Posttranslationally modified tyrosines from galactose oxidase and cytochrome C oxidase

POSTTRANSLATIONALLY MODIFIED TYROSINES FROM GALACTOSE OXIDASE AND CYTOCHROME C OXIDASE BY MELANIE S. ROGERS AND DAVID M. DOOLEY Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Posttranslational A m i n o Acid Modifications . . . . . . . . . . . . . . . . . . . . . . . . . B, Posttranslationally Modified Tyrosine Residues with New Catalytic Roles . . . . C. Tyrosine Cross-linked Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Galactose Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Galactose Oxidase Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. A Two-Electron Oxidation Mediated by a Single Copper Ion? . . . . . . . . . . D. EPR Spectroscopy Reveals a New Type of Tyrosyl Radical . . . . . . . . . . . . . . E. The Electron Density Reveals a Novel T h i o e t h e r Bond . . . . . . . . . . . . . . . . E Spectroscopic and Theoretical Studies of the Cross-Link Perturbation . . . . . . (',. The Effect of the T h i o e t h e r Substituent in Galactose Oxidase Probed by Model Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Structure, Function, a n d Biogenesis of the T h i o e t h e r Bond in Galactose Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. O t h e r Proteins with a T h i o e t h e r Bond . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Cytochrome c Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Discovery of the Active-Site Cross-Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Chemical Evidence of the Covalent Bond . . . . . . . . . . . . . . . . . . . . . . . . . . D. Model Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Role of the Cross-Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E Biogenesis of the Tyrosine-Histidine Cross-Link . . . . . . . . . . . . . . . . . . . . . . IV. Final C o n n n e n t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

387 387 389 389 390 390 390 393 395 398 398 401 408 412 417 417 419 42(1 423 427 430 431 43~

I. INTRODUCTION

A. Posttranslational Amino Acid Mod!fications A s is a b u n d a n t l y c l e a r f r o m t h e r e v i e w s i n t h i s v o l u m e , a d i v e r s e a n d growing class of proteins with posttranslationally modified redox-active a m i n o a c i d s is n o w b e i n g d e f i n e d ( F i g . 1) ( O k e l e y a n d v a n d e r D o n k , 2000). Moreover, the interest in examining the structures, functions, a n d b i o g e n e s i s o f t h e s e n e w a m i n o a c i d s is e x p a n d i n g r a p i d l y . T h e u n u s u a l n a t u r e o f m a n y o f t h e d e f i n e d m o d i f i c a t i o n s p o s e s fascinating questions about the mechanisms by which such cofactors are generated. Recently, several aspects of the biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor of amine oxidase 387 A.DVAN('t~.'; L\' I~I¢O'I'I~:IN CtfliMISTI~}:

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388

M E L A N I E S. R O G E R S AND DAVID M. D O O L E Y

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FIG. 1. The structural diversity created by the posttranslational modifications discussed in Okeley and van der Donk (2000) are shown, as well as some model compounds that have been studied to understand the physicochemical or catalytic properties of some of these novel cofactors. Compound 14 catalyzes amine oxidation and has contributed to understanding of TPQ-dependent enzymes. Compounds 1.~17 have been used to probe the modulation of pKA and redox potential of phenols when substituted with a thioether as in galactose oxidase, or with imidazole, as in cytochrome c oxidase. Reprinted from Chemistry and Biology, Vol. 7, Okeley, N. M., and van der Donk, W. A. Novel cofactors via posttranslational modifications of enzyme active sites, pp R159-R171, Copyright (2000), with permission from Elsevier Science.

have b e e n d e f i n e d (Dooley, 1999; Cai a n d Klinman, 1994; Dove et al., 2000; Schwartz et al., 2000), a n d hypotheses a d v a n c e d r e g a r d i n g the significance o f these m e c h a n i s m s for r e d o x e n z y m e evolution. T h e oxidation o f tyrosine to T P Q requires only c o p p e r ions a n d dioxygen (Matsuzaki et al., 1995; Ruggiero et al., 1997), a n d is a self-processing event, n o t requiring any accessory proteins. T P Q biogenesis a n d its role in a m i n e oxidase catalysis are t h o r o u g h l y reviewed elsewhere in this volume, a n d o u r u n d e r s t a n d i n g o f T P Q biochemistry has r e a c h e d a satisfying level o f m o l e c u l a r sophistication (Su a n d Klinman, 1998; W i l m o t et al., 1999).

P O S T T R A N S L A T I O N A L I X MODIFIED TYROSINES

389

B. Posttranslationally Modified Tyrosine Residues with New Catalytic Roles During the same period the structure and biochemistry of T P Q were being elucidated, new findings about the redox roles of tyrosine residues and modified variants emerged. Structural biology and protein biochemistry have d o c u m e n t e d the functionality and chemical roles of unmodified tyrosines within numerous enzymes and proteins. Generally, the tyrosine phenol group has been shown to play two major roles: as a metal ligand and in acid/base reactions. For example, tyrosine 188 is a key iron-binding residue in human serum transferrin N-lobe, where mutation of this residue abolishes metal binding (He et al., 1997). The phenol ring of tyrosine can also act as a proton donor, as has been seen in epoxide hydrolase. Steady-state kinetics of Y152F and Y215F epoxide hydrolase provided supporting evidence for the role of the tyrosines as proton donors. The crystal structure provides strong corroboration for this idea (Rink et al., 1999). Tyrosine may also act in a redox capacity,. The tyrosyl radical Yz° of photosystem II reaction center complex, for example, is directly involved in oxygen evolution (Stubbe and van der Donk, 1998). A tyrosyl radical is also seen to participate in catalysis in ribonucleotide reductase (RNR) (Stubbe and Riggs-Gelasco, 1998) and one is observed in prostaglandin H synthase that likely abstracts a hydrogen from the C 13 of arachidonic acid (Tsai and Kuhnacz, 2000). The possible role in catalysis for the radical has been demonstrated by chemical modification of tyrosine (Shimokawa et al., 1990), site-directed mutagenesis (Tsai et al., 1994), and changes in the peroxide-generated tyrosyl radical electron paramagnetic resonance (EPR) spectrum when anaerobically adding arachidonate or octa-deuterated arachidonate that were reversed on exposure to oxygen (Tsai et al., 1995). The repertoire of roles tyrosine plays may be greatly, extended by the finding that covalent cross-linking of tyrosine's phenol group to another amino acid side chain perturbs the properties of the phenol side chain, tbr example, its pKa or redox potential. Thus, covalently cross-linked tyrosine residues, which have now been identified in two proteins, may represent a new family of cofactors, most likely with a redox role. C. Tyrosine Cross-Linked Proteins Galactose oxidase was identified as an enzyme containing a modified tyrosine in 1991 when the crystal structure was determined at 1.9 A resolution (Ito et al., 1991). This was the first identification of a modified tyrosine functionally important in catalysis (Whittaker and Whittaker, 1988, 1990). More recently, cytochrome c oxidase has also been found to con-

390

MELANIE S. ROGERS AND DAVID M. DOOLEY

rain a modified tyrosine that may also be a new type of cofactor (Ostermeier et al., 1997). Similar cofactors have been identified in other metallooxidases. Catechol oxidase (Klabunde et al., 1998) and certain tyrosinases (Lerch, 1982) have been identified as containing a Cys-His cross-link, where the histidine also functions as a copper ligand. However, as yet, the formation and role of the Cys-His cross-link are not clean Cross-linked tyrosines have also been identified in two other proteins: Escherichia coli catalase HPII (C[LTyr-415 to N ~- of the proximal heme ligand His-392) (Bravo et al., 1997, 1999) and Nitrosomonas europaea hydroxylamine oxidoreductase (5-meso carbon of the porphyrin and the C3 ring carbon of Tyr-467) (Igarashi et al., 1997). Both cross-links have been confirmed via crystallography. However, in this chapter we will focus on galactose oxidase and cytochrome c oxidase, for which more information is available.

II. GALACTOSEOXIDASE A. Introduction

Galactose oxidase (E. C. 1.1.3.9) is an extracellular copper-containing oxidative protein secreted by Fusarium sp. (Knowles and Ito, 1993) and is part of the radical copper oxidase family. There is a single active site where primary alcohols (e.g., the hydroxyl group at C6 in D-galactose) are oxidized to aldehydes with the concomitant reduction of dioxygen to hydrogen peroxide [Eq. (1)]. R-CH2OH + 02 ~ R-CHO + H202

(1)

Although a broad range of substrates are utilized by the enzyme, there is a strict stereospecific requirement (Amaral et al., 1963). For example, although B-galactose is a substrate, L-galactose and B-glucose are not (Avigad et al., 1962). The substrate range may be broad to allow the organism to use any of a n u m b e r of c o m p o u n d s to produce H202 rapidly. A relatively high turnover n u m b e r (800 sec -1) is observed with D-galactose (Wachter and Branchaud, 1996). The function of galactose oxidase may be to rapidly produce peroxide as a defense against bacteria (Whittaker and Whittaker, 1998). B. Galactose Oxidase Structure

Galactose oxidase is a single polypeptide of 639 amino acids (McPherson et al., 1992) with a molecular mass of 68,000 Da. In 1991, the structure of galactose oxidase was solved using multiple isomorphous

POSTTRANSLATIONAL1X MODIFIED TYROSINES

391

b Domain I

Fl(;. 2. Overall t h r e e - d i m e n s i o n a l structure o f galactose oxidase as r i b b o n diagrams drawn using the p r o g r a m MOLSCRIPT. (a) Side view o f the m o l e c u l e with d o m a i n s I a n d III shaded. (b) View o f d o m a i n II a p p r o x i m a t e l y along the p s e u d o - s e v e n f o l d axis. T h e Cu is shown as a s h a d e d s p h e r e in b o t h cases (Ito et al. 1995).

r e p l a c e m e n t a n d r e f i n e d to a resolution o f 1.9 A (Ito et al., 1991). Crvstals were grown at p H 4.5 in an a c e t a t e - c o n t a i n i n g b u f f e r a n d later transf e r r e d to a PIPES-containing b u f f e r to obtain a s e c o n d structure at p H 7.0. Subsequently, the crystals were t r e a t e d with DDC to r e m o v e c o p p e r so that the a p o p r o t e i n structure c o u l d b e d e t e r m i n e d . T h e m o n o m e r i c p r o t e i n is a r r a n g e d into t h r e e d o m a i n s that are m a i n l y [~ structure; t h e r e is only o n e s h o r t 0t helix (residues 327 to 332) (Fig. 2a). T h e first d o m a i n (residues 1 to 155) has a ~-sandwich structure a n d is linked to d o m a i n II by a stretch o f a m i n o acids that are well-

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FIG. 3. (a) T h e active site of mature galactose oxidase. Reprinted with permission from Rogers et al., 2000. Copyright (2000) American Chemical Society. Schematic diagram of copper coordination at (b) p H 4.5 a n d (c) p H 7.0 (Ito et al., 1995).

TABLE I Bond Lengths of the Copper Active Site of Galactose Oxidase a Bond length (Jk)

pH 4.5

pH 7.0

Cu-O n (Y272) Cu-NE2 (H496) Cu-Ne2 (H581) Cu-O~1 (Y495) Cu-O (Acetate) Cu-O (Water)

1.9 2.1 2.2 2.7 2.3 --

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a From Ito et al. (1994).

