n o t l o c a l i z e d o n N i c e n t e r s i n e i t h e r c a s e . 2

3

E v i d e n c e o f s p i n density o n t h e S - d o n o r atoms has b e e n o b t a i n e d from V E N D O R spectroscopy (Gurbiel, R. and Hoffman, B . ML, N o r t h w e s t e r n U n i v e r s i t y , u n p u b l i s h e d r e s u l t s ) . I n t h e case o f r a d i c a l c a t i o n o f m o d e l c o m p o u n d 4, a r e s o n a n c e w i t h a c o u p l i n g c o n s t a n t o f 1 2 - 1 4


2.

MARONEY E T AL.


45

M H z is o b s e r v e d ( F i g u r e 1 2 A ) . G i v e n t h a t t h e o n l y p r o t o n s t h a t w o u l d b e e x p e c t e d to give rise to significant c o u p l i n g w i t h the s p i n are the m e t h y l e n e protons o n the c a r b o n a t o m a to the S-donors, this resonance m u s t a r i s e f r o m t h e s e p r o t o n s (or a subset o f t h e s e p r o t o n s ) , a n a l o g u e s o f c y s t e i n e 0 - C H p r o t o n s . I n fact, n e a r l y i d e n t i c a l ^ - E N D O R s p e c t r a a r e o b s e r v e d f o r T. roseopersicina H a s e ( f o r m C ) ( F i g u r e 1 2 B ) (88). T h i s r e s u l t suggests t h a t t h e c o v a l e n t i n t e r a c t i o n b e t w e e n t h e c y s t e i n e d o n o r s a n d N i i n t h e e n z y m e is s i m i l a r t o t h a t e x i s t i n g b e t w e e n t h e N i a n d t h e t h i o l a t e l i g a n d s i n 4. 2

2

A n o t h e r o x i d a t i o n r e a c t i o n o f N i t h i o l a t e c o m p l e x e s is o f p o t e n t i a l r e l e v a n c e to t h e d e a c t i v a t i o n o f H a s e s u p o n e x p o s u r e t o 0 . R e a c t i o n of the Ni(II) d i m e r i c c o m p l e x e s w i t h t w o equivalents o f C N " leads to the formation of planar, frans-dithiolato complexes. These mononuclear c o m p l e x e s r e a c t w i t h 0 o r w i t h a i r u n d e r a m b i e n t c o n d i t i o n s (79, 89). M a n o m e t r i c m e a s u r e m e n t s s h o w t h a t t h e r e a c t i o n s t o i c h i o m e t r y is 1 N i : l 0 . T h e reaction products are planar, diamagnetic Ni(II) complexes featuring one thiolato a n d one sulfinato l i g a n d . T h i s r e a c t i o n corresponds to a f o r m a l f o u r - e l e c t r o n o x i d a t i o n of a Ni(II) c o m p l e x to give a p r o d u c t reflecting oxidation of a thiolate ligand. Structurally c h a r a c t e r i z e d ex a m p l e s o f t h e m o n o n u c l e a r f r Y m s - d i t h i o l a t e o b t a i n e d f r o m 1 a n d its o x i d a t i o n p r o d u c t a r e s h o w n i n F i g u r e 5 (79, 89).


2

2

2

2

T h e oxidation can be f o l l o w e d s p e c t r o p h o t o m e t r i c a l l y ( F i g u r e 13). T h e spectra of the f r a n s - d i t h i o l a t o c o m p l e x e s are c h a r a c t e r i z e d b y ab-

Figure 12. Ή-ENDOR spectra (35 GHz) of 4 · (A) taken at g = 2.208 and of the nonexchangeable protons in Thiocapsa roseopersicina hydrogenase-form C (B) taken at g = 2.19. +


46


2.4

ï

_ ι. a e υ

|l.2 M


ω

0.6

0

250

2.4

2.4 μ

0

350

450

550

750

650

Wavelength (nm) Figure 13. Electronic absorption spectra taken during the oxidation of Ni(MeSCH CH N(CH CH S) CN)by 0 in DMF. Spectrum 1 is 0.30 mM 1. Spectrum 2 is following the addition of 2 equiv of Et (CN) under N . Spectrum 3 is following the oxidation of the CN~ complex by 0 . The inset describes the reaction with 0 at 30 °C at times t = 7, 30, 90, 150, 210, 270, 330, 390, 450, 510, 570, 630, 720, and 750 min. Extinction coefficients are for a constant [Ni] = 0.60 mM. (Reproduced from reference 89. Copyright 1989 American Chemical Society.) 2

2

2

2

2

2

4

2

2

2

sorption maxima near 290 and 310 n m i n their electronic

absorption

spectra. U p o n exposure to 0 , n e w bands associated w i t h the f o r m a t i o n 2

of the monosulfinato complex appear near 2 6 5 and 3 2 5 n m . T h e spectral changes p r o c e e d w i t h the formation of isosbestic points, i n d i c a t i n g that no stable intermediates are f o r m e d i n the o x i d a t i o n process. B e c a u s e t h e final s p e c t r a o b t a i n e d a r e i d e n t i c a l t o i s o l a t e d s a m p l e s o f t h e m o n o s u l f i n a t e s , t h e r e is n o e v i d e n c e t h a t a b i s - s u l f i n a t e , a d i s u l f i d e , o r a n y other oxidation product

forms.

