Understanding the Mechanisms in Bioinorganic Chemistry - Advances

1 Understanding the Mechanisms in Bioinorganic Chemistry

Downloaded by NORTHERN KENTUCKY UNIV on April 9, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/ba-1995-0246.ch001

H . Holden Thorp Department of Chemistry, University of North Carolina, Chapel H i l l , N C 27599-3290

The field of bioinorganic chemistry has experienced tremendous growth in scope and level of understanding. Advances in physical methods and the constant discovery of new biological systems that rely on metal ions have created a field that is evolving to be both broader and more detailed. As a result, bioinorganic chemists are increasingly concerned with a broad range of different types of mechanisms. In this review, the broad nature of the subject of bioinorganic chemistry is illustrated by discussing examples of important mechanisms in which metal ions mediate electron transfer, hydrolytic catalysis, redox catalysis, and gene expression.

THE

F I E L D O F B I O I N O R G A N I C C H E M I S T R Y has g r o w n d r a m a t i c a l l y o v e r

t h e past 2 0 y e a r s ( J ) . F r o m b e g i n n i n g s i n s t u d i e s o f h e m e p r o t e i n s (2) a n d p l a t i n u m a n t i t u m o r c o m p o u n d s (3) t o r e c e n t a d v a n c e s i n s u c h n e w areas as Z n finger p r o t e i n s (4), m e t a l - r e s p o n s i v e g e n e e x p r e s s i o n (5), R N A c a t a l y s i s (6), a n d m e t a l l o p r o t e i n e l e c t r o n t r a n s f e r (7), t h e field h a s b e c o m e a h a v e n f o r p r a c t i t i o n e r s o f s u c h d i v e r s e areas as e n z y m o l o g y , coordination chemistry, spectroscopy, and molecular biology. W h a t att r a c t s t h e s e s c i e n t i s t s f r o m s u c h s e e m i n g l y d i v e r s e areas t o b i o i n o r g a n i c chemistry? T h e answer can lie only i n the unique abilities of transition metals to c o m p l e m e n t a n d mediate biological processes a n d t h e c h e m i c a l transformations of biological molecules. Transition metals can e x c e e d the constraints o f organic functionalities and assume c o o r d i n a t i o n n u m b e r s greater t h a n four i n a w i d e array o f s t r u c t u r a l g e o m e t r i e s . F o r t h i s r e a s o n , m a n y t r a n s i t i o n m e t a l s are p r e s e n t i n b i o l o g i c a l s y s t e m s t o s e r v e (at least p a r t l y ) a s t r u c t u r a l r o l e , a l l o w i n g a m a c r o m o l e c u l e to assume a p a r t i c u l a r c o n f o r m a t i o n that w o u l d b e impossible without a transition-metal structural element. A n o t h e r useful 0065-2393/95/0246-0001 $08.00/0 © 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

f e a t u r e o f t r a n s i t i o n m e t a l s i n b i o l o g i c a l s y s t e m s is t h a t t h e s e e l e m e n t s m a y exist i n m u l t i p l e o x i d a t i o n states a n d o f t e n a r e f o u n d as i o n s o r i n o r g a n i c clusters that facilitate m u l t i e l e c t r o n oxidations or r e d u c t i o n s o f s u b s t r a t e s . B e c a u s e t h e n u m b e r o f s u i t a b l e d o n o r s t o m e t a l s is q u i t e l a r g e , t h e c h e m i c a l p o t e n t i a l o f a s y s t e m c a n b e fine t u n e d o v e r a n i m pressive range. B i o l o g i c a l systems fully exploit transition-element c h e m i s t r y b y c o u p l i n g t h i s t u n a b l e (in t e r m s o f both s t o i c h i o m e t r y a n d potential) r e d o x a c t i v i t y w i t h the capacity to c o o r d i n a t e n u c l e o p h i l e s . In this w a y a r e m a r k a b l e range of c h e m i c a l transformations, f r o m n i t r o g e n fixation t o w a t e r o x i d a t i o n , a r e u n d e r t a k e n i n a s e e m i n g l y effortless process. A n o t h e r u s e f u l f e a t u r e o f t r a n s i t i o n m e t a l s i n b i o l o g i c a l s y s t e m s is t h e u n i q u e b a t t e r y o f p h y s i c a l t e c h n i q u e s t h a t c a n b e u s e d to s t u d y t h e i r s t r u c t u r a l a n d e l e c t r o n i c p r o p e r t i e s (8). M a n y o f t h e s e t e c h n i q u e s a r e dependent on the unusual geometric and electronic properties of transition-metal centers a n d a l l o w for study of the metal i o n w i t h o u t i n t e r ference from other material i n the system. I n assuming f u n c t i o n a l roles, t r a n s i t i o n metals use one or b o t h o f t h e a b i l i t i e s o f e x i s t i n g i n m u l t i p l e o x i d a t i o n states a n d c o o r d i n a t i n g nucleophiles. In electron-transfer enzymes, the ability of transition metals to exist i n m u l t i p l e o x i d a t i o n states a l l o w s f o r t h e storage o f a n e l e c t r o n at a l o c a l i z e d site i n t h e e n z y m e , c r e a t i n g t h e n e e d f o r t r a n s f e r o f e l e c t r o n s across r e l a t i v e l y l a r g e d i s t a n c e s (9). T h e s e e l e c t r o n - t r a n s f e r r e actions are responsible for the t r a n s d u c t i o n of b i o c h e m i c a l e n e r g y , i n i tiation of photosynthesis and other catalytic processes, and maintenance o f p r o t o n g r a d i e n t s (JO, J J). I n h y d r o l y t i c e n z y m e s a n d r i b o z y m e s , t h e ability o f t r a n s i t i o n metals to c o o r d i n a t e w a t e r a n d h y d r o l y z a b l e s u b strates is r e s p o n s i b l e f o r c a t a l y t i c a c t i v i t y (6, 12). T h e s e t w o u n i q u e features are c o m b i n e d i n r e d o x catalysis e n z y m e s , b e c a u s e i n t h i s p r o c e s s m e t a l s b i n d s u b s t r a t e s o f i n t e r e s t a n d c h a n g e t h e o x i d a t i o n state o f t h e m e t a l c e n t e r t o effect a n e t a c t i v a t i o n o f t h e b o u n d s m a l l m o l e c u l e , w h i c h can then be released i n an altered form. B i o i n o r g a n i c c h e m i s t r y has b e e n c a l l e d a " m a t u r i n g f r o n t i e r " (J). W i t h this m a t u r i n g , the l e v e l of understanding of the structures a n d r e a c t i v i t y o f m e t a l i o n s i n b i o l o g i c a l s y s t e m s has b e e n r a i s e d c o n s i d e r ably. T h e availability of X - r a y absorption spectroscopy and other spectroscopic techniques a n d the i n c r e a s i n g database of results f r o m m a c romolecular X - r a y crystallography have p r o v i d e d an increasingly detailed p i c t u r e of the structural properties of metal ions i n b i o l o g i c a l s y s t e m s . W h e r e a s m a n y issues o f s t r u c t u r e r e m a i n u n r e s o l v e d , k e y structural features o f m a n y m e t a l l o e n z y m e systems are n o w k n o w n . A s a r e s u l t , b i o i n o r g a n i c c h e m i s t s find t h e m s e l v e s c o n t e m p l a t i n g m e c h a n i s t i c issues m o r e c a r e f u l l y . T h e m e c h a n i s t i c e m p h a s i s i n p h y s i c a l s t u d i e s o f m e t a l l o e n z y m e s is i n c r e a s i n g (8); t h e d e v e l o p m e n t o f s y n t h e t i c m o d e l