POSTTRANSLATIONALLY MODIFIED TYROSINES

393

ordered. Domain II (residues 156 to 532) has a structure reminiscent of a propeller or flower. There are seven blades (or petals) as seen in Fig. 2b. Each blade comprises a four-stranded antiparallel ~ sheet. The stability of this ~ structure is illustrated by the observation that activity can be detected in up to 6 M urea (Kosman et al., 1974). The third domain (residues 533 to 639) sits on top of domain II and two of the seven [3 strands reach down through the middle of the pseudo-sevenfold axis of domain II to provide a ligand to the copper ion. The active site (Fig. 3a) is located on the solvent-accessible surface of domain II, close to the sevenfold axis. The active site copper is ligated by two equatorial histidine residues (H496, H581) and two tyrosine residues, one equatorial (Y272) and one axial (Y495). The axial tyrosine is considered a "weak" ligand because it is located at 2.69 ~ from the copper ion. This tyrosine apparently functions as the active-site base, abstracting a proton from the b o u n d alcohol (Reynolds et al., 1997). The fourth equatorial position is proposed to be the substrate binding site (Knowles et al., 1995). Tryptophan 290 is positioned over the equatorial tyrosine, the indole side chain "stacking" over the thioether bond. O n e side of the indole ring is exposed to solvent; thus it has been proposed that W290 protects the thioether (or tyrosyl radical) from the solvent. Since removal of the copper ion resuhs in little structural change (Ito et al., 1994), the site appears to be somewhat rigid. The crystal structures determined at pH 4.5 and 7.0 showed significant difference in the copper coordination geometry shown in Fig. 3b-c and Table I. When the crystal was transferred to a non-acetatecontaining buffer at neutral pH, the coordinating acetate was replaced by a water. Although the water is located in a similar equatorial position, the Cu(II)-O distance is 2.8 A. This is too long for a strong Cu(II)-OH~ bond, suggesting that u n d e r these crystallographic conditions the active-site Cu(II) may have a distorted tetrahedral, pseudo-three-coordinate geometry. Spectroscopic results are not entirely consistent with the geometry inferred from the crystal structure at pH 7.0 (Knowles et al., 1995), although in most respects the agreement is satisfyingly close. C. A Two-Electron Oxidation Mediated by a Single Copper Ion ?

As seen in Eq. (1), galactose oxidase catalyzes a two-electron oxidation of an alcohol. In the late 1980s, research efforts were centered on determining the mechanism by which a mononuclear copper site. where Cu(II) ~ Cu(I) are the redox states normally accessible under physiological conditions, could catalyze such a reaction. It is frequently asserted that metalloenzyme mechanisms follow a "one-metal, one-electron rule" (Whittaker and Whittaker, 1988). In the absence of addi-

394

MELANIE S. ROGERS AND DAVID M. DOOLEY

+ 02

I Cu(I)-tyr

+ D-galactose / -O 2

~

substrate-reduced (anaerobic)

+ ferricyanide

,

Cu(ll)-ty r

-

semi-reduced+ ferrocyanide

"

Cu(ll)-tyr"

oxidized

SCHEME 1. O x i d a t i o n states o f t h e c o p p e r - t y r o s i n e c e n t e r in g a l a c t o s e oxidase.

tional metal ions or exogenous cofactors, several proposals were made to rationalize the two-electron reaction of galactose oxidase. In a detailed mechanistic study using both kinetic techniques and EPR spectroscopy, Hamilton and co-workers demonstrated that the active state of galactose oxidase was one oxidation state higher than Cu(II). They defined this state as "Cu (III)" and suggested it participated in the reaction (Hamilton et al., 1978). It was noted that a resonance hybrid, such as Cu (II) - Xo <--> Cu(III) - X- (where X is some enzymic group), could explain the data but the participation of the Cu(III) state was favored. Prior to 1988, the properties of galactose oxidase on isolation were variable and this hindered investigation of the enzymatic mechanism. Whittaker, in a key paper in 1988 (Whittaker and Whittaker, 1988), showed that, as isolated, the enzyme was a mixture of two oxidation states, oxidized (now known to be Cu(II)-Tyro) and resting [Cu(II)-Tyr, also known as semi-reduced]. Treatment of "as-isolated" enzyme with ferricyanide and ferrocyanide, respectively, generated oxidized and semi-reduced (reductively inactivated) galactose oxidase. A third redox form, Cu(I)-Tyr, was obtained by anaerobic substrate reduction. The identification of the three oxidation states of the metalloenzyme, as shown in Scheme 1, was a breakthrough that allowed for the preparation of h o m o g e n e o u s solutions of the enzyme. EPR studies of the various forms of the protein showed that the ferrocyanide-reduced protein had a typical Cu(II) spectrum, which disappeared after treatment with ferricyanide. The EPR spectrum of ferricyanide-treated (oxidized) protein also revealed a free-radical signal, which when quantitated, represented 1% of the protein. Whittaker p r o p o s e d that this signal, generated by a one-electron oxidation, was probably a paramagnetic species that interacted with the copper ion. In the absence of additional metal ions, or cofactors, he proposed this species to be a redox-active amino acid. Candidates included tyrosine, cysteine, and tryptophan. An interesting suggestion for the two-electron reactivity was the presence of covalently b o u n d pyrroloquinoline quinone (PQQ) (Van der Meer et al., 1989). However, the crystal structure unambiguously ruled out the presence of P Q Q in galactose oxidase.

POSTTRANSI.ATIONALLYMODIFIED I~t'ROSINES

395

D. EPR Spectroscopy Reveals a New Type of 7~ros~l Radical Whittaker's discovery that galactose oxidase could be prepared in three homogeneous oxidation states opened the door to detailed study of the redox center of galactose oxidase. Circular dichroism spectroscopy, exogenous ligand probes of metal oxidation state (Whittaker and Whittaker, 1988), and X-ray absorption edge studies showed that both the semi-reduced and oxidized proteins were in a Cu(II) oxidation state (Clark et al., 1990), which confirmed that the copper ion was not the site of redox activity in the enzymatically competent protein. This led to the inference that, as opposed to Cu(III) as had pre~iously been suggest~d, ~t stable free radical must be present in the oxidized protein. Unfortunately, the oxidized holoenzyme species is difficult to study by EPR spectroscopy as it is EPR-silent owing to magnetic interactions between copper (II) and the putative radical, presumably a redox active amino acid. However, removal of copper from the active site, tollowed by treatment with ferricyanide, generated a stable radical with a detectable EPR signal (Whittaker and Whittaker, 1990). The EPR spectrum of this species had a gay of 2.005, characteristic of an aromatic radical, and typical of a tyrosyl radical (Fig. 4). To confirm the identity of the radical, isotopic labeling experiments were performed. Growing Dactylium dendroides in the presence of glyphosate suppressed the aromatic amino acid synthesis pathway. The growth media was supplemented with exogenously labeled tyrosine ([3,~-2H-labeled) that was incorporated into galactose oxidase. The EPR spectrum of isotopically labeled galactose oxidase is seen in Fig. 5b. This spectrum was perturbed in comparison to that of the native tmlabeled protein (Fig. 5a), and a labeled-unlabeled difference spectrum showed a collapsed hyperfine structure (Fig. 5c). The hyperfine structnve originates fiom a tyrosine methylene group proton. The perturbation of the EPR hyperline splittings in the labeled protein established that the radical species was derived from tyrosine. The electronic spectrum of the yellow colored oxidized apoprotein also had tmique features (Fig. 4). There was a broad band centered around 800 taxi and substantial UV absorbance. The appearance of the oxidized apoprotein electronic spectrum also ruled out the presence of pyrroloquinoline quinone (either as quinone, semiquinone, or hydroquinone), which had previously been suggested to occur in galactose oxidase (Vail der Meet et aL, 1989). Although these featm'es were proposed to result fiom a "tmique en~fironment of the radical in the protein, which modulates both its structure and reactivity" (Whittaker and Whittaker, 1990), the molecular factors responsible for the differences remained murky. Whittaker and Whittaker (1990) proposed either the formation of :~

0,4

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WAVELENGTH(nrn)

r

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2100

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2500

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MAGNETIC FIELD(GAUSS)

FIG. 4. Optical absorption (top) and EPR spectra (bottom) for oxidized apogalactose oxidase (Whittaker and Whittaker, 1990). Protein concentration was 15 mg/ml (top) and 30 mg/ml (bottom). Instrumental parameters for EPR spectrum: microwave power, 0.03 microwatts; microwave frequency, 9.220 GHz; modulation amplitude, 5 G; temperature, 8.3 K (Whittaker and Whittaker, 1990). Reprinted with permission of the author and the Journal of Biological Chemistry.

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MAGNETIC FIELD(GAUS~

FIG. 5. EPR spectra of the radical site (A) oxidized apogalactose oxidase, (B) oxidized apogalactose oxidase prepared from [2H]tyrosine-labeled protein, (C) difference EPR spectrum obtained by subtracting 30% of spectrum A (corresponding to the unlabeled tyrosine fraction) from spectrum B. Instrumental parameters for EPR spectrum: microwave power, 20 microwatts; microwave frequency, 9.34 GHz; modulation amplitude, 1 G; temperature, 130 K (Whittaker and Whittaker, 1990). Reprinted with permission of the author and the Journal of Biological Chemistry.

397

POSTTRANSLATIONALLY MODIFIED ~ISzROSINES

knO~OH

b

Rea4°

SCHEME2. Galactose oxidase turnover cycle. The hydroxylic substrate is modeled in the galactose oxidase active site by conservative replacement of ctTstallographic water in the metal coordination sphere with a primary alcohol in an orientation consistent with stereospecific pro-S hydrogen abstraction from the methylene carbon. Predicted hydrogen-bonded distances from the hydroxylic proton to the Y495 phenolate oxygen and tIom the methylene pra-S hydrogen to the Y272 phenoxyl oxygen are indicated (Whittaker et al.. 1998). Reprinted with permission from %~hittaker el al. (1998). Copyright (1998) American Chemical Society.

charge transfer between the tyrosyl radical and a n o t h e r protein g r o u p OF possibly a radical sandwich charge transfer complex. T h e critical finding o f this work was to identify a radical species, which a p p e a r e d unusually stable a n d h a d a lowered r e d o x potential (as j u d g e d by its g e n e r a t i o n with ferricyanide t r e a t m e n t ) . Finally, t h e r e was a possible solution to the o n e - m e t a l - t w o - e l e c t r o n reaction puzzle. Extensive spectroscopic studies (Whittaker et al., 1988; Whittaker and Whittaker, 1993; B r a n c h a u d et al., 1993) and crystallographic characterization (Ito et al., 1991) ofgalactose oxidase enabled a catalytic m e c h a n i s m to be p r o p o s e d (Scheme 2; Whittaker et al., 1998). Alcohol substrate binds to the c o p p e r inn in fully oxidized enzyme, Cu(II)-Tyro, via the C6 oxygen.