T h e spectral changes observed d u r i n g oxidation provide a means for m o n i t o r i n g t h e k i n e t i c s o f t h e o x i d a t i o n p r o c e s s . T h e r e a c t i o n s f o l l o w a r a t e l a w t h a t is first o r d e r =

fc[Ni(L)CN]"[0 ] 2

i n [Ni] a n d

first

order

in [0 ]: 2

Rate

(79). T h e r e a c t i o n is r e l a t i v e l y s l o w a n d n o t v e r y

s e n s i t i v e to t h e n a t u r e o f t h e N - s u b s t i t u e n t (k = 1 . 4 - 3 . 1 Χ 1 0 "

2

M "

1

s"

1

i n d i m e t h y l f o r m a m i d e ( D M F ) at 3 0 ° C ) . T h e r e a c t i o n rates a r e i n d e pendent of the presence of a singlet oxygen scavenger or radical traps. T h e a c t i v a t i o n p a r a m e t e r s ΔΗ* a n d AS* w e r e m e a s u r e d b y u s i n g t h e t e m p e r a t u r e d e p e n d e n c e of the s e c o n d - o r d e r rate constants i n D M F


2.


MARONEY E T AL.

and have values respectively.

of 13.1-14.7

47

k c a l / m o l a n d - 2 4 . 2 to - 1 8 . 7 e u ,

R e a c t i o n s t h a t u s e i s o t o p i c a l l y l a b e l e d 0 a n d mass s p e c t r o m e t r y r e v e a l t h a t t h e s o l e s o u r c e o f t h e Ο a t o m s i n t h e s u l f i n a t e p r o d u c t is 0 (89) a n d t h a t b o t h a t o m s o f a s i n g l e m o l e c u l e o f 0 a r e i n c o r p o r a t e d i n t o ~ 8 0 % o f t h e p r o d u c t (79). In light o f these results, h y p o t h e t i c a l mechanisms for t h e oxidation of N i thiolate complexes b y 0 may be discussed. T h e only well-char a c t e r i z e d m e c h a n i s m for t h e o x i d a t i o n o f thiolates to sulfinates i n t r a n s i t i o n m e t a l c o m p l e x e s i n v o l v e s 0 ~ as a n o x i d a n t a n d p r o c e e d s v i a t h e s t e p w i s e f o r m a t i o n o f s u l f e n a t e s ( S c h e m e 1) (90, 91). 2

2

2

2


2

T h e r e a c t i o n m e c h a n i s m h a s b e e n i n t e r p r e t e d as i n v o l v i n g n u c l e o p h i l i c attack o f the c o o r d i n a t e d thiolate S atom o n the p e r o x i d e ( H 0 , H 0 ) a n d follows the rate l a w : Rate = fc [Nuc][H 0 ]. Although the rate l a w a n d the k i n e t i c barriers d e t e r m i n e d for this m e c h a n i s m i n a n u m b e r o f complexes are similar to those observed for the oxidation o f [ N i ( L ) C N ] " (e.g., f o r [ ( e n ) C r ( S C H C H N H ) ] : ΔΗ* = 9 . 7 ( 2 ) k c a l / m o l , AS* = —26 e u ) , t h e s t o i c h i o m e t r y o f t h e r e a c t i o n s s t u d i e d h e r e ( I N i : 1 0 ) rules out a stepwise mechanism for the oxidation of [ N i ( L ) C N ] ~ c o m p l e x e s . S u c h a m e c h a n i s m w o u l d also b e e x p e c t e d t o g i v e r i s e t o complete scrambling of labeled oxygen i n reactions involving 0 a n d 0 , i n contrast to the observation that the oxidation o f [ N i ( L ) C N ] ~ proceeds mostly w i t h the i n c o r p o r a t i o n of b o t h atoms o f a single 0 molecule. 2

3

2

2

2

2

2

2

2

2

2

2

1

1 8

6

2

2

2

D e s p i t e the differences i n m e c h a n i s m , the expectation that thiolate l i g a n d s w i l l act as n u c l e o p h i l e s a p p e a r s t o b e a f e a t u r e o f t h e o x i d a t i o n of [ N i ( L ) C N ] ~ b y 0 . T h e strong tendency o f N i thiolates to f o r m dimers a n d h i g h e r p o l y m e r s (80, 92-94) a n d t h e fact t h a t t h e p r e s e n c e o f a tightly b o u n d anionic l i g a n d appears to b e r e q u i r e d to cleave the d i n u c l e a r c o m p l e x e s (e.g., C N ~ o r t h i o l a t e (85)) is e v i d e n c e o f t h e n u cleophilicity of terminal thiolate ligands i n planar Ni(II) complexes. 2