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Understanding the Mechanisms in Bioinorganic Chemistry

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c o m p l e x e s is p r o g r e s s i n g from i n i t i a l goals o f m i m i c k i n g s p e c t r o s c o p i c o r s t r u c t u r a l features to m o r e s o p h i s t i c a t e d goals o f f u n c t i o n a l m o d e l i n g (13); t h e c h e m i s t r y o f D N A c l e a v a g e has p r o g r e s s e d t o w a r d a c h i e v i n g a n u n derstanding of the precise molecular m e c h a n i s m of cleavage reactions o n b o t h t h e m e t a l a n d D N A sides o f t h e r e a c t i o n , i n c l u d i n g t h e p r e c i s e sites o f D N A r e a c t i v i t y (14-17). A s a r e s u l t o f this n e w i n t e r p l a y , t h e l i n e s b e t w e e n s t r u c t u r a l a n d m e c h a n i s t i c studies are b e c o m i n g b l u r r e d . In this chapter, the u n i q u e features of transition metals i n b i o l o g i c a l s y s t e m s are d i s c u s s e d f r o m t h e p o i n t o f v i e w o f s t r u c t u r a l r o l e s , s p e c troscopic properties, electron transfer, h y d r o l y t i c a n d redox catalysis, and metal-responsive gene expression. T h e following chapters provide m o r e detail o n these subjects. Several i m p o r t a n t examples not discussed elsewhere i n this v o l u m e w i l l be presented. T h e goal of this chapter ( a n d t h i s v o l u m e ) is to a c q u a i n t t h e r e a d e r w i t h t h e w i d e r a n g e o f r o l e s p l a y e d b y m e t a l ions i n b i o l o g i c a l systems a n d t h e r e b y to d e m o n s t r a t e w h y metals are such useful cofactors a n d w h y scientists f r o m such b r o a d d i s c i p l i n e s a r e d r a w n to s t u d y t h e i r p r o p e r t i e s .

Structural Roles for Transition

Metals

T h e ability o f transition metals to assume a v a r i e t y o f c o o r d i n a t i o n geo m e t r i e s is l a r g e l y r e s p o n s i b l e f o r t h e i r u n i q u e b i o l o g i c a l f u n c t i o n . M o s t m e t a l c e n t e r s i n b i o l o g y a r e e i t h e r f o u r - , five-, o r s i x - c o o r d i n a t e ; h o w ever, the recent discovery of unusual three-coordinate iron centers i n n i t r o g e n a s e (18) i l l u s t r a t e s t h e a p p a r e n t l y l i m i t l e s s d i v e r s i t y o f c o o r d i n a t i o n e n v i r o n m e n t s for m e t a l centers i n b i o l o g i c a l systems. I n the f o u r coordinate e n v i r o n m e n t s , t e t r a h e d r a l a n d square-planar geometries are most c o m m o n . E x a m p l e s of the tetrahedral g e o m e t r y i n c l u d e i r o n c e n ters i n i r o n - s u l f u r clusters a n d m a n y c o p p e r centers, all of w h i c h are discussed i n this volume. A large n u m b e r of z i n c - b i n d i n g enzymes called Zn-finger proteins have b e e n d i s c o v e r e d (J9). I n these e n z y m e s , the Z n c e n t e r is a s t r u c tural element that organizes t w o histidine a n d t w o cysteinate residues o n e i t h e r e n d o f a p e p t i d e s t r a n d to f o r m a c y l i n d r i c a l s t r u c t u r e t h a t has b e e n t e r m e d a z i n c finger (20, 21). T h e s e p r o t e i n s t y p i c a l l y c o n t a i n m u l t i p l e fingers t h a t b i n d t o D N A c o o p e r a t i v e l y , as s h o w n i n F i g u r e 1. In the absence of Z n , these peptides do not b i n d to D N A ; h o w e v e r , o n c e t h e finger s t r u c t u r e is i m p o s e d b y c o o r d i n a t i o n t o t h e m e t a l c e n t e r , t h e r e s i d u e s i n t h e finger s e c t i o n a r e p o s i t i o n e d p r o p e r l y f o r D N A r e c ognition. O t h e r z i n c - b i n d i n g domains, i n c l u d i n g ones i n v o l v i n g d i n u c l e a r z i n c c o m p l e x e s (4), h a v e b e e n i d e n t i f i e d i n r e l a t e d t r a n s c r i p t i o n a l factors (22). T h e d i s c o v e r y o f t h e z i n c - b i n d i n g t r a n s c r i p t i o n factors s h o w s t h a t n a t u r e has t a k e n a d v a n t a g e o f t h e a b i l i t y o f m e t a l i o n s t o o r g a n i z e relatively small peptides into highly specific structures. 2 +

2 +



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Figure 1. Sketch of the complex of thefive-fingerhuman GLI protein with a 21-base pair DNA fragment. (Reproduced with permission from reference 20. Copyright 1993 American Association for the Advancement of Science.) T h e s q u a r e - p l a n a r g e o m e t r y is less c o m m o n t h a n t e t r a h e d r a l g e o m e t r y a m o n g four-coordinate m e t a l ions i n b i o l o g i c a l systems. T h e a n t i c a n c e r d r u g c i s p l a t i n , d 5 - P t ( N H ) C l , is a s q u a r e - p l a n a r c o m p l e x a n d is a n e f f e c t i v e a g e n t a g a i n s t t e s t i c u l a r a n d o v a r i a n c a n c e r s (3). T h e c o m p l e x b i n d s p r i m a r i l y to t w o adjacent g u a n i n e bases o n a single D N A strand of a double helix. Because of the square-planar geometry of the platinum center, the guanines are forced out of the usual stacked, parallel a r r a n g e m e n t t o o n e w h e r e t h e y a r e d i s p o s e d at r i g h t a n g l e s ( F i g u r e 2) (23). T h e c o o r d i n a t i o n s t r u c t u r e o f t h i s i n t r a s t r a n d a d d u c t i n d u c e s a k i n k i n t h e D N A t h a t is r e c o g n i z e d b y s t r u c t u r e - s p e c i f i c r e c o g n i t i o n proteins (SSRPs), suggesting that these proteins play a role i n c i s p l a t i n 3