398

MELANIE S. ROGERS AND DAVID M. D O O L E Y

The semi-reduced form of the protein, Cu(II)-Tyr, is devoid of catalytic activity (Whittaker and Whittaker, 1988), although substrate binding by this form has been demonstrated (Knowles et aL, 1995). The axial tyrosine phenol abstracts a proton from the coordinated oxygen, activating the substrate for hydrogen atom abstraction by the radical equatorial tyrosine, resulting in a reduced copper/tyrosine species. A large kinetic isotope effect (kH/kD = 21.1), associated with reduction of enzyme, supports hydrogen atom abstraction by the tyrosyl radical (Whittaker et al., 1998). The aldehyde product dissociates from the copper ion, with dioxygen subsequently reoxidizing Cu(I)-Tyr to the enzymatically active form, Cu(II)Tyro, with the concomitant production of hydrogen peroxide. E. The Electron Density Reveals a Novel Thioether Bond

An unexpected finding in the structure of galactose oxidase (Ito et al., 1991) was the presence of a covalent bond between the equatorial copper ligand, tyrosine 272, at C~1 and the sulfur atom of cysteine 228. During the model building and refinement process, there were not any special restraints (e.g., bond length) placed on C ~ and S~' except for the removal of the Van der Waals interactions between the two side chains. The electron density and model are shown in Fig. 6 (See color insert). The distance between C ~1 and S~' (1.84 ~) and the bond angles at C E1 (119 ° for CS1-CE1-S v and 120 ° for C~-C~I-Sv) were almost ideal for o-substitution. The dihedral angle of the bond (7 ° for CS1-CE1-Sv-C~) is almost linear, and this geometry suggests the thioether bond may have partial double bond character (Ito et al., 1994). The thioether bond, when revealed for the first time in the crystal structure, was so novel that i n d e p e n d e n t evidence for its existence was presented (McPherson et al., 1992). E Spectroscopic and Theoretical Studies of the Cross-Link Perturbation

The effect of the cross-link on the tyrosyl radical's magnetic properties has been the subject of debate over the past few years. In 1992, Babcock et al. (Babcock et al., 1992) studied the magnetic properties of the cysteine-substituted tyrosyl radical in apogalactose oxidase using EPR and ENDOR spectroscopy with comparisons made to radicals derived from substituted phenol compounds. Briefly, model tyrosyl radicals have the unpaired electron in odd-alternant ~ highest occupied molecular orbital (HOMO) with major spin concentrations in the p~ orbitals of carbons 1, 3, and 5 (Fig. 7). The EPR spectra are mostly defined by the hyperfine couplings to two ring protons at carbons 3, 5 and one

•

O

FIG. 6. T h e unusual t h i o e t h e r b o n d between Tyr-272 a n d Cys-228. (a) T h e m a p shown is the 2.5/~ m.i.r, m a p after solvent flattening, which is n o t biased by the molecular model. (b) T h e same view of the b o n d b u t with the refined 1.7 A 2Fo-Fc map. T h e blue c o n t o u r corresponds to 26 level a n d the red to 5~ level. T h e r e f i n e m e n t was carried out without assuming existence of the Cys-Tyr b o n d a l t h o u g h the n o n b o n d e d contact restraints between the two side chains were removed. T h e b o n d length between Ce a n d Sv was refined to 1.84 A, b o n d angles C ~ - C r S r to 119.4 °, C;-C~-S v to 120.6 ° a n d CE-Sy-C[~ to 105.1 °. T h e c/s conformation of the b o n d (the torsion angle C~-Ce-S~-C~ is 7 °) results in the formation of a large planar group a n d suggests that it has partial d o u b l e - b o n d character. O t h e r side chain torsion angles are Zl = -83° a n d ~2 = -99° (using Ce of Tyr-272 as the fourth atom) for Cys-228, ~1 - 6 8 ° a n d Z2 = -166° for Tyr-272. Reprinted by permission from Nature (lto et al., 1991) © 1991, MacMillan Magazines Ltd. =

FIG. 15. (a) "Unbiased" electron density map at the lff level of the binuclear heme asCUB site and the atomic model. A simulated annealing omit map, omitting all residues (including heme as) with an atom closer than 4.5/k to CuB, was calculated. The heine as iron atom is shown in red, CUB in blue, nitrogen atoms in blue, oxygen atoms in red, carbon atoms in yellow, and the electron density in purple. The figure was prepared using the program SETOR. From Ostermeier et al., (1997). Proc. Natl. Acad. Sci. USA. 94, 10547-10553. Copyright (1997) National Academy of Sciences, USA. (b) Crystal structure of Fe as-CuB site of the fully oxidized form at 2.3/~ resolution. The (Fo - Fc) difference Fourier map of the oxidized form calculated by omitting His-240, Tyr-244, and any ligand between Fe as and CUB from the Fc calculation. Contours are drawn at 7~ level (1 = 0.0456 e - / A s) (Yoshikawa et al., 1998). Reprinted with permission from Yoshikawa et al. (1998). Science 280, 1723-1729. Copyright (1998) American Association for the Advancement of Science.

POSTTRANSLATIONALIX MODIFIED TYROSINES

399

O

H~R2 H~"""],'/ ~H R1 FI(;. 7. N u m b e r i n g convention used ff)r tyrosine in Babcock et al. (1992). R 1 is tile CH2CH(NHe)COOH side chain. R2 is a hydrogen atom in unsubstituted tyrosine, whereas in the galactose oxidase tyrosine 272 radical, Re is the-SCH2- bridge to cysteine 228. Reprinted with permission from Babcock et al. (1992). Copyright (1992) American Chemical Society.

methylene proton on carbon 1. There is nominal spin located on carbons 2 and 6, and minimal hyperfine interaction originating from their protons. The spectra of odd-alternant tyrosyl radicals display variations in line shape and width, resulting from the strong modulation of hyperfine couplings of ~-methylene protons by, the conformation of the methylene group relative to the ring. The g#,,value of the cross-linked tyrosine in apogalactose oxidase, where the sulfur atom has replaced the carbon 3 proton, is 2.0055, which is appropriate for an odd-alternant radical. The low-field-peak to high-fieldtrough width is 33 G, a value that indicates relatively strong coupling of two or more protons in the radical. ENDOR studies showed strong hype> fine couplings to two classes of protons with kiso = 14.6 G, having a tensor characteristic of a 13 proton and a second Ai~,, = 8 G corresponding to a hyperfine anisotropy characteristic of an R proton. Simulation of the EPR spectra using the ENDOR parameters indicated that one proton is in each class, one [3 proton and one 0~ proton (Babcock et al., 1992). Thus this group concluded that the major hyperfine interactions arise from one of the methylene protons and the carbon 5 proton. The apogalactose oxidase EPR spectrum could not be simulated using unperturbed tyrosyl radical parameters, providing additional evidence that the radical was indeed located on the modified Tyr-272 residue and not Tyr-495 or any other unmodified tyrosine. The odd-alternant EPR radical spectra, as well as the optical spectrum and the redox potential of the apogalactose oxidase tyrosyl radical are readily distinguishable from the radicals of ribonucleotide reductase and photosystem II. An interesting point is that while the two tyrosines of PSII have identical EPR spectra (Babcock et al., 1992), their redox potentials differ by 250 mV (Yn°, 720-760 mV; Yz', -1000 mV, Stubbe and van der Donk, 1998) (Table 1I).

400

MELANIE S. ROGERSAND DAVIDM. DOOLEY

TABLE II Redox Potentials for the Generation of a Tyrosyl Radical Tyrosine

Redox potential (mV)

YD" and Yz° of photosystem II

720-760, -1000

Stubbe and van der Donk, 1998

Ribonucleotide reductase (class I)

1000

Sjoberg, 1997

Free tyrosine

940

Stubbe and van der Donk, 1998

Cu-phenolate complex Galactose oxidase tyrosine

800-1100 400

Wang et al., 1998 Johnson et al., 1985

Reference

Thus factors other than spin delocalization may influence the chemical properties of the radical in a complex and not fully understood manner. The g value (2.0055) of apogalactose oxidase, compared to the glso for a cysteine radical is low, suggesting that a relatively minor fraction of the spin density has delocalized onto the S atom of C228, implying that the O-cys substitution does not significantly perturb the observed H O M O symmetry (Babcock et al., 1992). This was supported by the model c o m p o u n d data that indicated the thioether substitution only slightly perturbed the g tensor or spin density distribution of the phenol. ENDOR data were consistent with a 25% decrease in spin density at the para position on thioether substitution. High-field EPR spectroscopy and molecular orbital calculations have also b e e n used to p r o b e the apogalactose oxidase radical (Gerfen et al., 1996) b u t resulted in different, and as yet unreconciled, conclusions. The advantage of this combination of techniques is that the effects of individual atoms, such as oxygen and sulfur, can be determined. The calculations confirm a basic conclusion of the earlier study (Babock et al., 1992), that the radical has odd-alternant spin density with delocalization of some spin density onto the sulfur atom. However, the hyperfine splittings were assigned to methylene protons, one from Tyrosine 272 and one from Cysteine 228, and not to an 0~ and ~ p r o t o n as had previously b e e n described (Babcock et al., 1992). The high-field EPR spectroscopy study could not assign an 0~ proton splitting due to inconsistencies in the MO calculations p e r f o r m e d as part of the study, and the inability to satisfactorily simulate the (methylthio)cresol radical spectra as an unsubstituted radical at 9 or 139.5 MHz. Gerfen et al. inferred that thioether substitution induced significant perturbation of the g r o u n d state electronic structure (0.28 spin density is located at the sulfur atom, with reductions at the phenoxyl oxygen and ring carbons), which molecular orbital calculations

POSTTRANSLATIONALLY MODIFIED qDt'ROSINES

401

assigned to a heavy atom effect arising from the spin-orbit coupling associated with the cysteine side chain. The axial g tensors observed for the sulfur-substituted radical result from transfer of substantial unpaired spin density onto exocyclic sulfur through ~ covalency in the highest occupied molecular orbital. In fact, Gerfen notes that sulfur provides the largest single atomic orbital contribution to the SOMO of the (methylthio)cresol radical. The crossqink may eftiect a decrease in the redox potential of the tyrosyl radical via the delocalized spin density onto the tyrosine-sulfur unit. To address the ambiguities in the hyperfine assignment tbr the apogalactose oxidase radical, density functional calculations have been p e r f o r m e d (Himo et al., 1999; Engstrom et al., 2000). The hyperfine coupling constants and spin density distributions of the sulfur-substituted radical and an unsubstituted radical were calculated using fouldensity functional methods. The earlier proton coupling assignments by Babcock (1992), Also = 14.6 G (~ proton of tyrosine) and 8 G (carbon-5 0~-proton) were supported, and the 0.28 spin density that highfield EPR spectroscopy assigned to Cys-228 sulfilr could not be reproduced by the calculations. These studies suggest that the crosslink does not significantly perturb the odd-alternant spin pattern of the radical with the small amount of spin density localized on the sultilr substitution resulting only in minor shifts in the g tensor. Density, functional calculations with molecular mechanics have also been used to probe the catalytic mechanism (Himo et al., 2000). The results of these studies indicate that the cross-link has little effect on the energetics of the turuover cycle of galactose oxidase, shovm in Scheme 2, although oxidative generation of the radical was not considered in their calculations. The energetics and structures of the models were almost identical with or without the cross-link, and the sulfur substitution stabilized the radical by only 1.7 kcal/mol. Himo et al. suggest that the role of the cross-link is primarily structural, as there appears to be no major electronic effect on tyrosine. Even though others have suggested that the cross-link is responsible for a 0.5 to 0.6 V drop in the redox potential of the radical (Itoh et al., 1993), some data suggest that the cross-link results in only minor changes in the electronic structm'e and energetics of the ty'rosyl radical (Whittaker et aL, 1993; Halfen et al., 1997). G. The E[/ect of the Thioether Substituent in Galactose Oxidase Probed by Model Chemist U

A variety of biomimetic models have been prepared to examine the basic structural and reactivity features of the galactose oxidase active

402

MELAN1E S. ROGERS AND DAVID M. DOOLEY

site (Itoh et al., 2000; Jazdzewski and Tolman, 2000). Studies most germane to the effects of the cross-link on the radical and active-site structure and function have reached differing conclusions. The general approach has been to prepare the one-electron oxidized form of the thioether-substituted compound, and then investigate its optical and EPR properties, as well as the electrochemical behavior with a view to understanding the influence of the thioether substitution. The first study to probe the role of the thioether linkage was perf o r m e d by Whittaker in 1993 (Whittaker et al., 1993) using (methylthio)cresol (1, MTC) (Fig. 8). MTC is a relatively simple model for the thioether b o n d and has also been explored by Itoh (Itoh et al., 1997), both studies making comparisons to p-cresol (2, Itoh et al., 1997). Addition of [Cu (PMDT) ] (CIO4) 2-2CH3CN to MTC formed 1:1 copper complexes, where, unlike the enzyme, sulfur was coordinated to the copper ion. A more extensive model of the active site with a thioether substituent was subsequently described by Whittaker based on a synthetic tripod chelate, duncamine (3, dnc) (Whittaker et al., 1996a) (Fig. 8). A (methylthio)phenol group represented the crosslink, with dimethylphenol mimicking the axial tyrosine and 2(aminomethyl)pyridine coordinating a copper ion. A monomeric unit was obtained by addition of pyridine to the dimer complex. To further probe the electronic effects of the thioether bond, MTC (1), 2-(methylthio)-4,6-dimethylphenol (4), and 2-(methylthio)-4methyl-6- [ [bis [2-(2-pyridyl) ethyl] amino] methyl] p h e n o l (5), 2[[bis[2-(2-pyridyl)ethyl]amino]methyl]-4-methylphenol (6) were examined by Itoh et al. (1997) (Fig. 8). To probe the effects of the thioether group on the copper ion center, CuC12.2H20 and pyridine were added to 5 (containing SMe) and 6 resulting in the formation of m o n o m e r i c copper complexes [CuII (5-)(py)] (PF6), 8 and [CuH(6 -) (py] (PF6), 9, respectively. An isostructural series of copper and zinc bis(phenolato) complexes were examined, for the effects of thioether substitution on the equatorial tyrosinate ligand (Halfen et al., 1997). The copper complexes, including biomimetic 0-thioethers (alkythio groups), were assembled a r o u n d a 1,4,7-triazacyclononane frame with various phenolate substituents. The c o m p o u n d s were designed to provide a square-pyrimidal Cu(II) geometry with a phenolate d o n o r in the equatorial position, which could be converted to a radical. The complex LMeSMeCu(O2CCH3) (7) most closely resembles the structure of galactose oxidase (at pH 4.5) (Fig. 8). There is an exogenous cisequatorial acetate ligand, and the sulfur substitution lies in the plane of the equatorial phenolate, as in the enzyme active site. The S-Carene