B e c a u s e r e a c t i o n o f t h i y l r a d i c a l s w i t h 0 is k n o w n t o f o r m s u l f i n y l r a d i c a l s (95), m e c h a n i s m s i n v o l v i n g r a d i c a l s w e r e also c o n s i d e r e d . A l though such a mechanism w o u l d have the correct stoichiometry a n d account for the lack of s c r a m b l i n g i n the studies i n v o l v i n g l a b e l e d 0 , r e a c t i o n s i n v o l v i n g t h e f o r m a t i o n o f free r a d i c a l s w e r e r u l e d o u t b e c a u s e 2

2

H0

H0

2

2

Scheme 1


48


n o E P R a c t i v e s p e c i e s w e r e o b s e r v e d a n d t h e rates o f r e a c t i o n a r e n o t affected b y t h e presence o f radical scavengers. T h e a b i l i t y o f t r a n s i t i o n m e t a l s t o f o r m c o m p l e x e s w i t h 0 is w e l l k n o w n . Ni(II) c o m p l e x e s are k n o w n to r e v e r s i b l y b i n d 0 (96, 9 7 ) . T h e N i - 0 c o m p l e x e s are p o w e r f u l o x i d i z i n g agents a n d h a v e b e e n d e s c r i b e d as i n v o l v i n g f o r m a l N i ( I I I ) a n d 0 c e n t e r s . T h e f o r m a t i o n o f a N i - 0 c o m p l e x as a p r e c u r s o r t o t h e l i g a n d o x i d a t i o n o f [ N i ( L ) C N ] ~ is u n l i k e l y because no intermediates are detected i n the oxidation process and the s p e c t r u m o f [ N i ( L ) C N ] " t a k e n u n d e r 1 a t m o f 0 at 1 5 ° C , a t e m p e r a t u r e t h a t is b e l o w t h a t r e q u i r e d t o h a l t t h e l i g a n d o x i d a t i o n , is i d e n t i c a l t o those o f anaerobic samples o f [ N i ( L ) C N ] " a n d does not change i n 6 h . M e c h a n i s m s i n v o l v i n g t h e p r o d u c t i o n o f free 0 ~ v i a oxidation o f t h e m e t a l c e n t e r , f o l l o w e d b y o x i d a t i o n o f t h i o l a t e l i g a n d s b y t h e 0 ~ (or 0 " from reaction w i t h a second N i center) are inconsistent w i t h the l a c k o f E P R s i g n a l s f r o m N i ( I I I ) a n d t h e a b s e n c e o f a n effect o n t h e r a t e s o f r e a c t i o n i n t h e p r e s e n c e o f r a d i c a l t r a p s . F u r t h e r m o r e , these r e a c t i o n s w o u l d b e e x p e c t e d t o p r o c e e d s t e p w i s e , as i n S c h e m e 1, l e a d i n g t o t h e formation o f intermediates and to c o m p l e t e scrambling o f labeled 0 . 2

2

2

2

2

2


2

2

2

2

2

A m e c h a n i s m t h a t is c o n s i s t e n t w i t h t h e d a t a g a t h e r e d i n t h i s s t u d y f o r t h e o x i d a t i o n o f [ N i ( L ) C N ] ~ b y 0 is s h o w n i n S c h e m e 2 . T h i s m e c h a n i s m a c c o u n t s f o r t h e s t o i c h i o m e t r y o f t h e r e a c t i o n , has a m a j o r p a t h w a y i n v o l v i n g i n c o r p o r a t i o n o f b o t h atoms o f a single 0 m o l e c u l e , a n d fea tures a r a t e - d e t e r m i n i n g step that involves o n e N i c o m p l e x a n d o n e 0 molecule. 2

2

2

T h e proposed mechanism involves the formation of persulfoxide or thiadioxirane intermediates. These intermediates have b e e n suggested t o b e i n v o l v e d i n t h e o x i d a t i o n o f t h i o e t h e r s b y 0 (98-100) a n d h a v e 1

2

b e e n p o s t u l a t e d f o r t h e o x i d a t i o n o f N i d i t h i o l e n e s t o b i s - s u l f i n a t e s (101). T h e r a t e - d e t e r m i n i n g s t e p i n t h e p r o p o s e d m e c h a n i s m is t h e c l e a v a g e o f t h e O - O b o n d i n t h e t h i a d i o x i r a n e i n t e r m e d i a t e . T h i s c o n c l u s i o n is based on the similarity between the enthalpy and entropy of activation f o r a n u m b e r o f o x i d a t i o n s i n w h i c h O - O b o n d c l e a v a g e is r a t e d e t e r m i n i n g (e.g., S c h e m e 1). T h e m e c h a n i s m o f t h i o e t h e r o x i d a t i o n is d e s c r i b e d as i n v o l v i n g e l e c t r o p h i l i c attack o f 0 o n t h e s u l f u r a t o m (or c o n v e r s e l y n u c l e o p h i l i c attack o f t h i o e t h e r S o n * 0 ) l e a d i n g to t h e formation o f sulfoxides a n d 1

2

2

ΊRN—Ni-CN

Scheme 2


2.

MARONEY E T AL.