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Figure 2. X-ray crystal structure of the cis-{Pt(NH ) [d(pGpG)]} complex showing the displacement of the adjacent guanines from the usual stacked arrangement. (Reproduced with permission from reference 23. Copyright 1985 American Association for the Advancement of Science.) 3

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c y t o t o x i c i t y (24, 25). T h e s e p r o t e i n s d o n o t r e c o g n i z e a d d u c t s o f i n a c t i v e platinum complexes,

s u c h as £ r a n s - P t ( N H ) C l ; t h u s , t h e a n t i c a n c e r 3

2

2

p r o p e r t i e s o f c i s p l a t i n a r e a d i r e c t r e s u l t o f its s t e r e o c h e m i s t r y a n d s q u a r e - p l a n a r g e o m e t r y , w h i c h is c h a r a c t e r i s t i c o f a n u m b e r o f t r a n s i t i o n m e t a l s i n t h e a p p r o p r i a t e o x i d a t i o n state. T h e r e c e n t

findings

on the

role of SSRPs i n cisplatin cytotoxicity demonstrate the breadth of chall e n g e s i n b i o i n o r g a n i c c h e m i s t r y : C i s p l a t i n s t a r t e d o u t as a n e x c i t i n g p r o b l e m i n c o o r d i n a t i o n c h e m i s t r y a n d is n o w a n e q u a l l y e x c i t i n g p r o b lem in molecular biology.


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Spectroscopic Signatures of Transition

Metals

A n a l l u r i n g f e a t u r e o f t r a n s i t i o n m e t a l s i n b i o l o g i c a l s y s t e m s is t h e w i d e array o f p h y s i c a l t e c h n i q u e s that are u n i q u e to the c h a r a c t e r i z a t i o n o f metal centers. In particular, a n u m b e r of methods allow the metal center to b e u n i q u e l y e x a m i n e d i n t h e p r e s e n c e o f o t h e r o p t i c a l l y t r a n s p a r e n t o r d i a m a g n e t i c m a t e r i a l i n t h e s y s t e m (8). P e r h a p s t h e m o s t o b v i o u s l y u n i q u e f e a t u r e o f s y s t e m s t h a t c o n t a i n t r a n s i t i o n m e t a l s is t h a t m a n y o f t h e m a r e s t r o n g l y c o l o r e d . T h i s t r a i t arises b e c a u s e o f t h e u n i q u e e l e c tronic structures of transition-metal chromophores. F o r example, the characteristic optical properties of b l u e c o p p e r proteins, h e m e cofactors, a n d i r o n - s u l f u r clusters are all discussed i n this v o l u m e . D e t a i l e d study o f t h e s e o p t i c a l p r o p e r t i e s has b e e n p a r t i c u l a r l y r e v e a l i n g w i t h r e g a r d to u n d e r s t a n d i n g the g e o m e t r i c a n d e l e c t r o n i c structures of t r a n s i t i o n metals i n b i o l o g i c a l systems. Continuous-wave and pulsed electron paramagnetic resonance (EPR) methods p e r m i t the characterization of only paramagnetic metal centers without complications arising from other diamagnetic centers elsewhere i n t h e s y s t e m (8). W h e n t h e m e t a l c e n t e r o r c e n t e r s c a n b e c y c l e d t h r o u g h d i f f e r e n t o x i d a t i o n states, signals c a n b e t u r n e d off a n d o n i n a controlled manner, permitting detailed characterization of individual signals a n d t h e i r a s s o c i a t e d p a r a m a g n e t i c c e n t e r s (26). T h e use o f p u l s e d E P R m e t h o d s p e r m i t s i n m a n y cases t h e d e t e r m i n a t i o n o f t h e n u m b e r a n d n a t u r e o f ligands b o u n d to the m e t a l c e n t e r that gives a p a r t i c u l a r s i g n a l (27). T h e use o f E P R m e t h o d s t o c h a r a c t e r i z e m e t a l l o e n z y m e s is d e s c r i b e d i n many chapters i n this v o l u m e , a n d the c o m b i n a t i o n of i n f o r m a t i o n f r o m E P R w i t h t h a t f r o m o p t i c a l s p e c t r o s c o p y is o f t e n p a r t i c ularly powerful. X - r a y a b s o r p t i o n s p e c t r o s c o p y ( X A S ) has also h a d a p r o f o u n d i m p a c t o n b i o i n o r g a n i c c h e m i s t r y (28). T h e a d v a n t a g e o f X A S is t h a t i t is e l e ment-specific because using m o d e r n synchrotron radiation, the X A S spectrum of any element of interest can be obtained without interference f r o m o t h e r t y p e s o f e l e m e n t s i n t h e s y s t e m (29). T h i s r e s u l t m e a n s t h a t even diamagnetic, optically inactive metal centers can be characterized i n d e p e n d e n t l y for each transition e l e m e n t i n a g i v e n system. T h u s , the properties of one element can be studied independently i n the presence of another element that m a y have o p t i c a l or magnetic properties. A n a l ysis o f t h e s o - c a l l e d a b s o r p t i o n e d g e is r e v e a l i n g w i t h r e g a r d t o t h e electronic structure of the metal center, w h i c h can permit many conc l u s i o n s r e g a r d i n g t h e o x i d a t i o n state a n d c o o r d i n a t i o n e n v i r o n m e n t t o b e d r a w n . E v e n m o r e p o w e r f u l is t h e a n a l y s i s o f 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 ) . T h i s a n a l y s i s p e r m i t s d e t e r m i n a t i o n o f b o n d lengths to p a r t i c u l a r d o n o r atoms a n d e s t i m a t i o n o f c o o r d i n a t i o n n u m b e r s f o r e a c h o f t h e s e d o n o r s . T h e s p e c i a l a d v a n t a g e o f E X A F S is


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that the b o n d lengths d e t e r m i n e d are often of q u i t e h i g h p r e c i s i o n . C a l culations i n v o l v i n g the b o n d valence s u m m e t h o d show that E X A F S b o n d lengths can b e u s e d to estimate e i t h e r c o o r d i n a t i o n n u m b e r s or o x i d a t i o n states i f o n e o f t h e t w o is k n o w n (30, 31). A l l of the special techniques u s e d for m e t a l l o e n z y m e s are most useful w h e n analogous small molecules have b e e n p r e p a r e d b y inorganic synthesis a n d c h a r a c t e r i z e d b y X - r a y c r y s t a l l o g r a p h y . T h e p r e p a r a t i o n o f these m o d e l c o m p l e x e s allows a g i v e n p h y s i c a l p r o p e r t y to b e associated w i t h a p a r t i c u l a r c o o r d i n a t i o n s t r u c t u r e o r o x i d a t i o n state. S y n t h e s i s o f m o d e l complexes w i t h structural, functional, or spectroscopic properties r e l e v a n t t o a p a r t i c u l a r m e t a l l o e n z y m e is a v i t a l a s p e c t o f b i o i n o r g a n i c c h e m i s t r y (13) a n d is d e s c r i b e d i n n u m e r o u s c h a p t e r s i n t h i s v o l u m e .