POSTTRANSLATIONALLY MODIFIED ~I~'P.OSINES

4()~

~HH3 S~..CH3 1

2

3

CH3

HsC4SICH3

s--

OH 3

OH N

4

5

N

IS

t)

T o-q, Fl(;. 8. Model compounds for the thioether cross-link in galactose oxidase: 1-2, 4-6 (ltoh et al., 1997). Reprinted with permission from Itoh et al. (1997). Copyright (1997) American Chemical Society. Cu-duncainine, 3 (Whittaker et al., 1996a). Reprinted with permission from Whittaker et al. (1996a). Copyright (1996) American (;hemical Society. LM"sM"Cu(O2CCH:~), 7 (Halfen et al., 1997). Reprinted with permission from Halfen el ol. (1997). Copyright (1997) American Chemical Socimy.

d i s t a n c e is 1.767 A, a n d is (in c o n t r a s t to the e n z y m e ) t h o u g h t to have little d o u b l e b o n d c h a r a c t e r . T h e radical f o r m s o f p-cresol a n d M T C were g e n e r a t e d using two m e t h o d s , UV i r r a d i a t i o n ( W h i t t a k e r et al., 1996a) a n d pulse radiolysis (Itoh et al., 1997). L o w - t e m p e r a t u r e optical spectra o f the radical f o r m o f p-cresol r e v e a l e d b a n d s at 388 n m a n d 408 n m (400 n m ; I t o h el al., 1997), typical o f a p h e n o x y l radical ( W h i t t a k e r et al., 1993), w h e r e a s the ( m e t h y l t h i o ) c r e s o l radical h a d a b a n d at 400 n m a n d a b r o a d b a n d at

404

MELANIE S. ROGERS AND DAVID M. DOOLEY

c-

o

,~oo

eoo

aoo

Wovelength

~ooo

~2oo

(rim)

FIG. 9. Low-temperature absorption spectra for the products of UV irradiation of (top) 5 mM cresol, and (bottom) 7 mM (methyhhio)cresol,in propionitrile:butyronitrile at 77°C (Whittaker et al., 1993). Reprinted with permission from Whittaker et al. (1993). Copyright(1993) AmericanChemicalSociety.

830 nm. Itoh observed strong absorbances a r o u n d 350 n m to 400 nm and a broad absorption band between 600 nm and 900 nm for this species. Interestingly, the spectral features of the irradiated MTC (Fig. 9) closely resembled those of apogalactose oxidase (see Fig. 4). Not only is this evidence that the radical species is located on the Tyr-Cys unit in the enzyme, it suggests the radical is not appreciably delocalized over a larger unit (e.g., on the stacking tryptophan) in the enzyme. The optical spectrum of the pyridine-liganded Cu (dnc) (Whittaker et al., 1996a) exhibited an intense band at 475 nm (1430 M-~ cm-1), which was consistent with equatorial phenolate coordination in a pyrimidal complex. The band seen at 705 n m was representative of the Cu(II) ligand field. The appearance of the spectrum suggests that the thioether substitution makes little contribution to the LMCT. However, a large red shift in the LMCT of 8 was observed compared to 9, 5, or 6 (Itoh et al., 1997). Itoh attributed the shift to delocalization of an electron from the phenolate moiety into the sulfur atom of the SMe group. The optical spectra and electrochemical properties of the Cu(II)phenolates with ortho-substituted ligands (Halfen et al., 1997) provided insight into possible consequences of the modification of the equatorial tyrosine in galactose oxidase. Comparison of the ~max of the isostructural series, L RR'Cu(O2CCH3) and L RR'CuCI, shows the energy of the transition within each series increases as the energy of the filled ligandbased orbitals decreases according to the order L MeOMe (most electron rich) = L MeSMe ~ L tBuSMe > L Me2 ~ L tBu2 (least electron rich). The

POSTTRANSLATIONALIX MODIFIED TYROSINES

4OD

alkylthio substitution seems to induce only a small shift in the Pho- --~ Cu(II) LMCT. The small range of energies implies, by extrapolation, a minor role for the thioether cross-link in tuning the redox potential of Y272 of galactose oxidase, in line with the increased electron density on the ring compared to the alkyl-substituted reference complexes used in this study. The electron-releasing effect of the 0-SMe group results in a greater electron density on the ring compared to alkyl-substituted con> plexes (this is also seen by the lower energy of the LMCT of L musmt'Cn compared to L tBuZCu). This conclusion is supported by the cyclic voltammetl T data for L tBuSMeCuC1 compared to L a~U2CuCl: the El ,,_, decreased by only ~ 50 m V in the thioether a p p e n d e d model (alkylthio). There are also suggestions that tile a~yl-thioether substituents contribute significantly to low-energy absorbance features (800 nm to 1200 nm) in oxidized galactose oxidase (Mahadevan et o1., 2000; Itoh et al., 1999; Halcrow et al., 1999). Helium-temperature EPR spectroscopy of the radical forms of methylthiocresol (MTC.) and p-cresol cresol have an average gvalue of 2.005 (1., 2.0060; 4., 2.0052, Itoh et al., 1997), which is characteristic of a phenoxyl radical (Whittaker and Whittaker, 1993). These values are similar to the apogalactose oxidase tyrosyl radical, which has an average g value of 2.0055. Surprisingly, MTC. did not exhibit a significant shift in the g value, which would have been expected to occur if there were substantial sulfur atom contribution to the electronic ground state. These data correlate with E N D O R analysis (Babcock et al., 1992) that showed MTCo to have odd alternant spin distribution resuhing in unpaired electron density on the oxygen and the ortho and para carhons but not the adjacent sulfur. The increase in g value for the methylthio-substituted compounds, compared to the radical forln of 2,4,6,trimethylphenol (g 2.0036), is attributed to the electronic effect of the sulfilr atom, which has a larger spin-orbit coupling constant. The hyperfine coupling constants show a decrease in the total spin density at positions 3,4,5, and 6 of models 1. and 4. compared to p-cresol, which was interpreted in terms of a substantial degree of spin densily being delocalized into the snltur atom of the methyhhio group (Itoh el cd., 1997). This, however, is inconsistent with the interpretation of the gWllues advanced by Babcock et al. ( 1 9 9 2 ) . EPR spectroscopy of the copperduncamine complexes in the presence of pyridine reveal a simple Cu(ll) complex (g~, 2.02; &, 2.05; &, 2.25; a,, (Cu) 150 G). The EPR spectrum of 8 (compared to 9) showed it possessed a distorted squarepyramidal geomeuT, typical of a tetragonal Cu (II) complex. Similar values were reported for galactose oxidase and other reported model complexes with distorted square-pyramidal geometry,

406

MELANIE S. ROGERS AND DAVID M. DOOLEY

Formation of the radical species in the model compounds is facilitated by a lowered, albeit moderately, phenolate/phenoxyl radical redox potential (Halfen et al., 1997). In contrast, electrochemical measurements by Itoh (Itoh et aL, 1997) showed that the methythio substitution caused a significant decrease in the E°ox. Cyclic voltametry data of 8 (+415 mV) showed that it could be more readily oxidized than the unsubstituted compound 9 (+623 mV) (Itoh et aL, 1997). Comparison to 5 and 6 showed a consistent reduction of the redox potential by around 200 mV when SMe was incorporated. Both the electron-donating nature and the radicalstabilization effect of the methylthio group (electron-sharing conjugafive effect) were implicated in this decrease. The reduction in redox potential mediated by a sulfur group is corroborated by Tagaki (Tagaki, 1977), who notes the electron-donating nature of the thioether group. The electrondonating nature of the thioether substitution may be reflected in an upfield chemical shift of the aromatic protons at the 3 and 5 positions in 1 and 4. (Itoh et aL, 1997). However, the shift in redox potential seen in the models is substantially smaller than the N 500 mV decrease seen in galactose oxidase. Hence, other factors may be operating in the enzymatic system to stabilize the [Cu(II)-Tyr.] state, such as the stacking tryptophan (as yet not incorporated into any model) or variations in the polarity around the redox-active groups. Semi-empirical molecular orbital calculations (PM3) on 1H, 1-, and 1. indicate conversion of 1H to 1. results in b o n d order changes (Itoh et al., 1997) consistent with the occurrence of an 0-quininoid canonical form, which pardy contributes to the stability of the radical species (Scheme 3). The calculated spin density of 1- supports the 0-quininoid formulation. The net atomic charges on sulfur in I H and 1- are +0.064 and -0.04, respectively, which suggests that sulfur can stabilize the negative charge on the phenolate oxygen atom in 1-. The C4, C6, and 0 8 positions had decreased density when the methylthio substituent was incorporated into the model, and the unpaired electron density was

CH3

CH 3

SCHEME 3. T h e m o d e l e d o-quinonoid canonical form of the galactose oxidase tyrosyl radical. Reprinted with permission from Itoh et al. (1997). Copyright (1997) American Chemical Society.