49


sulfones. A recent study u s i n g 0 r e v e a l e d that t h e p r o d u c t sulfone c o n t a i n s t w o a t o m s o f o x y g e n d e r i v e d f r o m t h e same 0 m o l e c u l e (98). B e c a u s e a d d i t i o n o f t h e 0 s c a v e n g e r l , 4 - d i a z a b i c y c l o [ 2 . 2 . 2 ] o c t a n e has n o effect o n t h e r a t e o f r e a c t i o n , s i n g l e t o x y g e n is n o t d i r e c t l y i n v o l v e d . H o w e v e r , n u c l e o p h i l i c attack o f the c o o r d i n a t e d thiolates o n 0 does account for the stoichiometry o f the reaction, the derivation o f the major product from a single 0 molecule, a n d the absence o f radical i n t e r m e d i a t e s , a n d d o e s n o t i n v o l v e N i - c e n t e r e d r e d o x c h e m i s t r y . It is p o s s i b l e that t h e i n c r e a s e d n u c l e o p h i l i c i t y o f t h i o l a t e s u l f u r v e r s u s t h i o e t h e r s u l f u r is e n o u g h t o c a u s e t h e r e a c t i o n t o o c c u r at a s l o w r a t e w i t h t h e weaker electrophile. 1

8

2

2

l

2

3

2

2

N i c k e l - c o n t a i n i n g H ases are deactivated u p o n exposure to air i n a p r o c e s s t h a t is d e p e n d e n t o n 0 a n d r e s u l t s i n a w e a k i n t e r a c t i o n o f t h e S = fa c e n t e r i n t h e e n z y m e w i t h 0 . T h i s d e a c t i v a t i o n suggests t h a t 0 b i n d s n e a r t h e N i site (26). I n a d d i t i o n t o d i f f e r i n g i n t e r m s o f t h e i r E P R s p e c t r u m , t h e r e d u c t i v e a c t i v a t i o n k i n e t i c s u n d e r H also d i f f e r f o r forms A and B . E x p o s u r e to H results i n t h e r a p i d r e d u c t i o n o f f o r m B , w h e r e a s f o r m A e x h i b i t s a l a g t h a t has b e e n a s s o c i a t e d w i t h t h e r e m o v a l o f 0 f r o m t h e s a m p l e (3). S e v e r a l o b s e r v a t i o n s p o i n t t o a p o s s i b l e r o l e for S-oxidation i n the deactivation process. F i r s t , i n a d d i t i o n to the c o m pounds discussed here, t h e oxidation o f N i thiolate ligands to sulfinate l i g a n d s has b e e n o b s e r v e d i n o t h e r c o m p o u n d s (102). T h i s o x i d a t i o n o f N i t h i o l a t e l i g a n d s suggests that t h e o x i d a t i o n is t y p i c a l o f n i c k e l t h i o l a t e s a n d t h e r e is n o a p p a r e n t r e a s o n w h y i t w o u l d n o t o c c u r i n a n e n z y m e site t h a t is a c c e s s i b l e t o 0 . S e c o n d , i n m a n y cases f o r m Β h a s b e e n s h o w n t o r e a c t i n a i r t o p r o d u c e f o r m A . F u r t h e r m o r e , t h e r e a c t i o n is s l o w , w i t h ti/2 o n t h e o r d e r o f s e v e r a l h o u r s t o d a y s d e p e n d i n g o n t h e


2

2

l

1 7

2

2

2

2

2

2

e n z y m e i n v o l v e d a n d t h e t e m p e r a t u r e (3). T h i s is a t i m e scale s i m i l a r t o w h a t is o b s e r v e d f o r t h e 0 o x i d a t i o n i n N i t h i o l a t e c o m p l e x e s . L a s t , the substitution o f Se for t h e S center that reacts w i t h 0 m i g h t b e e x p e c t e d t o l e a d t o a n e n z y m e t h a t is m o r e o x y g e n t o l e r a n t , b e c a u s e oxides o f selenium are m o r e difficult to f o r m than sulfur oxides a n d are m o r e r e a d i l y r e d u c e d (103). T h i s a p p e a r s t o b e t h e case f o r H a s e s t h a t contain a selenocysteine ligated to the N i center. These enzymes can b e i s o l a t e d i n a i r i n a n E P R s i l e n t state w i t h o u t o x i d a t i o n t o f o r m A o r Β (5). 2

2

2

i s Ni the H -Binding 2

Site?

A n o t h e r p o t e n t i a l r o l e f o r t h e N i c e n t e r i n H a s e s is as a site i n v o l v e d i n H a c t i v a t i o n . A c a t a l y t i c a l l y v i a b l e f o r m o f t h e e n z y m e ( f o r m C ) is characterized b y a rhombic E P R signal, N i - C ( g = 2.19, 2.15, 2.02), t h a t has b e e n a t t r i b u t e d t o a N i ( I I I ) o r N i ( I ) c o m p l e x o f H " o r H , b a s e d o n E P R a n d E N D O R s p e c t r o s c o p i c studies (24, 26, 27). I n D. gigas H a s e , 2