Hydrolytic

Catalysis

M e t a l ions are vital to the f u n c t i o n o f m a n y e n z y m e s that catalyze h y d r o l y t i c reactions. C o o r d i n a t i o n o f a w a t e r m o l e c u l e to a m e t a l i o n alters its a c i d - b a s e p r o p e r t i e s , u s u a l l y m a k i n g i t e a s i e r t o d e p r o t o n a t e , w h i c h c a n offer a r e a d y m e a n s f o r c a t a l y z i n g a h y d r o l y t i c r e a c t i o n . A l s o , t h e p l a c e m e n t o f a m e t a l c e n t e r i n the active site o f a h y d r o l y t i c e n z y m e c o u l d p e r m i t efficient d e l i v e r y o f a catalytic w a t e r m o l e c u l e to the h y d r o l y z a b l e s u b s t r a t e . I n f a c t , t h e first e n z y m e d i s c o v e r e d , c a r b o n i c a n h y d r a s e , is a m e t a l l o e n z y m e t h a t r e q u i r e s a Z n c e n t e r f o r its c a t a l y t i c a c t i v i t y (32). T h e f u n c t i o n o f c a r b o n i c a n h y d r a s e is t o c a t a l y z e t h e h y drolysis o f c a r b o n d i o x i d e to b i c a r b o n a t e : 2 +

C0

+ H 0 ^ HC0 - + H

2

2

3

(1)

+

w h i c h plays an i m p o r t a n t role i n r e g u l a t i n g respiratory processes i n a n i m a l s , p l a n t s , a n d b a c t e r i a (12). T h e X - r a y c r y s t a l s t r u c t u r e s h o w s t h a t t h e z i n c c e n t e r is c o o r d i n a t e d t o t h r e e h i s t i d i n e r e s i d u e s a n d a w a t e r m o l e c u l e (33), a n d t h e c a t a l y t i c m e c h a n i s m has b e e n p r o p o s e d t o i n v o l v e formation of a Z n O H complex and insertion of C 0 into the Z n - O bond: 2

(His) Zn-OH 3

(His) Zn-OH + C 0 3

2

— (His) Zn-OH + H 3

(His) Zn-OC0 H

2

3

(His) Zn-OC0 H + H 0 3

2

(2)

+

(3)

2

(His) Zn-OH + H C 0 "

2

3

2

(4)

3

L o o n e y a n d c o - w o r k e r s p r e p a r e d a h y d r o x o c o m p l e x of z i n c that r e v e r s i b l y b i n d s C 0 (34, 3 5 ) : 2

[HB(3-*-Bu-5-Mepz) ]ZnOH + C 0 3

2

^

[HB(3-f-Bu-5-Mepz) ]ZnOC0 H 3

w h e r e p z is p y r a z o l y l .


2

(5)

8


H y d r o l y t i c c a t a l y s i s b y m e t a l i o n s is also i m p o r t a n t i n t h e h y d r o l y s i s o f n u c l e i c a c i d s , e s p e c i a l l y R N A (36). M o l e c u l e s o f R N A t h a t c a t a l y z e h y d r o l y t i c reactions, t e r m e d r i b o z y m e s , r e q u i r e divalent m e t a l ions to effect h y d r o l y s i s efficiently. T h u s , a l l r i b o z y m e s are m e t a l l o e n z y m e s (6). T h e r e is s p e c u l a t i o n t h a t r i b o z y m e s m a y h a v e b e e n t h e first e n z y m e s t o e v o l v e (37), so t h e v e r y first e n z y m e s m a y h a v e b e e n m e t a l loenzymes! R e c e n t l y , substitution of sulfur for the 3'-oxygen atom i n a substrate o f t h e t e t r a h y m e n a r i b o z y m e has b e e n s h o w n to g i v e a 1 0 0 0 - f o l d r e d u c t i o n i n rate of hydrolysis w i t h M g but no attenuation of the hydrolysis rate w i t h M n and Z n (38). B e c a u s e M n and Z n have stronger affinities for s u l f u r t h a n M g has, this feature p r o v i d e s strong e v i d e n c e for a true catalytic role of the divalent cation i n the h y d r o l y t i c m e c h a n i s m , i n v o l v i n g c o o r d i n a t i o n o f t h e m e t a l t o t h e 3'oxygen atom. O t h e r examples of metal-ion catalyzed hydrolysis of R N A i n v o l v e lanthanide complexes, w h i c h are discussed i n this v o l u m e . 2 +


2 +

2 +

2 +

2 +

2 +

Electron

Transfer

T h e a b i l i t y t o exist i n m o r e t h a n o n e o x i d a t i o n state a l l o w s t r a n s i t i o n m e t a l c o m p l e x e s t o s e r v e as t h e a c t i v e s i t e o f e n z y m e s w h o s e f u n c t i o n is t o t r a n s f e r e l e c t r o n s (39). A g r e a t d e a l o f effort has b e e n d i r e c t e d at understanding the mechanisms of electron transfer i n metalloproteins, s u c h as c y t o c h r o m e s a n d b l u e c o p p e r p r o t e i n s (40). O f p a r t i c u l a r i n t e r e s t is t h e m e c h a n i s m b y w h i c h a n e l e c t r o n c a n t u n n e l f r o m a m e t a l c e n t e r t h a t is i m b e d d e d i n a p r o t e i n m a t r i x t o a s i t e o n t h e o u t e r s u r f a c e o f t h e p r o t e i n (7). A d i s c u s s i o n o f c u r r e n t t h e o r i e s is g i v e n i n t h i s v o l u m e . I n n a t u r a l s y s t e m s , r e d o x p r o t e i n s s u c h as c y t o c h r o m e c (cyt c) f u n c t i o n n o t o n l y t o t r a n s f e r e l e c t r o n s , b u t t o t r a n s f e r e l e c t r o n s specifically to a p a r t i c u l a r r e d o x p a r t n e r , u s u a l l y a n o t h e r m a c r o m o l e c u l e . T r a n s f e r of electrons between subunits of modified hemoglobins and w i t h i n c o m plexes of cyt c w i t h cyt b and cyt c w i t h cyt c peroxidase (Ccp) have t h e r e f o r e b e e n s t u d i e d e x t e n s i v e l y (41,42). T h e s e s t u d i e s h a v e r e v e a l e d the f u n d a m e n t a l r e q u i r e m e n t s for the r e c o g n i t i o n process l e a d i n g to t h e f o r m a t i o n o f t h e p r o t e i n - p r o t e i n c o m p l e x as w e l l as t h e t h e r m o d y n a m i c features of the electron-transfer r e a c t i o n itself. T h i s r e a c t i o n , o u t l i n e d i n e q u a t i o n 6, c o n s i s t s o f t h r e e f u n d a m e n t a l p r o c e s s e s : r e c o g n i t i o n t o f o r m a c o m p l e x (Ki), e l e c t r o n t r a n s f e r w i t h i n t h e c o m p l e x , and dissociation of the redox-altered complex ( K ) . F o r the cyt c - C c p c o m p l e x , Fe(II) cyt c corresponds to P and oxidized C c p corresponds to P ! . 5