I'OSTTRANSLATIONALINMODIFIED [5'ROSINES

407

distributed among C2 and $9. The altered b o n d order and the spin density illustrate the electron sharing conjugative effect of sulfide groups as described by Tagaki (Tagaki, 1977). The optimized geometry of 1., determined from PM3 calculations, showed the methylthio group in the same plane as the phenol ring, while the methyl group moves out of the plane. The differences in 1H, 1% and 1. suggest an increase in the sp 2 character of the sulfur atom in the radical and thus an increase in the contribution of the 0-quininoid canonical form. Partial double-bond character was also suggested for the thioether bond (C-S) in galactose oxidase based on geometi T seen in the crystal structure (Ito et al., 1994). Itoh argues that the results may be understood in terms of both the electron-donating and electron-sharing conjugation properties of the methylthio group. The lowered pK~ (of 1H and 4H compared to 2H) suggests a 2/m-3drt electron conjugative effect. This results from orbitals on the sulfide stabilizing the negative charge on the phenolate oxygen when connected to a conjugated system (so called 2 / m - 3 d r t conjugation). Another factor contributing to the lowered pK~, value may also be electron donation (radical stabilization eft~ct of the sulfide). Both tile EPR spectra and the semi-empirical molecular orbital calculations support the electron-sharing conjugative effect of the the substituent in tt~e radical state. Thioether substitution of the phenolate of MTC effected a decrease in the pKa of the hydroxyl group by about 1 pH unit (9.5) compared to p-cresol (10.2) (Whittaker et al., 1993). These values were confirmed by Itoh (Itoh et al., 1997). Although model chemistry has provided much useful information on the thioether cross-link, no single model simultaneously reflects all the features of the galactose oxidase active site. For example, none has both an equatorial and axial tyrosine with the en@'me's copper-site geometQ'. Whittaker (Whittaker et al., 1993) concluded that the role of the crossqink is to provide selective accessibility and stabilization of the one-electron oxidized product [Cu(II)-Tyro]. It has been suggested that absence of the cross-link in most catalytic galactose oxidase models (operating via a galaclose H-atom abstraction mechanism) is not consistent with the purported electronic stabilization role of the cross-link in the enzyme (Mahadevan el al., 2000). kal alternative role in forming a rigid and oxidatively robust ligand enviromnent was proposed. Such an environment might ensme a lower energetic cost of substrate binding, effectively increasing the Cu (lI) bonding affinity of the active site by retarding the metal dissociation rate. A contrasting view of the effect of the thioether bond has been presented by Itoh et al. (2000) who emphasize the 2/m-3drc electron conjugative effect of the thioether group. In addition, according to this view, the elec-

408

MELANIE S. ROGERS AND DAVID M. DOOLEY

won-donating nature of the thioether substituent stabilizes the negative charge on the phenolate oxygen while increasing delocalization in the radical of the thioether. H. Structure, Function, and Biogenesis of the Thioether Bond in Galactose Oxidase

Notwithstanding the somewhat conflicting results from model chemistry, one of the possible consequences of the thioether bond is to reduce the redox potential of Tyr-272. As the radical is catalytically active, the modulation of its redox potential is critical to the enzymatic function. The range of redox potential for tyrosyl radicals seen in biology is shown in Table II. Clearly, the potential of Tyr-272 in galactose oxidase is the lowest measured to date. This is reflected by the observation that the radical can be generated by potassium ferricyanide treatment. The thioether bond may be expected to contribute to the lowered redox potential of the tyrosine, perhaps resulting in an accessible tyrosyl radical, although numerous other factors may also influence the redox potentials of active-site gro.ups. The radical, once formed, may be stabilized by the electron-donating properties of the C228 sulfur atom (Klinman, 1996). Tagaki has suggested that the lowered redox potential of tyrosine is lowered due to the electron-donating nature of the thioether group (Tagaki, 1977). An indication of the role that the thioether bond plays in the reactivity of galactose oxidase may be seen in the mutational variant C228G (Baron et al., 1994). The crystal structure shows little structural change with no change in structure of the main chain, copper ion and copper ligands. The side chains of Trp-290, Phe-227, and Phe-194 have moved slightly to fill the space left by the loss of the sulfur atom. The minimal changes in the crystal structure seem to suggest that only a minor role is played by the thioether bond in forming the active site. Nonetheless, this mutant shows a 3761-fold decrease in ~cat from the wild-type (Baron et al., 1994). The copper occupancy of C228G was 0.25 Cu/protein, so the cross-link may also affect copper affinity of the active site. Heterologous expression (Baron et al., 1994) of the Fusarium protein in AspergiUus nidulans u n d e r copper-limited conditions has resulted in the appearance of multiple protein forms (Fig. 10). The molecular weights of the SDS-PAGE bands, established to be galactose oxidase by Western blotting (Baron et al., 1994), were estimated as 70,200, 68,500, and N 65,500 (Rogers et al., 2000). N-terminal sequencing confirmed that the fastest migrating protein (Fig. 10a, left lane, lower band, 65,500) corresponded to mature, wild-type galactose oxidase. This

POSTTRANSLATIONALLYMODIFIED°IS'ROSINES

409

Wild

GO P r o - s e q u e n c e form I - 70.2 k D a U n m o d i f i e d form I I - 68.5 k D a M a t u r e form - 65.5 k D a

b) P r o - s e q u e n c e form I - 70.2 k D a M a t u r e form 65.5 k D a

FIe;. 10. Ten percent SDS-PAGE of unprocessed galactose oxidase obtained under both (a) limited and (b) metal-free conditions (Rogers et al., 2000). C228G and wild-type galactose oxidase samples were prepared in the presence of copper. Reprinted with permission h'om Rogers et al. (2000). Copyright (2000) American Chemical Society.

migrated on SDS-PAGE with an anomalous molecular weight (65,500 as compared to 68,500 predicted by the sequence) owing to the thioether bond, which produces a stable loop, thus preventing full unfolding on treatment with SDS (Baron et al., 1994). The middle band (Fig. 10a, left lane) has an estimated Mr that correlates with the mass of the mature galactose oxidase amino acid sequence, suggesting that it is a form of galactose oxidase lacking the thioether bond. This behavior is mirrored by the variant C228G, which is unable to generate a thioether bond (Baron et al., 1994). Finally, the upper band (Fig. 10a, left lane), having an estimated M,- of 70,200, corresponds to the form with a 17 amino acid pro-sequence attached, which was confirmed by the N-terminal sequence data (Rogers et al., 2000). This suggests that both prosequence cleavage and thioether bond formation may be conveniently monitored via SDS-PAGE. As perhaps expected, the visible spectrum of metal-free unprocessed protein was featureless prior to the addition of Cu(II) (Rogers et al., 2000). On aerobic exposure to copper sulfate, new transitions were observed at 410 n m and 750 nm (Fig. 11), suggesting that initial Cu(II) incorporation is relatively rapid. With time, the band seen initially at 410 n m shifts to 445 nm, and the broad band initially observed at 750 nm shifts to 800 nm, with an increase in its

410

MELANIE S. ROGERS AND DAVID M, DOOLEY

0.06 0.05 0.04

i

0.03 0.02

0.01 0.00 -0.01 Wavelength (nm)

FIG. l 1. Absorbance spectral changes accompanying the aerobic addition of copper to "metal-free" unprocessed galactose oxidase. Copper sulfate (28 ~M) was added aerobically to unprocessed galactose oxidase (28 [tM) (Rogers et al., 2000)• Reprinted with permission from Rogers et al. 2000. Copyright (2000) American Chemical Society.

intensity. The characteristic bands at 445 nm and 800 nm unambiguously confirm the generation of the tyrosine radical. These transitions are diagnostic for the radical state of the Tyr-Cys unit and are assigned as follows: 445 nm, p h e n o l a t e --~ Cu(II) charge-transfer and Tyro n -->n* (Whittaker et al., 1989); 800 nm, tyrosinate to tyrosyl ligand-to-ligand charge-transfer mediated by the d~z orbitals on Cu(II) (McGlashen et al., 1995). H e r e these results unequivocally establish that the formation of the Tyr--Cys redox cofactor (pro-sequence cleavage being spectroscopically silent in this instance) in galactose oxidase is a self-processing reaction requiring only the apoprotein, Cu(II), and dioxygen; no other proteins or enzymes are required for the processing and assembly of the catalytically active enzyme. This is perhaps not u n e x p e c t e d as a similar reaction has been n o t e d in the amine oxidases where T P Q is f o r m e d from tyrosine via a self-processing event d e p e n d e n t on c o p p e r and oxygen only (Matsuzaki et al., 1994; Ruggiero et al., 1997; Cai et al., 1997). An overall scheme for the generation of the thioether b o n d is outlined in Scheme 4. Whittaker et al. (1993) suggested that the crosslink is f o r m e d nonenzymatically as a result of free radical coupling between tyrosine and cysteine within a proenzyme complex. Rogers et aL (2000) suggested that a c o p p e r ion initially coordinates to the two histidine ligands ( H 4 9 6 / 5 8 1 ) , in analogy to suggestions for T P Q biogenesis in Arthrobacter globiformis amine oxidase (Wilce et al., 1997). Activation of the p h e n o l ring is likely to occur via coordina-

POSTTRANSLATIONALLYMODIFIED1WROSINES

41 1

® Tyr 27~2 ~

"~

~H~

Cys 228

*

~u (II)

Cu(I)

© Tyr2 ~ 6.

Cys 228

~U (II) SG|IEME 4. Generation of the thioether bond in galactose oxidase (Rogers el al., 2t)00). Reprinted with permission from Rogers et al. (2000). Copyright (2000) American Chemical Society.

tion ofY272 to Cu(II) [A, Cu(II)-O-Y272], as illustrated by the resonance form B [Cu(I)-O-Y.] where the tyrosine ring may be expected to be electron deficient. On addition of molecular oxygen, the reaction may be envisioned to proceed via either a radical or ionic mechanism. Interestingly, the final species (C) was the enzymatically active [Cu(II)Tyr.] form. Additional spectroscopic and mechanistic studies will be n e e d e d to fully elucidate the mechanism of thioether b o n d formation. The observation that biogenesis of the cross-link concurrently generates a tyrosyl radical is worth noting. The enzyme purified from either the native organism or from the Aspergillus overexpression system is a mixture of semi-reduced and oxidized protein; thus it may be that the presence of the enzymatically inactive semi-reduced form is a result of protein purification. Structural studies provided key information in the biogenesis studies of amine oxidase (Wilce et aL, 1997). In order to elucidate the mechanism of thioether bond formation, it will be necessary to know the structure of the enzyme prior to the chemical modification. The structural characterization of the unprocessed form of galactose oxidase to 1.4 fk has recently been reported and will provide a detailed view of the precursor (Firbank et al., 2000). A final, and quite interesting, mechanistic issue is that of the

412

MELANIE S. ROGERS AND DAVID M. DOOLEY

relationship between thioether bond formation and cleavage of the 17 amino acid pro-sequence from the precursor form of galactose oxidase. The cleavage reaction also appears to require only copper and dioxygen. However, the order of events for maturation of the protein has not yet been definitively determined. SDS-PAGE analysis of copper-limited preparations of galactose oxidase (Fig. 10) suggests that pro-sequence cleavage may occur before thioether bond formation since a band corresponding to a protein with both the pro-sequence and thioether bond has not been identified (Rogers et al., 2000). I. Other Proteins with a Thioether Bond

Glyoxal oxidase has been isolated and characterized and is also suggested to possess the tyrosine-cysteine cross-link. Glyoxal oxidase, which appears to be another m e m b e r of the radical copper oxidases, is an extracellular protein secreted by Phanerochaete chrysosporium, a lignocellulosic filamentous fungi. The enzyme converts an aldehyde, such as glyoxal, to a carboxylic acid, with the concomitant reduction of molecular oxygen to hydrogen peroxide (Whittaker et al., 1996b). This catalytic reaction [Eq. (2)] is similar to that catalyzed by galactose oxidase, [Eq. (1)]. RCHO + H20 + 02 ----)RCO2H + H202

(2)

The protein is an acidic m o n o m e r i c glycoprotein of 57 kDa. Whittaker suggests that ~150 residues are missing from the N-terminal d o m a i n c o m p a r e d to galactose oxidase. Like galactose oxidase, the protein contains one g-atom of copper for full activity (0.7-0.8 equivalent Cu2+/mol have b e e n d e t e r m i n e d ) . Although the sequence identity between glyoxal oxidase and galactose oxidase is only 28%, the key active-site residueswtwo histidines and two tyrosines--are present. The stacking tryptophan in galactose oxidase is not conserved in the glyoxal oxidase sequence. An RMYHS motif (where Y is the thioether tyrosine) is proposed as diagnostic of the thioether b o n d and is present in glyoxal oxidase. Crystallographic studies of glyoxal oxidase have been h a m p e r e d by twinning disorder in the crystals. In the absence of a crystal structure, a detailed spectroscopic study by Whittaker has led to the proposal that this enzyme may possess an active-site structure nearly identical to that of galactose oxidase (Whittaker et al., 1996b). The redox and spectroscopic behavior of this enzyme also suggests the presence of a thioether linkage. Glyoxal oxidase is isolated in an inactive form, but the protein can be treated with Na2IrC16 (0.892 V; Margerum et al., 1975) to generate an

POSTTRANSLATIONALLYMODIFIEDTYROSINES

41 3

oxidized catalytically active species. This contrasts to the radical in galactose oxidase, which can be generated by potassium ferricyanide (0.424 V; Hawkridge and Kuwana, 1973) treatment. The glyoxal oxidase radical is relatively unstable, and Whittaker reports the h a l f life of the radical to be 4 hours, compared to 7 days in galactose oxidase u n d e r similar conditions. The redox potential of the protein as determined by spectroelectrochemical titration is 0.64 V versus NHE (compared to galactose oxidase 0.4 V; Johnson et al., 1985). This suggests that if the thioether bond is indeed present in glyoxal oxidase, then it alone cannot be responsible for lowering the redox potential of the galactose oxidase tyrosyl radical. Other factors, for example, the stacking tryptophan seen in galactose oxidase, must be important as well. Assmning the radical species in glyoxal oxidase is a tyrosyl radical, the finding is that its potential is significantly lowered compared to unmodified tyrosyl radicals (see Table II), a result that perhaps supports the presence of the thioether bond in glyoxal oxidase. The spectroscopic features of glyoxal oxidase are veo' similar to galactose oxidase, as is seen in Fig. 12. The optical spectra of active and inactive

~'~

4000

3000

CO

"/j

2000 \.J/

1000

0

-

~

4O0

. . . . . .