2

l f 2 f 3

2

2


50


t h r e e sets o f * H E N D O R s i g n a l s h a v e b e e n o b s e r v e d . A set o f n o n e x changeable protons w i t h a c o u p l i n g constant o f 12 M H z was observed and attributed to cysteine 0 - C H protons. T h e observation o f protons w i t h t h e s a m e c o u p l i n g c o n s t a n t i n b o t h o x i d i z e d (forms A a n d B ) a n d r e d u c e d e n z y m e ( f o r m C ) is d i r e c t e v i d e n c e o f d e r e a l i z a t i o n o f s p i n d e n s i t y o n t o N i c y s t e i n e l i g a n d s a n d suggests that t h e e l e c t r o n i c s t r u c t u r e o f t h e E P R a c t i v e s p e c i e s is s i m i l a r i n b o t h t h e o x i d i z e d a n d r e d u c e d f o r m s o f t h e e n z y m e (i.e., t h a t N i d o e s n o t c h a n g e o x i d a t i o n state). I n a d d i t i o n t o t h e n o n e x c h a n g e a b l e p r o t o n s , t w o sets o f s o l v e n t e x c h a n g e a b l e p r o t o n s w e r e o b s e r v e d . T h e first is w e a k l y c o u p l e d (4 M H z ) a n d is a s s i g n e d t o a p r o t o n a t e d N i l i g a n d (e.g., H 0 ) . T h e s e c o n d set o f p r o t o n s , m o r e s t r o n g l y c o u p l e d ( 1 7 M H z ) , is d u e t o a p r o t o n t h a t is d e r i v e d f r o m H . A l t h o u g h t h e c o u p l i n g c o n s t a n t f o r t h i s p r o t o n is r e l atively large, it does not resemble t h e couplings t y p i c a l o f k n o w n p a r a magnetic N i - H hyperfine interactions, w h i c h are i n the 3 0 0 - 5 0 0 - M H z r a n g e a n d a r e v i s i b l e i n E P R s p e c t r a (104,105). T h e n a t u r e o f t h e p r o t o n g i v i n g r i s e t o t h e 1 7 - M H z c o u p l i n g i n H a s e is n o t c l e a r , a l t h o u g h a h y d r i d e b o u n d to N i using an orbital containing t h e u n p a i r e d electron is r u l e d o u t b y t h e E P R a n d E N D O R s p e c t r a . It w a s s u g g e s t e d t h a t t h i s proton c o u l d b e a h y d r i d e ligand b o u n d to a Ni(III) center through a d o r b i t a l i n t h e x,y p l a n e t h a t d o e s n o t c o n t a i n s p i n d e n s i t y (27). T h i s p o s s i b i l i t y is b o l s t e r e d b y E P R s p e c t r a o b t a i n e d o n N i ( C N ) ( H 0 ) " , in w h i c h no hyperfine splitting d u e to equatorial C N " is o b s e r v e d (106). T h e p o s s i b l e i n v o l v e m e n t o f a N i d i h y d r o g e n l i g a n d (107) r e m a i n s an o p e n question. 2


2

2

2

m

1

4

2

2

3

F o r m C is l i g h t - s e n s i t i v e a n d is k n o w n t o b e c o n v e r t e d t o a n E P R a c t i v e p h o t o p r o d u c t b y e x p o s u r e t o v i s i b l e l i g h t at l o w t e m p e r a t u r e (3, 24, 108). T h e p h o t o p r o d u c t e x h i b i t s a u n i q u e E P R s i g n a l , N i - L ( g i , , = 2 . 2 9 , 2 . 1 3 , 2 . 0 5 ) ( F i g u r e 1 4 ) . T h e p h o t o c h e m i s t r y is r e v e r s i b l e , a n d a n n e a l i n g t h e s a m p l e at a h i g h e r t e m p e r a t u r e r e g e n e r a t e s t h e N i - C E P R s p e c t r u m . I n t h e case o f T. roseopersicina H a s e , t h e t r a n s i t i o n f r o m N i - L t o N i - C is v e r y s h a r p a n d o c c u r s at 1 9 4 Κ ( F i g u r e 14) (88). I t h a s b e e n s u g g e s t e d t h a t f o r m C c o r r e s p o n d s t o a h y d r i d e (or d i h y d r o g e n ) N i a d d u c t (26, 27) a n d t h a t t h e p h o t o c h e m i s t r y i n v o l v e s d i s s o c i a t i o n o f t h e h y d r i d e f r o m t h e N i c e n t e r (24). W e e x a m i n e d t h i s p r o p o s a l u s i n g a c o m b i n a t i o n o f E N D O R a n d X A S measurements a n d f o u n d that a l t h o u g h t h e p h o t o c h e m i s t r y d o e s i n d e e d c o r r e s p o n d t o t h e loss o f a b o u n d p r o t o n ( s ) , t h e r e is n o c o n c l u s i v e e v i d e n c e t o s u p p o r t t h e a s s i g n m e n t o f N i as t h e b i n d i n g s i t e . 2

3

2

T h e ^ - E N D O R s p e c t r a o b t a i n e d o n T. roseopersicina H a s e r e v e a l resonances attributable to t w o o f the three types o f protons observed f o r t h e D. gigas e n z y m e , t h e n o n e x c h a n g e a b l e c y s t e i n e / 3 - C H p r o t o n s (A = 1 2 M H z ) a n d t h e m o r e s t r o n g l y c o u p l e d e x c h a n g e a b l e p r o t o n (A = 2 1 . 5 M H z ) . T h e w e a k l y c o u p l e d e x c h a n g e a b l e p r o t o n (4 M H z ) is n o t 2

2


2.