2

2

r e d

0 X

-±

? i

red

+

p < ox 2

(6)

T h e r a t e o f e l e c t r o n t r a n s f e r w i t h i n t h e c o m p l e x is a f u n c t i o n o f t h e t h e r m o d y n a m i c d r i v i n g force of the reaction, the conformation of the


1.

THORP


9


p r o t e i n - p r o t e i n c o m p l e x , a n d t h e i n t e r v e n i n g m e d i u m (41, 42). O r i g i nally, an invariant p h e n y l a l a n i n e r e s i d u e i n cyt c was t h o u g h t to m e d i a t e its e l e c t r o n - t r a n s f e r r e a c t i o n s , b u t ( i n o n e o f t h e e a r l y a p p l i c a t i o n s o f the t e c h n i q u e ) s i t e - d i r e c t e d mutagenesis o f the P h e r e s i d u e to Ser, G l y , a n d T y r s h o w e d t h a t t h e P h e w a s n o t e s s e n t i a l f o r f u n c t i o n (43). T h e recent crystal structure of the cyt c - C c p c o m p l e x shows that e l e c t r o n transfer p a t h w a y s do i n d e e d exist that do not i n v o l v e the P h e r e s i d u e ( F i g u r e 3) (7, 44). I n d i v i d u a l residues f r o m each p r o t e i n that are vital for c o m p l e x form a t i o n h a v e also b e e n i d e n t i f i e d t h r o u g h s i t e - d i r e c t e d m u t a g e n e s i s (9). T h u s , f o r m a t i o n of the p r o t e i n - p r o t e i n c o m p l e x occurs t h r o u g h a series of site-specific interactions of amino acids o n the surface of one p r o t e i n w i t h particular amino acids on the surface of the other p r o t e i n . T h e crystal structures o f the c o m p l e x e s o f C c p w i t h cyt c f r o m yeast a n d h o r s e c o n f i r m t h e s i t e - s p e c i f i c i n t e r a c t i o n s (44). H a k e et a l . (45) p r e p a r e d mutants of C c p i n w h i c h k e y aspartate residues r e q u i r e d for c o m p l e x

Figure 3. Possible electron-transfer pathway between the hemes of CCp and yeast cyt c determined from the X-ray crystal structure of the proteinprotein complex. (Reproduced with permission from reference 44. Copyright 1992 American Association for the Advancement of Science.)


10


formation w e r e c h a n g e d to lysine, d i s r u p t i n g any h y d r o g e n b o n d s f o r m e d b y the aspartate i n the c o m p l e x . Affinity c h r o m a t o g r a p h y

experiments

c o n f i r m t h e i m p o r t a n t r o l e p l a y e d b y t h e s e r e s i d u e s a n d also s h o w t h a t t h e r e a c t a n t i n e q u a t i o n 6, F e ( I I ) c y t c, b i n d s m o r e s t r o n g l y t o C c p t h a n t h e p r o d u c t , F e ( I I I ) c y t c, at p h y s i o l o g i c a l i o n i c s t r e n g t h . O f

course,

t h i s s i t u a t i o n is d e s i r e d f o r e f f i c i e n t c a t a l y t i c e l e c t r o n t r a n s f e r i n t h e natural system because strong b i n d i n g of the Fe(III) cyt c p r o d u c t

to

C c p w o u l d inhibit further reaction. Surprisingly, the order of b i n d i n g


affinities for t h e v a r i o u s m u t a n t s w a s d i f f e r e n t f o r F e ( I I I ) c y t c a n d F e ( I I ) c y t c. T h u s , t h e b i n d i n g p r o b a b l y o c c u r s at d i f f e r e n t sites o n t h e p r o t e i n s u r f a c e s f o r t h e t w o o x i d a t i o n states. T h e s e e x p e r i m e n t s s h o w t h a t t h e simple presence or absence of an electron on a single metal center b u r i e d i n a l a r g e p r o t e i n c a n d r a m a t i c a l l y affect t h e i n t e r a c t i o n s o f t h e e n t i r e m a c r o m o l e c u l e w i t h its p r o t e i n p a r t n e r . T h i s r e s u l t u n d e r l i n e s t h e p r o f o u n d i m p a c t that f u n d a m e n t a l i n o r g a n i c c h e m i s t r y , s u c h as t h e o x i d a t i o n state o f a s i n g l e m e t a l i o n , c a n h a v e o n t h e b e h a v i o r o f c o m p l e x b i o l o g i c a l systems.

Redox Catalysis E n z y m e s t h a t c a t a l y z e t h e r e d o x r e a c t i o n s o f s m a l l m o l e c u l e s , s u c h as the r e d u c t i o n o f n i t r o g e n to a m m o n i a , the o x i d a t i o n o f w a t e r to o x y g e n , o r t h e o x i d a t i o n o f m e t h a n e t o m e t h a n o l , h a v e l o n g c a p t u r e d t h e fascination of inorganic chemists a n d biochemists, a n d these a n d other r e l a t e d processes are discussed e l s e w h e r e i n this v o l u m e . P e r h a p s these s y s t e m s a r e so a t t r a c t i v e b e c a u s e t h e r e a c t i o n s c a t a l y z e d a r e d i f f i c u l t t o achieve u s i n g e i t h e r small i n o r g a n i c catalysts or a p r o t e i n w i t h o u t a metal center. Therefore, only through the marriage of protein and transition-metal chemistry can these complex

t r a n s f o r m a t i o n s o c c u r effi-

c i e n t l y w i t h h i g h catalytic t u r n o v e r . T h e s e systems thus present

the

t a n t a l i z i n g possibility that a d e t a i l e d u n d e r s t a n d i n g of structure a n d m e c h a n i s m w i l l l e a d to l o g i c a l means of s y n t h e s i z i n g small