L

.........

6O0

~ . _ _

eO0

--

I

lOOO

_

_

1200

Wavelength (nm) Fie;. 12. Optical absorption spectra of glyoxal oxidase. Solid line, active (top) and native (bottom) glyoxal oxidase [0.15 mM enzyme, in 50 mM sodium phosphate buit~r, pH 6.5 (active) or 8.34 (native)]. Dotted line, active (top) and reductively inactivated (bottom) galactose oxidase shown for comparison (Whittaker et al., 1996b). Reprinted with permission of the author and the Journal of Biolo~eal Chemist*3,.

TABLE III Comparison of Spectral Data for Glyoxal Oxidase and Galactose Oxidase a Glyoxal oxidase

Complex Active Active + N3-

Inactive Inactive + N3-

(nm)

(M-lcm-1)

Galactose oxidase

(nm)

(M-lcm-1)

448

(5700)

444

(5194)

851

(4300)

800

(3211)

380

(5532)

383

(4218)

509 882 451 678 370 564 728

(3822) (1788) (1875) (1365) (2670) (1248) (888)

499 890 438 625 375 559 747

(3654) (1218) (1000) (1167) (1880) (642) (500)

a From Whittaker et al. (1996b). Reprinted with permission of the author and the Journal of Biological Chemistry.

protein (and azide complexes) are summarized in Table Ill. On oxidation of glyoxal oxidase, there is a red band present at 850 nm, as in galactose oxidase. In galactose oxidase, this band has been assigned as a ligand-toligand charge transfer (via the Cu dxz orbitals) associated with the presence of both a Cys-Tyr free radical and an unmodified tyrosinate (McGlashen et al., 1995). The resonance Raman spectra of the oxidized, native, and azide complexes of glyoxal oxidase look remarkably like those of galactose oxidase (Fig. 13, Table IV). Axial tyrosinate vibrations at 1170 cm -1, 1249 cm -1, and 1604 cm -1 were assigned based on the similarity to phenolate vibrations in other metal-tyrosine proteins and model complexes (McGlashen et aL, 1995). These bands disappeared on addition of azide to the sample, which has been demonstrated to lead to the axial tyrosine dissociation in galactose oxidase, consistent with their assignment to the tyrosinate ligand of glyoxal oxidase. Resonance Raman spectroscopy has provided the strongest evidence for a thioether-substituted tyrosine in glyoxal oxidase. A key vibration in identifying the galactose oxidase cross-link is the 1382 cm -1 mode, which was assigned as an in-plane ring stretch (VlOa) in the 2thiomethoxy-4-methyl phenoxyl radical (McGlashen et aL, 1995). A similar band is seen at 1375 cm -I in glyoxal oxidase. The similarities in the vibrational frequencies and intensities of glyoxal oxidase to galactose oxidase provide compelling evidence that a Tyr-Cys phenoxyl radical is present as a copper ligand in glyoxal oxidase. The persistence of vibra-

POSTTRANSLATIONALLY MODIFIED TYROSINES

4]5

A c t i v e Oxidase ¢o ¢0

~

A

,~

=~

°

"

•

q.

°

,

IIil .,

I

',

t

IS

,

•

~t=-

,,

.

¢,

,

,

Azide Adduct

C

!,

,,

•

=

oO

u~

"

*0

i ..,

,

is •

~

I

1200

-

,

,

Jt it

t

•

,

,s

t

I

1300

s

,

~" •

-I

i s

a t

I

1400

.~

•

,

i

t L

=

t

,,

ii

t

I

1500

# •

=

I

1600

F r e q u e n c y , c m "1 FIG. 13. Resonance Raman spectra obtained with 647 nm excitation. - - , glyoxal oxidase (1.5 mM enzyme, in 25 mM sodium phosphate buffer pH 7) in presence of 1.2 equivalents of Na,)IrCla (A) and on addition of 10 mM NaN:~ (C). - - -, corresponding spectra for active galactose oxidase ir~ absence (B) or presence (D) of NaN3 for comparison (Whittaker et al., 1996b). Reprinted with permission of the author and the Journal

of Biological Chemist~.

416

MELANIE S. ROGERS AND DAVID M. DOOLEY

TABLEIV Resonance Roman Data for Glyoxal Oxidase Complexes a YOgi

N .~c,," c-v

N

"'OH2

y

N.. I .., C.Y

N~"~C)F,II

YOH

N,...,. C.-Y N

J'CU~N-N-N

p H 6.1

p H 6.1

Azide

)~maxfor C-Y 3 540 n m

660 n m

570 n m

1240 cm -1

1267 cm -1

1272 cm -1

1486

1481

1483

1596

1596

1599

y

N..l.. (C-Y).

N"~ ~ ' O~4~ Active

C-Y ligand Cu CT RR modes

Y ligand )~maxf o r Y 3

450 n m

450 n m

Cu CT RR modes

1171 cm -1 -1260 1609

1170 cm -1 1249 1604

a From Whittaker et al. (1996b). Reprinted with permission of the author and the Journal of Biological Chemistry.

tions in glyoxal oxidase spectra at both pH 6.1 (1240 cm -1, 1486 cm -1, and 1596 cm -1) and pH 8.1 (1267 cm -1, 1481 cm -1, and 1596 cm -1) and in the presence of azide (1272 cm -1, 1483 cm -1, and 1599 cm -1) indicated that these modes are not associated with the axial tyrosine, which dissociates from copper at both low pH and on addition of azide in galactose oxidase. The decrease in the vibrational frequency of the CysTyr unit at 1267 cm -1 to 1240 c m - 1 at low pH was attributed to the increase in the C u - O bond strength on axial tyrosine dissociation. Electron paramagnetic resonance (EPR) spectroscopy of glyoxal oxidase does not provide conclusive evidence for a thioether bond in glyoxal oxidase, but conclusions regarding the Cys-Tyr unit may be drawn based on the similarity of the glyoxal and galactose oxidase spectra as summarized by Whittaker (Whittaker et al., 1996b). On oxidization, the Cu(II) signal was lost, and a small radical signal was seen, as in galactose oxidase. A small amount of radical is attributed to limited amounts of apoprotein. The radical g~v is 2.0055, indicative of a thioether-modified phenoxyl n radical.

POSTTRANSLATIONALLY MODIFIED TYROSINES

417

Whittaker identified some metal binding and catalytic residues by site-directed mutagenesis, based on sequence alignments with galactose oxidase. Active-site variants (Whittaker et al., 1999) Y377F (the axial tyrosine in galactose oxidase), Y135F (the equatorial tyrosine in galactose oxidase), and C70A (the cross-link cysteine in galactose oxidase) were characterized. The results may provide evidence for a functionally important thioether b o n d in glyoxal oxidase. Loss of enzymatic activity in Y135F and C70A (0.04% and 0.02% activity relative to the wild-type protein, respectively) suggests a key role for these residues. The EPR spectrum of C70A was interesting in that there was no clearly resolved ligand hyperfine structure, which Whittaker ascribed to "a degree of heterogeneity in the metal-binding site that is consistent with a greater mobility afforded by an uncrosslinked tyrosine residue." The optical spectrum of Y135F, although containing 1:1 Cu:protein, has lost the equatorial Tyr --+ Cu transition. In a study investigating the fruiting body formation of StigmateUa aurantiaca, fofB was identified as a gene that encodes a polypeptide with sequence homologies to the galactose oxidase of Dactylium dendroides. The predicted protein FbfB (526 residues) has a molecular mass of 57.8 kDa and shows a significant homology to the Fusarium galactose oxidase (Silakowski et al., 1998). The motif, RXYXSS, suggested by Whittaker to indicate the presence of the cross-link, is conserved in FbfB (RGYHSSS) c o m p a r e d to RVYHSI in galactose oxidase. The thioether b o n d residues of galactose oxidase are conserved in FbfB, allowing for the possibility that such a feature may be present in this protein. The finding that two additional proteins may contain the cross-link opens up the possibility that more examples of a Tyr-Cys modification may be discovered. It will be of great interest to further probe the formation and tractions of this type of cross-link.

III. CYTOCHROME C OXIDASE

A. Introduction Cytochrome c oxidase (E.C. 1.9.3.1) is a m e m b e r of the heme-copper oxidase superfamily found in both eukaryotes and bacteria. It is the terminal enzyme of the m e m b r a n e respiratory electron-transfer chain where it has a key role in reducing dioxygen to water, [Eq. (3)]. This process requires eight protons, four of which are used in the reduction of oxygen (known as chemical, substrate, or scalar protons) and four of which are p u m p e d from the matrix side (Hi) (cytoplasm, Paracoccus) to

418

MELANIE S. ROGERS AND DAVID M. DOOLEY

the cytosolic side (Ho) (periplasm, Paracoccus) of the mitochondrial m e m b r a n e (vectorial protons). Transport of those four protons generates proton motive force used for ATP biosynthesis. 4 cyt C2+ + 02 + 8H+i ---->4 cyt c a+ + 2H20 + 4H+o

(3)

Owing to its enormous physiological importance, the enzyme has been the focus of broad and detailed studies for decades (Yoshikawa, 1997). A major breakthrough in the research efforts came in 1995 with the independent determination of the crystal structure of the enzyme from two different sources, Paraccocus denitrificans (Iwata et al., 1995) and bovine heart (Tsukihara et al., 1995). Due to the large size of the membrane protein and its hydrophobic nature, crystallization and subsequent structure determination proved extremely difficult. Although the enzyme has up to 13 subunits, depending on its source, the critical redox and H + translocation functions are located in subunits I and II (Fig. 14). The enzyme contains three redox-active catalytically important metal centers: Cun, heme a, and a binuclear site, heme aa-CuB. A dinuclear copper center, CUA, is located in subunit II and is the point of electron

"

Subunit I

v fragment

Subunit II

FIG. 14. Ribbon representation of the structure of the two-subunit cytochrome c oxidase from P. denitrificans complexed with the antibody Fv f r a g m e n t 7E2. T h e programs MOLSCRIPT a n d RASTER 3D were used to prepare the figure. Adapted from Ostermeier et al., (1997). Proc. Natl. Acad. Sci. USA. 94, 10547-10553. Copyright (1997) National Academy of Sciences, USA.

POSTTRANSI.AT1ONALIX MODIFIED ISqa,OSINES

419

entry. F e r r o c y t o c h r o m e c delivers electrons via electrostatic-mediated docking to the enzyme's surface close to the location of CUA. Electrons are transferred to the binuclear site via heine a, located in subunit I. T h e binuclear site is com pos ed of heine a and a single m o n o n u c l e a r c o p p e r ion, CUB. Heine a3 is able to bind ligands, and this is the site where 02 binds and is reduced. In addition to the heine and c o p p e r centers, a magnesium and a zinc site were also present in the crystal structure (Tsukihara et al., 1995) although they are not presently considered catalytically important.