MARONEY ET AL.


51


Form C

Form C + hv

!

2800

3000 32Ω0 3400 Magnetic Field (Gauss)

3600

Figure 14. EPR spectra o/Thiocapsa roseopersicina hydrogenase, Ni-C (top) and Ni-L (bottom). Spectra were taken at 77 K, at a microwave fre quency of 9.62 GHz, a microwave power of 20 mW, and a modulation amplitude of 4 G.

observed. T h e resonance for the exchangeable p r o t o n that c o m e s f r o m d i h y d r o g e n is c l e a r l y s h o w n i n H - E N D O R s p e c t r a o b t a i n e d b y p r e p a r i n g t h e sample i n D 0 buffer (Figure 15). E x p o s u r e o f this sample to l i g h t results i n t h e c o n v e r s i o n o f the E P R s p e c t r u m f r o m N i - C to N i L a n d i n t h e loss o f t h e s t r o n g l y c o u p l e d H - E N D O R r e s o n a n c e . T h i s r e s o n a n c e is r e s t o r e d u p o n a n n e a l i n g t h e s a m p l e , t h u s d e m o n s t r a t i n g that t h e c o n v e r s i o n o f N i - C to N i - L i n v o l v e s t h e p h o t o l y s i s o f a p r o t o n d e r i v e d f r o m H a n d that a n n e a l i n g t h e sample gives rise to t h e r e c o m b i n a t i o n o f t h e p r o t o n w i t h t h e active site. 2

2

2

2

I f the p h o t o l y s i s i n v o l v e d dissociating a h y d r i d e (or H ) l i g a n d f r o m the N i site, changes i n e l e c t r o n density, geometry, a n d structure that c o u l d b e revealed b y X A S w o u l d result. T h e N i K-edge spectra taken o n samples d i s p l a y i n g E P R signals N i - C a n d N i - L are s h o w n i n F i g u r e 2



52


-20

-10 Δν

1 Η

0 10 (MHz)

20

Figure 15. H-ENDOR spectra of the solvent exchangeable protons asso ciated with Thiocapsa roseopersicina hydrogenase in form C (A) and its photoproduct (B). 2

1.2H

8300

8320

8340 8360 8380 X-ray Energy (eV)

8400

Figure 16. Ni K-edge XAS spectra obtained for Thiocapsa roseopersicina hydrogenase form C ( ) and its photoproduct ( ). (Reproduced from reference 88. Copyright 1993 American Chemical Society.)


2.

MARONEY E T AL.


53

16. These spectra show n o change i n t h e edge energy, p r e - e d g e features t h a t a r e s e n s i t i v e t o g e o m e t r y , o r X A N E S s p e c t r a ( T a b l e I) a n d suggest t h a t t h e p h o t o d i s s o c i a t i o n o f t h e h y d r o g e n i c p r o t o n ( s ) has n o s t r u c t u r a l c o n s e q u e n c e f o r t h e N i s i t e . S i m i l a r l y , t h e E X A F S s p e c t r a ( F i g u r e 17) reveal no evidence for a change i n b o n d length for any of the nonhyd r o g e n l i g a n d s o f t h e N i c e n t e r (88). A l t h o u g h i t is p o s s i b l e t h a t X A S is n o t s e n s i t i v e t o t h e s t r u c t u r a l c h a n g e s t h a t o c c u r , t h e l a c k o f any c h a n g e i n t h e X A S s p e c t r a suggests t h a t N i is n o t t h e H - b i n d i n g s i t e . T h e fact t h a t C O b o u n d to the e n z y m e reveals a strong C hyperfine interaction ( A = 85.3, 88.0, 9 0 . 3 M H z ) (26) a n d t h a t p a r a m a g n e t i c N i h y d r i d e s also h a v e l a r g e c o u p l i n g s t o t h e H ~ l i g a n d , b u t H " (or H ) s h o w s a m u c h w e a k e r c o u p l i n g i n N i - C p r o v i d e s a n o t h e r a r g u m e n t against a s t r o n g b o n d t o H " o r H at t h e N i s i t e . 1

2

1

3

3

x y z


2

2

O t h e r p o s s i b l e b i n d i n g sites i n c l u d e F e , S c l u s t e r s , w h i c h a r e p r e s e n t i n a l l H a s e s a n d a r e t h e l i k e l y b i n d i n g sites f o r H i n F e - o n l y H a s e s (2), a n d s u l f i d e o r t h i o l a t e l i g a n d s . T h e p o s s i b l e p r e s e n c e o f a N i , F e , S c l u s t e r i n t h e a c t i v e site i n F e , N i H a s e s , as s u g g e s t e d b y E P R (2, 3, 108) a n d E X A F S s t u d i e s (34), c o u l d g i v e r i s e t o a m e c h a n i s m b y w h i c h 2

2

2

2

1

2

1

1

1

1

4

6

8

10

k(A" )

r

12

1

Figure 17. Ni K-edge Fourier-filtered EXAFS spectra from hydrogenase in form C (top) and its photoproduct (bottom). Two shell fits are shown as small dashed lines. Form C. (1-2)N,0 1.92 (2) A + (3 ± 1)S at 2.22(2) A; form C + light: (1-2)N,0 at 1.92 (2) A+(4±l)Sat 2.22(2) A. Three-shell fits incorporating a long Ni-S interaction are shown as large dashed lines. Form C: (1-2)N,0 at 1.92(2) A+ (3±l)Sat 2.22(2) A, + (1)S at 2.75(2) A; form C + light: (1-2)N,0 at 1.94 (2) A + (3 ± 1)S at 2.23(2) A + (1)S at 2.70(2) A.