molecules

capable o f m e d i a t i n g the same transformations. A s m e n t i o n e d

above,

t h i s i n t e r p l a y o f e n z y m e c h e m i s t r y a n d i n o r g a n i c s y n t h e s i s has l e d t o s e v e r a l n e w s y s t e m s c a p a b l e o f a c t i n g as f u n c t i o n a l m o d e l s f o r a n u m b e r o f e n z y m e r e a c t i o n s (13), m a n y o f w h i c h a r e d i s c u s s e d h e r e . I n a d d i t i o n to c a t a l y z i n g the o x i d a t i v e or r e d u c t i v e t r a n s f o r m a t i o n o f s m a l l m o l e c u l e s , r e d o x m e t a l l o e n z y m e s c a n also effect t h e t r a n s l o c a t i o n o f p r o t o n s against a c h e m i c a l g r a d i e n t across a m e m b r a n e . C o n ceptually, this m o v e m e n t can be u n d e r s t o o d from the point of v i e w of the fundamental coordination chemistry of m e t a l - o x o complexes

(46,

47). R e v e r s i b l e r e d u c t i o n o f m e t a l - o x o c o m p l e x e s o f t e n o c c u r s c o n c o m i t a n t l y w i t h p r o t o n a t i o n , as s h o w n i n e q u a t i o n s 7 a n d 8.


1.

THORP

Understanding the Mechanisms in Bioinorganic Chemistry [M(n + 2 ) 0 ]

+ H

+

+ e"

[M(n + l ) O H ]

[M(n + l j O H ] ^ " ^ + H

+

+ e"

[M(n)OH ] "

n +


1

2

( n

( n

'

11

(7)

1 ) +

(8)

2 ) +

These reactions can occur w i t h terminal oxo complexes or b r i d g i n g o x o c o m p l e x e s (48), as w e l l as w i t h c o m p l e x e s o f o t h e r l i g a n d s t h a t c a n a c c e p t p r o t o n s , s u c h as t h i o l a t e s o r a l k o x i d e s . T h i s p r o c e s s is d r i v e n b y the c o u p l i n g of the reduction o f the metal center to an increase i n the basicity o f the oxo l i g a n d . F r o m this elementary m o d e l , a simple p r o t o n p u m p can b e devised i f protons are taken u p from one side o f the m e m brane u p o n m e t a l r e d u c t i o n a n d released to t h e other side o f the m e m brane u p o n reoxidation. T o achieve this r e q u i r e m e n t , t h e p u m p must exist i n t w o d i f f e r e n t states, e a c h o f w h i c h is i n p r o t o n i c c o n t a c t w i t h a different side o f t h e m e m b r a n e . I n this manner, c y c l i n g o f t h e redox state o f t h e m e t a l c e n t e r l e a d s t o p r o t o n t r a n s l o c a t i o n , a n d e l e c t r o n transfer c a n b e u s e d to d r i v e t h e p u m p i n g o f protons against a n elect r o c h e m i c a l gradient. T h i s process has b e e n discussed i n terms o f a n e i g h t - s t a t e c u b i c m o d e l , i n w h i c h t h e e i g h t states c o r r e s p o n d t o t h e f o u r f o r m s ( t w o r e d o x a n d t w o p r o t o n i c ) o f b o t h states o f a c c e s s i b i l i t y t o e a c h s i d e (49). T h e respiratory e n z y m e complex c y t o c h r o m e c oxidase (Ceo) cata l y z e s t h e o x i d a t i o n o f c y t c b y d i o x y g e n (10, 11, 50): 4 cyt c

2 +

+ 0

+ 4H

2

+

— 4 cyt c

3 +

+ 2H 0

(9)

2

T h e p r o t e i n resides i n the i n n e r m i t o c h o n d r i a l m e m b r a n e a n d receives electrons f r o m t h e cytosolic side w h i l e c o n s u m i n g protons f r o m t h e m a t r i x s i d e . I n t h i s w a y , a p r o t o n e l e c t r o c h e m i c a l g r a d i e n t is g e n e r a t e d . I n a d d i t i o n , t h e e n z y m e p u m p s as m a n y as f o u r a d d i t i o n a l p r o t o n s f o r each dioxygen molecule reduced. This latter mode of proton p u m p i n g , i n w h i c h p r o t o n t r a n s l o c a t i o n is n o t c o u p l e d t o s u b s t r a t e r e d u c t i o n , h a s b e e n t e r m e d " v e c t o r i a l " i n contrast to the " s c a l a r " p r o t o n p u m p i n g resulting from dioxygen r e d u c t i o n (equation 9). F o u r redox-active metal centers are present i n C e o : t w o copper c e n t e r s , C u a n d C u , a n d t w o h e m e s f r o m c y t o c h r o m e s a a n d a (10, 11, 50). T h e h e m e f r o m c y t a a n d t h e C u c e n t e r a r e s t r o n g l y (—/ > 2 0 0 c m ) a n t i f e r r o m a g n e t i c a l l y c o u p l e d a n d exist as a n S = 2 b i n u c l e a r c o m p l e x (51), w h i c h is t h e p r o b a b l e site o f d i o x y g e n r e d u c t i o n . T o achieve the strong antiferromagnetic coupling, the C u a n d heme a centers probably interact v i a some type o f b r i d g i n g ligand, possibly a u-oxo l i g a n d . T w o l a b o r a t o r i e s h a v e r e p o r t e d t h e s y n t h e s e s o f u-oxo c o p p e r - h e m e c o m p l e x e s (52,53), w h i c h a r e s h o w n i n F i g u r e 4. I n t h e s e complexes, the iron and c o p p e r centers are strongly antiferromagnetic a l l y c o u p l e d , as i n C e o . M o d e l s f o r h o w t h i s site m i g h t r e d u c e d i o x y g e n h a v e b e e n p r o p o s e d ( F i g u r e 5) (10), a n d t h e n e x t s t e p f o r t h e m o d e l i n g A

B

3

3

B

- 1

B


3

12


[(OEP)Fe-0-Cu(Me tren)r


6

Figure 4. a, X-ray crystal structure of an oxo-bridged heme-copper complex synthesized by Lee and Holm. (Reproduced from reference 52. Copyright 1993 American Chemical Society.) b X-ray crystal structure of an oxobridged heme-copper complex synthesized by Nanthakumar and co-workers. (Reproduced from reference 53. Copyright 1993 American Chemical Society.) y


1.