B. Discovery of the Active-Site Cross-link The initial crystal structures of bovine and Paraccocus cytochrome c oxidase were both refined to 2.8 A. In the four-subunit Paraccocus enzyme crystal structure, Ostermeier initially c o m m e n t e d that one of the CuB ligands, His-276, was in Van der Waals contact with Tyr-280, and the contact was modeled as a hydrogen bond. T he H-bond was suggested to stabilize the CuB site. In 1997, a second Paraccocus structure, containing only subunits I and II, was determined to 2.7 A. In their description of the new structure, Ostermeier (Ostermeier et al., 1997) noted a communication from Yoshikawa et al. (1998) describing the presence of a covalent linkage in the binuclear site. This bond was located between His-276 and Tyr-280 (corresponding to His-240 and Tyr-244 in the bovine enzyme), which Ostermeier had previously assigned as a hydrogen bond (Fig. 15a, see color insert). When modeled into the two-subunit Pa~accocus structure. the covalent b o nd was found to provide much better agreement with the unbiased electron density than the former hydrogen bond. In 1998, Yoshikawa pr es e nt e d a refined data set of bovine hearl enzyme at 2.3 A resolution (Yoshikawa et al., 1998). H e also confirmed the new cross-link feature that was not resolved in the original structure. T h e electron density of tyrosine 244 and histidine 240, determined by a (Fo-Fc) difference Fourier map at 2.3 A resolution, was consistent with a covalent b o n d between the C~-2of the tyrosine phenol side chain and the imidazole Ne9 of histidine (Fig. 15b). T h e r e is also a hydrogen b o n d from the hydroxyl group of Tyr-288 to the hydroxytormylethyl group of heine a~. T he electron density distribution between His-240 and Tyr-244 was also seen in three o t h e r protein forms: fully reduced, the carbon m o n o x i d e complex, and the azide structure. This provided additional evidence that the cross-link is an authentic chemical b o n d and does not result from synchrotron radiation dan> age. Th e covalent b o n d of both the Paraccocus and bovine binuclear sites are seen in Fig. 15a and b.

420

MELANIE S. ROGERS AND DAVID M. DOOLEY

Thus in both galactose oxidase and cytochrome c oxidase a copper ligand is part of the cross-link. And here again, the presence of the cross-link was not predicted prior to the availability of the crystal structure. Note though, that the cross-link b o n d is a C - N whereas in galactose oxidase it is a C-S bond. However, the occurrence of a redox-active amino acid during the catalytic cycle of cytochrome c oxidase had been proposed (Weng and Baker, 1991; Watmough et al., 1994). C. Chemical Evidence of the Covalent Bond It was widely recognized that chemical confirmation of this novel TyrHis feature was of great importance, as the results would establish that the cross-link modeled into the electron density was not as a result of X-ray irradiation damage or a crystallization artifact. Such reactions can occur during X-ray data collection in the presence of iron or copper and oxygen in proximity to tyrosine and histidine side chains (Buse et al., 1999). Buse et al. took up this challenge as described in a 1999 Protein Science article that reports a multifaceted study of four distantly related cytochrome c oxidases. The two crystallographically characterized cytochrome c oxidases, bovine heart (13 subunits) and Paracoccus denit~ificans (2 subunits) and two proteins from Thermus thermophilus, claa3 oxidase and ba3 oxidase, were examined. The crystal structure of ba3-oxidase has subsequently been solved (Soulimane et al., 2000). The Thermus two-subunit enzymes were produced as fused proteins, claa~ expressed as a cytochrome-c/subunit II and ba~ expressed as a subunit I/III complex with heme b substituting for heme a (Buse et al., 1989; Keightley et al., 1995). Amino acid sequence alignments (Figure 16) of helix VI (the location of the cross-link as seen in the crystal structures) of the four proteins, showed that both histidine 240 and tyrosine 244 (bovine numberings) were conserved. Comparison of Bradyrhizobiumjaponicum cbb3 (FixN) oxidase and Pseudomonas stutzeri nitric oxide reductase (NOR) sequences suggested the cross-link is not conserved in these proteins. The proposed absence of the cross-link in cbb~ and N O R proteins suggests that the difference in these proteins and the dioxygen reductases is at the level of the binuclear site and may determine the reactivity/selectivity for the substrates NO or O2 (Giuffr~ et al., 1999). The HPXVY motif, containing the modified amino acids, is present in all CcO and quinol oxidases of eukaryotes (mitochondria), eubacteria, and archaea (Buse et al., 1999). The Protein Information Resource (Barker et al., 2000) lists 172 proteins (cytochrome c oxidase, bo-type ubiquinol oxidase, and ba-type ubiquinol oxidase) as possibly containing the tyrosine-histidine cross-link (based on the presence of the HPXVY motif). The conservation of the residues suggests the cross-link might play an important structural or mechanistic role.

POSTTRANSLATIONALLYMODIFIED~I53ROSINES

227

240

244

421

270

$ Bh

aa 3

DPILYQHLFW

FFGHPEVYIL

ILPGFGMISH

IVTYYSGKKE

PFGY

Pd

aa 3

DPVLYQHILW

FFGHPEVYII

ILPGFGIISH

VISTFAKKPI

FGY

Tt

ca 3

DPVLFQQFFW

FYSHPTVYVM

LLPYLGILAE

VASTFARKPL

FGY

Tt

ba 3

DPLVARTLFW

WTGHPIVYFW

LLPAYAIIYT

ILPKQAGGKL

VSDP

Bd

fixN

GGIQDAMFQW

WYGHNAVGFF

LTAGFLAIMY

YFIPKRAERP

IYSY

NLSRDKFYWW

FVVHLWVEGV

WELIMGAMLA

FVLIKVTGVD

REVI

Ps N O - r e d

++

*

*+ *+

++

Flc;. 16. Comparison of membrane helix VI amino acid sequences with His-240 and Tvr-244 cross-link of subunits I from four cytochrome c oxidases with one FixN oxidase and nitric oxide reductase. Bovine heart aa3-oxidase (Bh aa3) (AOO464); P. denitr!ficans aa~-oxidase (Pd aa3) (C35121); T. thermophilus caas-oxidase (Tt ca3) (A46616); 7~ the~. mophilus bas-oxidase (Tt ba3) ( LO9121 ); Bradyrhizobium japonicum fixi~oxidase (Bd .fix~\) (A47468); Pseudomonas stutzeri NO-reductase (Ps NO-red) ($41117). -]. Asp@ro cleavage site. Accession numbers (Protein Identification Resource databank) in parentheses. *Conserved; +conserved in respiratory cytochrome c and quinol oxidases (Buse et al.. 1999). Reprinted with permission of Cambridge University Press.

Limited acid hydrolysis on the subunit I polypeptides affected cleavage between Asp-227 and Pro-228, which is invariably positioned 13 residues N-terminal of the cross-link histidine. An additional cleavage site was fortuitously present in Thermus ba:~ (Asp-269-Pro-270) resulting in a 42-mer, which could be purified in nmol amounts. The sequence of the C-terminal half of the bovine, Paraccocus and Thermus caa:~ cytochrome c oxidase fragments, having proline 228 (bovine numbering) at their C terminus, resulted in gaps in the sequence at residues 13 and 17 (Fig. 17). The absence of these residues in the sequencing implies they were unavailable to be derivatized by phenylthiohydantion, which most likely results from their involvement in the covalent crosslink. A similar situation was seen in galactose oxidase (McPherson et al., 1992). Peptide sequencing of both oxidized and reduced Thermus 42mers also lacked detectable histidine and tyrosine, suggesting the crosslink is a persistent feature during the catalytic cycle. The size of the Thermus 42-met was confirmed using electrospray mass spectrometry (ES-MS) with a resolution of_+ 2 mass units in a molecular mass of about 5000 Da. The peptide sequence indicates a 4816.76 Da chain although the ES-MS data determined 4814.8 _+0.4 Da,

12 13

5

~

10

15

20

25

Retention time (minutes)

FIG. 17. Amino acid sequence at the covalent His-Tyr cross-link shows the genededuced sequences but no (PTH-) histidine and tyrosine at cycles 13 and 17, respectively, of the peptide. The chromatogram of the previous cycle has been substracted from the actual using the Knauer "WinSeq" Program (Buse et aL, 1999). Reprinted with permission of Cambridge University Press.

8

R3

R:o2Me 9 FIG. 18. Model compounds for the Tyr-His cross-link in cytochrome c oxidase: 2-imidazol-l-yl4methylphenol (8, IMP) (reprinted with permission from McCauley et al., 2000). Copyright (2000) American Chemical Society) the Tyr-His side-chaln coupled dipeptide model (9) where R1 = (S)-CH2C(H) (NHBoc)CO2Bn, R2 = H, and R3 = OMe (reprinted with permission from Elliot and Konopelski, 2000. Copyright (2000) American Chemical Society).

POSTTRANSLATIONALIX MODIFIED TYROSINES

423

expected if a covalent b o n d had removed 2 hydrogens. These data exclude the possibility of an unmodified Tyr-His-containing peptide. Amino acid sequencing and ES-MS data provide strong evidence that the Tyr-His cross-link is an integral part of four cytochrome c oxidases: bovine, P. denitrificans, T. thermophilus claa3 oxidase, and ba3 oxidase (Buse et al., 1999). More recently, the crystal structure of T. thermophilu,s ba~ cytochrome c oxidase has confirmed the proposed presence of the cross-link (Soulimane et al., 2000). It seems unlikely that this feature, m o d e l e d into the electron density, arises from either a crystallization artifact or as a result of synchrotron radiation damage in the crystals in bovine and P. denitrificans enzyme.

D. Model Chemistry In an attempt to understand the role of the modified histidine-tyrosine unit in enzyme catalysis, McCauley et al. (2000) synthesized a model cross-link compound, 2-imidazol-l-yl-4-methylphenol (8, IMP) (Fig, 18), which was compared to p-cresol. The pKa of the hydroxyl group of both c o m p o u n d s was determined from the pH d e p e n d e n c e of the optical spectrum, as shown in Fig. 19. The pKA of the phenol group

1.4

¢,

o.8

.I , <

/

, , ,

0.4 0.2 0

4

6

8

pH

10

12

FIG. 19. Spectrophotornetric titrations of aqueous solutions of IMP (0.4 mM, closed circles) and p-cresol (0.56 raM, o p e n circles). T h e titration was m o n i t o r e d at 314 nm tot IMP at which wavelength only the d e p r o t o n a t e d torm absorbs. Inset shows pH depend e n c e of the absorbance at 230 nm. For p-cresol the titration was m o n i t o r e d at 297 nln (McCauley et al., 2000). Reprinted with permission from McCaulev et al. (2000). Col) > right (2000) American Chemical Society.