54

MECHANISTIC BIOINORGANICCHEMISTRY

H

* C p M o ^ l \ /

S

S

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2

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Cp M o ^ ^ ^ M o C p

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/

ps Scheme 3

weak c o u p l i n g b e t w e e n the h y d r o g e n i c proton(s) b o u n d to an F e atom a n d t h e N i c e n t e r c o u l d o c c u r . A n o t h e r p o s s i b i l i t y is t h a t a c t i v a t i o n o f H involves interaction w i t h a sulfide l i g a n d o f N i o r an F e , S cluster. It is n o t p o s s i b l e f r o m X A S a n a l y s i s t o d i f f e r e n t i a t e b e t w e e n t h i o l a t e a n d s u l f i d e l i g a n d s ; t h u s t h e p o s s i b i l i t y o f a N i s u l f i d e l i g a n d exists. F u r t h e r m o r e , h e t e r o l y t i c cleavage o f H (the reaction c a t a l y z e d b y H a s e ) i n v o l v i n g a b r i d g i n g sulfide has b e e n d e m o n s t r a t e d to occur i n o n e m o d e l s y s t e m ( S c h e m e 3) (109). I n t h i s m o d e l s y s t e m a b a s e ( p y r i d i n e ) gets p r o t o n a t e d , a n d t h e " h y d r i d i c ' ' p r o t o n e n d s u p c o n v e r t i n g a b r i d g ing sulfide l i g a n d to a b r i d g i n g hydrosulfide. Analogous chemistry c o u l d account for the presence o f a strongly c o u p l e d solvent exchangeable p r o t o n i n H a s e (hydrosulfide) a n d a m o r e w e a k l y c o u p l e d solvent exc h a n g e a b l e p r o t o n c o r r e s p o n d i n g t o a p r o t o n a t e d l i g a n d , as r e v e a l e d b y t h e E N D O R studies.


2

2

2

2

Y e t a n o t h e r p o s s i b i l i t y i n v o l v e s H a c t i v a t i o n at t h i o l a t e l i g a n d s . T h i s p o s s i b i l i t y is s u g g e s t e d b y t h e c h e m i s t r y o f a n F e t e t r a t h i o l a t e c o m p l e x ( S c h e m e 4) (110), w h i c h e v o l v e s H w h e n H is a d d e d t o t h e system. Intermediates p r o p o s e d for this reaction i n c l u d e t h i o l complexes d e r i v e d from the protonation o f the thiolate ligands. A role for a metal 2

1 1

2

+

c l u s t e r i n t h e catalysis is also s u g g e s t e d b y t h e m e c h a n i s m , w h i c h i n v o l v e s the formation o f d i m e r i c species i n order to p r o v i d e t h e t w o electrons necessary for the p r o d u c t i o n o f H . 2

crih£o] [cOlOo H

H

- I L

H

|-2H

Scheme 4


+

2.

MARONEY E T AL.


Summary and


55

Conclusions

T h e redox role of the N i center i n hydrogenase was e x a m i n e d using a combination of biophysical and synthetic model approaches. T h e lack o f a s i g n i f i c a n t shift i n t h e N i K - e d g e e n e r g y o r i n t h e N i - l i g a n d b o n d l e n g t h s i n h y d r o g e n a s e , as o b s e r v e d b y X A S o n r e d o x - p o i s e d e n z y m e samples, are inconsistent w i t h a redox role for N i . T h e b o n d lengths obtained f r o m E X A F S analysis a n d the S = 0 d e t e r m i n a t i o n for the N i c e n t e r f r o m m a g n e t i z a t i o n d a t a t h a t is a v a i l a b l e f o r a n E P R s i l e n t f o r m o f h y d r o g e n a s e f r o m D. baculatus a r e b o t h c o n s i s t e n t w i t h a n o x i d a t i o n state o f I I f o r t h e N i c e n t e r . C o m b i n e d w i t h t h e e d g e e n e r g y d a t a , t h e s e r e s u l t s suggest t h a t t h e o x i d a t i o n state o f N i is I I i n a l l r e d o x states o f t h e e n z y m e . A l t h o u g h t h i s c o n c l u s i o n is a p p e a l i n g i n v i e w o f t h e r e d o x c h e m i s t r y o f classical Ni(II) complexes, it does not e x p l a i n t h e E P R s p e c t r a a s s o c i a t e d w i t h t h e N i site o r p r o v i d e a n y i n s i g h t i n t o t h e n a t u r e o f t h e r e d o x a c t i v e s p e c i e s . It is c l e a r f r o m t h e o b s e r v a t i o n o f N i h y perfine splitting i n the E P R spectra that the r a d i c a l g i v i n g rise to the E P R signals o b s e r v e d at 7 7 Κ i n t i m a t e l y i n v o l v e s t h e N i c e n t e r . H o w e v e r , w e have a r g u e d that the E P R data are not u n e q u i v o c a l e v i d e n c e for N i centered redox chemistry or the presence of Ni(III) or Ni(I) i n the e n z y m e . T h e s e a r g u m e n t s a r e b a s e d o n t h r e e facts: 6 1