THORP


Fe

©

Cu£

a3

+02

FeL-0 3


13

N

0

,Cu£

Compound A

© Compound C (607 nm)

H • +

©

-e"

c F e

H + +

n

+e

_

_/

H

a -°N / 3

0

_n C u

Fea =0 E

B

Cupric hydroperoxide (CugEPR Signal)

Ferryl (580 nm)

3

HO

©

-e"

+e~

HO

Pulsed

Figure 5. Possible mechanism for reduction of 0 in cytochrome oxidase involving the binuclear heme-copper site. (Reproduced from reference 10. Copyright 1990 American Chemical Society.) 2

chemistry will b e to achieve dioxygen reduction using the coupled c o p p e r - h e m e models. In addition to the mechanism o f dioxygen reduction, an understandi n g o f h o w C e o p u m p s p r o t o n s is also d e s i r a b l e . M o d e l s h a v e b e e n p r o p o s e d that a l l o w for linkage o f the p r o t o n p u m p i n g to t h e d i o x y g e n r e d u c t i o n r e a c t i o n (50). O n e a t t r a c t i v e m o d e l i n v o l v e s t h e C u site a n d is s h o w n i n F i g u r e 6 (JO). I n t h i s m e c h a n i s m , t h e C u c e n t e r is l i g a t e d to t w o histidines a n d t w o thiolates a n d receives t h e initial e l e c t r o n f r o m A

A


14



Figure 6. Model for redox-linked proton pumping in cytochrome oxidase involving the Cu site. (Reproduced from reference 10. Copyright 1990 American Chemical Society.) A


1.

THORP


15

c y t c. T h e r e d u c e d c o m p l e x r e l e a s e s o n e t h i o l a t e a n d c o o r d i n a t e s a n e a r b y t y r o s i n e l i g a n d , w h i l e t h e p r o t o n f r o m t h e t y r o s i n e is c o n c u r r e n t l y t r a n s f e r r e d to the r e l e a s e d thiolate. T h e r e d u c e d p h e n o x i d e c o m plex t h e n transfers an e l e c t r o n to the o x y g e n - b o u n d C u - h e m e a site, a n d a p r o t o n is lost f r o m t h e s u l f h y d r y l t o t h e c y t o s o l s i d e o f t h e m e m brane. T h e r e s u l t i n g free thiolate t h e n displaces the alkoxide l i g a n d o n


B

3

t h e o x i d i z e d c o p p e r c e n t e r , a n d t h e f r e e p h e n o x i d e is t h e n p r o t o n a t e d f r o m t h e m a t r i x s i d e . T h i s m o d e l is a t t r a c t i v e b e c a u s e i t uses s i m p l e c o o r d i n a t i o n c h e m i s t r y , s u c h as t h a t s e e n i n m e t a l - o x o a n d r e l a t e d c o m p l e x e s w i t h a c i d - b a s e a c t i v e l i g a n d s , t o effect p r o t o n t r a n s l o c a t i o n .

Metal-Responsive

Gene Expression

A s discussed h e r e , b i o l o g i c a l systems r e l y o n metals for n u m e r o u s catalytic a n d structural functions. L i v i n g systems therefore have a t r e m e n dous n e e d for a r e g u l a t o r y system that w i l l ensure that a p a r t i c u l a r m e t a l i o n is a v a i l a b l e w h e n it is n e e d e d t o f u l f i l l o n e o f t h e s e c a t a l y t i c o r structural roles. O n the other h a n d , h i g h concentrations of m e t a l ions c a n b e t o x i c , a n d t h e r e g u l a t o r y s y s t e m m u s t also m a i n t a i n t h e c o n c e n trations of free m e t a l ions b e l o w toxic levels. T h e s e r e g u l a t o r y systems m u s t t h e r e f o r e b e v e r y finely t u n e d , a n d m o s t s y s t e m s r e l y o n p r o t e i n s , t e r m e d metalloregulatory proteins, that specifically b i n d the m e t a l i o n o f i n t e r e s t (5). T h e m e t a l - b o u n d m e t a l l o r e g u l a t o r y p r o t e i n t h e n b i n d s t o D N A a n d i n d u c e s t r a n s c r i p t i o n o f m e s s e n g e r R N A ( m R N A ) that c o d e s f o r a p r o t e i n t h a t c a n c a t a l y z e t h e r e m o v a l o r s t o r a g e o f t h e excess m e t a l ion. F o r example, w h e n H g concentrations reach toxic levels, the met a l l o r e g u l a t o r y p r o t e i n m e r R b i n d s a s i n g l e m e r c u r i c i o n (54, 5 5 ) . T h e H g - m e r R p r o t e i n distorts t h e D N A i n t h e p r o m o t e r r e g i o n a n d i n d u c e s t r a n s c r i p t i o n o f t h e g e n e f o r m e r c u r i c i o n r e d u c t a s e ( F i g u r e 7), w h i c h then renders H g h a r m l e s s b y c a t a l y z i n g its r e d u c t i o n t o t h e v o l a t i l e Hg° form. A related system regulates c o p p e r concentrations b y activating t r a n s c r i p t i o n of c o p p e r m e t a l l o t h i o n e i n , a c o p p e r storage p r o t e i n (56, 5 7 ) . 2 +

2 +

2 +

A s w i t h c o p p e r a n d m e r c u r y i o n s , c o n c e n t r a t i o n s o f i r o n m u s t also b e c a r e f u l l y r e g u l a t e d (58, 5 9 ) . T h e c h i e f i r o n s t o r a g e p r o t e i n is f e r r i t i n , w h i c h is c a p a b l e o f s t o r i n g u p t o 4 5 0 0 i r o n a t o m s i n a n i r o n - o x o m i n e r a l l a t t i c e (60). W h e n i n t r a c e l l u l a r i r o n c o n c e n t r a t i o n s a r e h i g h , s y n t h e s i s o f f e r r i t i n is r e q u i r e d , as s e e n w i t h c o p p e r m e t a l l o t h i o n e i n (56, 5 7 ) . H o w e v e r , t h e m e c h a n i s m b y w h i c h f e r r i t i n s y n t h e s i s is r e g u l a t e d is unique in metalloregulation. Rather than by activation of transcription, in w h i c h high metal ion concentrations induce activity of R N A polymerase and p r o d u c t i o n of m R N A , ferritin synthesis proceeds v i a r e g u l a t i o n at t h e translational l e v e l , t h a t i s , s y n t h e s i s o f p r o t e i n f r o m m R N A is r e g u l a t e d b y i r o n c o n c e n t r a t i o n . T h e m R N A f o r f e r r i t i n c o n t a i n s a


16


Hg(II)


• POISED (Closed) C O M P L E X

RNA polymerase

A C T I V A T E D (OPEN) C O M P L E X

Figure 7. Potential mechanism for Hg -responsive gene expression. Binding ofHg* to merR causes local underwinding of the DNA that optimizes contact of the DNA with RNA polymerase. (Reproduced with permission from reference 5. Copyright 1993 American Association for the Advancement of Science.) 24