1. S - c e n t e r e d r a d i c a l s m a y e x h i b i t g - v a l u e s i n t h e r a n g e o f those observed i n the e n z y m e a n d are therefore u n r e l i a b l e i n d i c a t o r s o f p r i m a r i l y m e t a l vs. p r i m a r i l y l i g a n d o x i d a t i o n . 2. O x i d i z e d m o d e l c o m p o u n d s f e a t u r i n g a l k y l t h i o l a t e l i g a n d s exhibit small hyperfine couplings with the

6

1

N icenter and

large hyperfine couplings w i t h ligand methylene protons. These hyperfine couplings are indicative of substantial l i gand oxidation a n d are similar to the hyperfine couplings observed for hydrogenase. 3. S t u d i e s o f t h e r e d o x c h e m i s t r y o f N i c o m p l e x e s w i t h a l k y l thiolate ligands, l i k e those e x p e c t e d i n the b i o l o g i c a l site, suggest t h a t t h e p r o d u c t s o f o x i d a t i o n s o f s u c h c o m p l e x e s w i l l l i k e l y reflect S-oxidation. I f t h e c h a n g e s t h a t o c c u r u p o n o x i d a t i o n w e r e l a r g e l y l o c a l i z e d o n S (or d e l o c a l i z e d over several S centers), this localization w o u l d p r o v i d e one p o s s i b l e e x p l a n a t i o n for t h e o b s e r v e d i n s e n s i t i v i t y o f t h e N i site s t r u c t u r e t o t h e r e d o x state o f t h e e n z y m e . A l t e r n a t i v e l y , a r e d o x site c o m p o s e d o f p r o t e i n s u b s t i t u e n t s n e a r t h e N i site c o u l d also a c c o u n t f o r t h e observations. T h e p o s s i b l e r o l e o f t h e N i c e n t e r as a h y d r o g e n - b i n d i n g site w a s also e x a m i n e d . E N D O R s p e c t r o s c o p y c l e a r l y s h o w s t h a t a p r o t o n f r o m


56


H b i n d s t o a n a t o m t h a t is i n p a r t r e s p o n s i b l e f o r t h e E P R s p e c t r u m o f f o r m C . F u r t h e r m o r e , studies o f t h e p h o t o c h e m i s t r y o f f o r m C using E N D O R spectroscopy demonstrate that t h e dissociation o f this p r o t o n o c c u r s d u r i n g t h e p h o t o c h e m i c a l p r o c e s s . T h i s p h o t o l y t i c c l e a v a g e is reversed u p o n annealing the sample. Understanding the structure of the E P R a c t i v e site i n f o r m C w i l l b e v e r y i m p o r t a n t t o u n d e r s t a n d i n g h o w t h e e n z y m e activates H . T h e a s s u m p t i o n that N i is t h e H (or H " ) b i n d i n g site i n t h e e n z y m e is n o t firmly e s t a b l i s h e d a n d , g i v e n t h e h i g h l y d e l o c a l i z e d n a t u r e o f t h e s p e c i e s g i v i n g r i s e t o E P R s i g n a l C a n d its a s s o c i a t e d E N D O R spectra, a n u m b e r of alternative possibilities remain to be addressed. 2


2

2

T h e m o s t s t r i k i n g r e s u l t f r o m t h e b i o p h y s i c a l s t u d i e s is t h e l a c k o f a n y c h a n g e s a s s o c i a t e d w i t h t h e N i site s t r u c t u r e . T h i s l e a d s t o a c o n c l u s i o n s i m i l a r t o o n e s u g g e s t e d f o r t h e M o site i n n i t r o g e n a s e (111): t h a t t h e r o l e o f t h e N i i n h y d r o g e n a s e is t o m o d i f y a n e x i s t i n g a c t i v e s i t e , r a t h e r t h a n t o s e r v e as a n e n t i r e l y n e w a c t i v e c e n t e r . T h e e n z y m o l o g y o f t h e h y d r o g e n a s e s suggests t h a t t h e s e m o d i f i c a t i o n s a r e as sociated w i t h greater oxygen tolerance or specificity for hydrogen uptake.

Acknowledgments This w o r k was supported b y N a t i o n a l Institutes o f H e a l t h G r a n t G M 3 8 8 2 9 for M i c h a e l J . M a r o n e y a n d National Science F o u n d a t i o n G r a n t M C B - 9 2 0 7 9 7 4 for Brian M . Hoffman.

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for review July 19, 1993. ACCEPTED revised manuscript March 15,

1994.


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