+

30-nucleotide stem-loop structure, termed the iron recognition element ( I R E ) (61). I R E s a r e p r e s e n t i n m R N A s f o r o t h e r i r o n - s t o r a g e a n d - t r a n s p o r t p r o t e i n s , s u c h as t r a n s f e r r i n a n d t r a n s f e r r i n r e c e p t o r , a n d also p l a y a r o l e i n r e g u l a t i n g synthesis o f t h e s e e n z y m e s (58). R e g u l a t i o n o f f e r r i t i n is a c h i e v e d v i a i n t e r a c t i o n o f t h e I R E w i t h t h e I R E - b i n d i n g p r o t e i n ( I R E B P ) , w h i c h inhibits translation b y b i n d i n g to the I R E . T h e I R E B P binds to t h e I R E at l o w i r o n c o n c e n t r a t i o n s , b u t w h e n i r o n l e v e l s a r e i n c r e a s e d , a n i r o n - s u l f u r c l u s t e r is a s s e m b l e d i n t h e I R E B P , i n d u c i n g a s t r u c t u r a l c h a n g e that p r o h i b i t s R N A b i n d i n g a n d p e r m i t s f e r r i t i n synthesis (62). T h u s , f e r r i t i n is s y n t h e s i z e d as d e s i r e d w h e n i r o n c o n c e n t r a t i o n s are h i g h .



1.

THORP


17

T h e b i o c h e m i s t r y o f t h e I R E r e g u l a t i o n s y s t e m f o r f e r r i t i n has a n u m b e r o f u n i q u e f e a t u r e s . A s a l r e a d y s t a t e d , t h e I R E s y s t e m is u n u s u a l i n o p e r a t i n g at t h e t r a n s l a t i o n a l l e v e l i n s t e a d o f t h e t r a n s c r i p t i o n a l l e v e l . I n a d d i t i o n , r e g u l a t i o n o f f e r r i t i n s y n t h e s i s i n v o l v e s negative r e g u l a t i o n because b i n d i n g of the regulatory protein i n the absence of the metal ion stimulates gene expression. T h e other m e t a l l o r e g u l a t o r y systems discussed here operate via positive regulation, because b i n d i n g of the m e t a l i o n to the m e t a l l o r e g u l a t o r y p r o t e i n stimulates g e n e expression. B e c a u s e t h e s y s t e m o p e r a t e s at t h e t r a n s l a t i o n a l l e v e l , i t r e l i e s o n t h e s t r u c t u r e o f a n R N A s e g m e n t , w h i c h is m u c h m o r e h e t e r o g e n e o u s t h a n the D N A structures that mediate t r a n s c r i p t i o n a l activation. R e c e n t s t u d i e s s h o w t h a t t h e s t r u c t u r e o f t h e I R E is s t r o n g l y c o r r e l a t e d w i t h the presence of h i g h l y conserved base pairs i n so-called flanking regions o n e i t h e r s i d e o f t h e a c t u a l I R E (63). A n u n d e r s t a n d i n g o f t h e c o m p l e x s t r u c t u r e o f t h e I R E is a n i m p o r t a n t g o a l i n u n r a v e l i n g t h e m e c h a n i s m s of i r o n homeostasis, a n d structural studies using transition-metal cleavage agents a p p e a r p a r t i c u l a r l y f r u i t f u l (64). In a d d i t i o n to a u n i q u e b i o c h e m i s t r y , the i n o r g a n i c c h e m i s t r y o f f e r r i t i n r e g u l a t i o n is also u n u s u a l . T h e c o o r d i n a t i o n c h e m i s t r i e s o f t h e m e t a l l o r e g u l a t o r y p r o t e i n a n d the r e g u l a t e d p r o t e i n are quite different (5). A s s e m b l y o f t h e f e r r i t i n c o r e i n v o l v e s s y n t h e s i s o f a m e t a l - o x o c o r e , w h e r e a s t h e a c t i v e site o f t h e I R E B P is a n i r o n - s u l f u r c l u s t e r , w h i c h involves a significantly different type of coordination chemistry. R e l a t e d m e t a l l o r e g u l a t o r y systems e x h i b i t similar c h e m i s t r y for b o t h the r e g u lated protein and the metalloregulatory protein. A l s o , w h e n the i r o n s u l f u r c l u s t e r is a s s e m b l e d i n t h e I R E B P , t h e p r o t e i n c a n f u n c t i o n as a n aconitase e n z y m e that catalyzes the c o n v e r s i o n o f citrate to isocitrate (62). T h i s e x a m p l e is t h e o n l y o n e k n o w n o f a separate e n z y m a t i c a c t i v i t y f o r a m e t a l l o r e g u l a t o r y p r o t e i n . T h e r o l e (if t h e r e is one) o f t h i s e n z y m a t i c a c t i v i t y i n t h e i r o n r e g u l a t i o n p r o c e s s is u n c l e a r ; h o w e v e r , t h e c o u p l i n g of m e t a l l o r e g u l a t i o n to m e t a l l o e n z y m a t i c catalysis b y a single e n z y m e is a n e x c i t i n g p o s s i b i l i t y .

Conclusions T h e f u t u r e o f b i o i n o r g a n i c c h e m i s t r y is filled w i t h p r o m i s e a n d e x c i t e ment. A s m o r e sophisticated p h y s i c a l methods are d e v e l o p e d for the study of b i o l o g i c a l systems, e v e n h i g h e r levels of structural characterization w i l l b e c o m e available a n d m o r e d e t a i l e d mechanistic studies w i l l be possible. G r e a t e r u n d e r s t a n d i n g of e n z y m e mechanisms w i l l be acc o m p a n i e d b y the d e v e l o p m e n t of synthetic m o d e l c o m p l e x e s that not o n l y m i m i c s p e c t r o s c o p i c a n d s t r u c t u r a l f e a t u r e s b u t also act as f u n c t i o n a l m o d e l s . S i g n i f i c a n t p r o g r e s s i n t h i s d i r e c t i o n is a l r e a d y b e i n g m a d e (13). This research w i l l p r o v i d e an increased understanding of e n z y m e m e c h -



18

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anisms and a host of synthetic catalysts with new reactivities and efficiencies. The new understanding of the mechanisms of hydrolytic and oxidative cleavage of nucleic acids will provide new techniques for understanding the structures of complex nucleic acids and clues for manipulating DNA and RNA in living systems (6, 65-67). As the fundamental properties of metal ions in biological systems become apparent, the ability of living systems to regulate metal ion concentrations becomes an important issue (5). An understanding of these regulatory mechanisms represents an important new frontier that may ultimately allow scientists to manipulate the concentrations of metal ions in vivo and to prepare pharmaceutical products with desirable properties (68).

Acknowledgments I thank the National Science Foundation for a Presidential Young Investigator award and the David and Lucile Packard Foundation for a Fellowship in Science and Engineering.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

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