424

MELANIE S. ROGERS AND DAVID M. DOOLEY

of IMP was determined to be 8.60 _+0.04. Closer inspection of the spectral changes with pH (at 230 nm) revealed a second pKA at 5.54 + 0.12, which was assigned to the imidazole group of the model compound. For comparison an analogous experiment with p-cresol produced a pKA at 10.23 + 0.09, thereby demonstrating that the cross-link has acted to decrease the hydroxyl group's pKA by 1.5-fold. The pKA of the substituted phenolate may be lowered via resonance stabilization of the conjugate anion by the N-linked imidazole, or by stabilization of the phenolate through an inductive electron withdrawing effect, which is the rationale preferred by McCauley et al. (2000). This inductive effect is more pronounced in the protein with a copper ion ligating to the histidine imidazole, and should depend also on the oxidation state of the metal ion as well as its coordination geometry, a~-p~ back-bonding may reduce the anticipated contribution to the inductive effect from copper ligation. The cross-link in the model has lowered the imidazole pKA to 5.54 (free histidine pKA 6.0; Barker, 1971), which may weaken the copper coordinating ability significantly. As suspected for galactose oxidase, the modification of the tyrosine in cytochrome c oxidase might alter the redox properties of this residue. Cyclic voltammograms (pH 11.5) displayed irreversible behavior for the model compound and p-cresol. However, the anodic peak of the IMP was 66 + 3 mV greater than that of p-cresol, which may be attributed to the electron-density withdrawing effect of the imidazole substituent. The pH dependence of the midpoint potential for a oneelectron oxidation of the cross-linked compound, determined by differential pulse voltammetry (DPV) measurements, revealed three dissociations constants (Fig. 20). Examination of both equilibrium acidity (pKA) (Table V) and the oxidation potential of the conjugate anions of IMP and p-cresol allowed the bond dissociation energy (BDE) of the phenol oxygen-hydrogen bond to be estimated. A decrease in the pKA for the dissociation of the hydrogen of the phenol hydroxy group of IMP and the increased oxidation potential result in a decrease in the oxygen-hydrogen bond of 0.7 kcal/mol compared to p-cresol. At first glance, these data support the suggestion that the tyrosine component of the cross-linked moiety can donate a hydrogen atom during the reduction of dioxygen; however McCauley et al. suggested the cross-link might act only in a structural capacity as the BDE of the hydroxyl group was only slightly perturbed. Other data indicate that the cross-link is important to the correct assembly of the binuclear copper center (Das et al., 1998). If H-atom (electron + proton) donation to bound oxygen is mechanistically important, a tyrosine-histidine radical may be generated dur-

425

POSTTRANSLATIONALLY MOD1FIED TYROSINES

1 0 0 0

i

i ,ll,

,,,,,,,,

,, ,, ,,,, ,,

,,,,,j

95O 90O

~, ¢,;o

uJ

750 700 650 6 0 0

....

4

I

I

5

6

.... ~

7

I

I

8

9

I

10

I

11

12

I)H FIG. 20. p H d e p e n d e n c e of the m i d p o i n t potential of IMP in aqueous bufter determ i n e d by differential pulse voltammetry. Potential values are versus NHE. Working electrode, glassy carbon; c o u n t e r electrode, Pt wire; reference electrode, Ag+/AgCI. All buffers were adjusted to identical ionic strengths using KNO~ electrolyte. Solid line p r o d u c e d by fitting the data to the following equation: b~n = Cst + 0.059 log {[H+] 2 + K~1 [H +] + KalKa 2} -0.059 log {[H +] } + K~3} (McCauley et al., 2000). R e p r i n t e d with permission from McCauley et al. (2000). Copyright (2000) American Chemical Society.

ing cytochrome c oxidase turnover. A recent report (MacMillan et al., 1999) detected a radical EPR signal at 80 K (glso ~ 2.0055) in a sample of fully oxidized cytochrome c oxidase treated with a 1 molar equivalent of H202 at pH 6.0. The identity of the radical was unequivocally assigned as a tyrosyl radical following perturbation of the radical EPR signal resulting from selective deuteration of tyrosine by supplementation of

TABLE V pK~ values of I M P and p-cresol" IMP

Optical PhOH ~ P h O - + H + hnH+-PhOH ~ ImPhOH + H + hnH+-PhO" ~-~ ImPhO" + H* "From McCauley et al. (2000).

8.60 _+0.04 5.54 _+0.12

(pK.) Electrochemical 8.90 ± 0.08 5.7 + 0.20 4.80 ± 0.24

#cresol (p/~d Optical 10.23±0.09

426

MELANIE S. ROGERSAND DAVIDM. DOOLEY

Field (G) FIG. 21. X-band EPR spectrum of the radical form of IME Instrument settings: frequency 9.27 GHz, power 100 I.tW, modulation amplitude 1G, time constant 0.032 s, and temperature 77°K (McCauley et al., 2000). Reprinted with permission from McCauley et al. (2000). Copyright (2000) American Chemical Society.

the growth media with [2,3,5,6-ZH]tyrosine. However, treating many enzymes, especially heine-containing proteins with H202, can generate tyrosyl radicals (Ortiz de Montellano and Catalano, 1985; Catalano et al., 1989; Wilks and Ortiz de Montellano, 1992), so the significance of this observation is not yet clear. A preliminary experiment to determine whether a tyrosyl radical could be generated in the IMP model by UV photolysis (pH 10) resulted in an EPR signal (Fig. 21) having a glso of 2.0058, which is remarkably similar to the enzymatic radical signal (in terms ofgvalue and linewidth). One important consideration is that, as in galactose oxidase, the EPR signal may be eliminated due to magnetic coupling between Yo and CuB via histidine (in galactose oxidase the tyrosyl radical coordinates to the copper ion). McCauley et al. point out that the radical signal in the enzyme, if not eliminated, may be significantly broadened. The tyrosyl radical generated during this process would need to be stable but not so much that subsequent one-electron reduction becomes unfavorable. McCauley et al. have shown that changes in pKa (lower) and redox potential (increase) are finely balanced for optimal enzyme activity and this is the case with the IMP model compound. Detailed characterization of structurally characterized Tyr-His models will be required to fully understand the role of this unit in

POSTTRANSLATIONALLY MODIFIED ~IWROSINES

427

cytochrome c oxidase. Recently a peptide mimic (Fig. 18, 9) of the TyrHis cross-link in cytochrome c oxidase was prepared (Elliot and Konopelski, 2000). The target Tyr-His dipeptide was formed as a single isomer in a 48% yield at room temperature without the addition of added base. These mild reaction conditions allowed for the N-1 regiospecificity. Investigation of the properties and coordination chemistry of this novel dipeptide might provide insight into the structure, function, and radical chemistry of this Tyr-His cross-link in cytochrome (: oxidase (Elliot and Konopelski, 2000) E. Role of the Cross-Link

In the absence of definitive mechanistic evidence, there have been sew eral suggestions for the role of the Tyr-His cross-link in cytochrome c oxidase. Initially, the generation of the cross-link was proposed to modulate one or more of the properties of either residue. Recent experimental dam and precedents in other enzymes suggest that the most important consequences of the modification are associated with ~rosine. One obvious possible role for the cross-link may be to stabilize a tyrosine-radical intermediate (Das et al., 1998; MacMillan et al., 1999). To ensure the complete reductive cleavage of dioxygen in the P state four electrons are required. H e m e a3 iron [Eq. (4)] and CuB [Eq. (5)] can provide three electrons, Fe (II) --->Fe (1V) + 2eCu (I) --->Cu (II) + e-

(4) (5)

The source of the fourth electron during reductive cleavage is less certain. There is no spectroscopic evidence for a porphyrin radical, and it seems unlikely that the metal ions can provide further electrons as this would implicate either a Fe(V) species (Ogura et al., 1996), which is u n p r e c e d e n t e d in a heme protein, or Cu(III). A trivalent copper state may be stabilized by the Tyr-His cross-link (Das et al., 1998), although there are no data to support this idea. Fabian and Palmer (1995) investigated the reaction of cytochrome c oxidase with hydrogen peroxide in an attempt to elucidate the nature of the P form and identify the source of the fourth electron. In the absence of a Cu(II) EPR signal in the P form, it was proposed that the electron came fl-om Cu(II), resulting in a Cu(III) state, [Fe(1V)=O, CUB(Ill)]. Trivalent copper had been proposed in the active site of galactose oxidase at one time (Hamilton et al., 1978). Although there are now no precedents for trivalent copper in nature, this oxidation state has been observed in model

428

MELANIE S. ROGERSAND DAVIDM. DOOLEY

[8~-O~ Ou~'-H-Y ]

18~-0 (Tu~-H-Y,,] [ ] "x . ~

[e~ Ou:+- H-~

..t

educe~ e'.H"

i-r

ta#=o c I'- M-v!

[@-OX }xidi; H" SCHEME 5. A simplified scheme for the reaction between cytochrome oxidase and 02. From Babcock, (1999). Proc. Natl. Acad. Sci. USA. 96, 12971-12973. Copyright (1999) National Academy of Sciences, U,S.A

compounds (Bossu et al., 1977; McDonald et aL, 1995, McDonald et al., 1997). Cu(III) is stabilized in complexes with multiple deprotonated peptide nitrogens as donor ligands but these have very different properties than the three histidine ligands in the CUB site. Another possible source of the fourth electron may be the tyrosine residue involved in the Tyr-His cross-link. Amino acid radicals have now been identified in a number of proteins as noted throughout this volume. A recent time-resolved resonance Raman study (Proshlyakov et al., 1998) probed the reaction of mixed-valence cytochrome c oxidase with dioxygen. Scheme 5 shows a simplified reaction cycle for cytochrome c oxidase. Electrons are initially donated from cytochrome c to the C u a site in the enzyme, followed by intramolecular electron transfer to heme a and then to the heme a3-CuB site. Reduction and protonation of the binuclear center produces the reduced state. Dioxygen binds at the binuclear site to generate the oxy state, which is subsequently converted to the "peroxy" (P) and oxo-ferryl (F) intermediates, before the oxidized form

POSTTRANSLATIONALLY MODIFIED ~[YROSINES

429

of the enzyme is regenerated. Reduction of the P and F intermediates is limited by proton transfer reactions as seen in Scheme 5. A specific role of tyrosine is implied by the scheme, which will be discussed later in this section. Species P in Scheme 5 was identified as an oxo-ferryl Fe (1V)=O species rather than the Fe-OO (H) species previously suggested in the literature (Varotsis et al., 1993). The presence of the oxo-ferryl intermediate implied that a fourth electron had already been donated, and it was suggested to have originated from the cross-linked tyrosine (Proshlyakov et al., 1998). Although an amino acid radical has not been directly observed during turnover, there is circumstantial evidence for its existence (Proshlyakov et al., 1996a, 1996b, 1998; MacMillan et al., 1999; Fabian et al., 1999; Chen et al., 1999). The covalent modification could facilitate hydrogen atom donation to b o u n d oxygen via modulation of the hydroxyl group pKa and oxidation potential of the cross-linked tyrosine (McCauley et al., 2000). Model studies indicate the calculated b o n d dissociation energy of the IMP phenol to be decreased compared to p-cresol. Himo et al. report a similarly small decrease of 1.7 kcal/mol in the sulfur-substituted tyrosine of galactose oxidase (Himo et al., 2000). Although the pK~ of a free tyrosine is 10.1 (Barker, 1971), the substitution of the phenol ring, the protein environment, and the proximity of metal ions may perturb the pK~ in an unpredictable manner. Evidence has also been presented that suggests the Tyr-His cross-link is important in the correct assembly of the binuclear site, providing rigidity and also a scaffold for the CuB (Das et al., 1998). The effects of mutagenesis ofY244 (bovine numbering) in an early study were interpreted in terms of a structural role for the tyrosine residue (Hosler et al., 1993). Moreover, mutagenesis of the cross-link tyrosine to phenylalanine in Rhodobacter sphaeroides cytochrome bo~ caused the loss of CuB, suggesting a role for the TyroHis b o n d in maintaining the architecture of the binuclear site of the protein (Mogi et al., 1998: Kawasaki et al., 1997; Thomas et al., 1994). The Rhodobacter sphaeroides cytochrome c oxidase mutant Y228F was designed to prevent cross-link formation and to abolish the hydrogen bonding ability (Y288-OH to the formyl group of heme a3) (Das et al., 1998). Resonance Raman spectroscopic studies showed that this enzvmatically inactive mutant suffered significant disruption to the binuclear site. For example, the resonance Raman spectrum of ferricyanide-treated Y288F is characteristic of six-coordinate low-spin heme, rather than the high-spin heine vibration normally expected for oxidized heine a3. Specifically, the 213 cm -1 vibration (diagnostic for five-coordinate Fe-histidine) seen in the wild-type enzyme, was absent in

430

MELANIE S. ROGERS AND DAVID M. DOOLEY

Y288E A water molecule may be modeled as a sixth ligand in the crystal structure of the Paracoccus enzyme. Inspection of the crystal structure revealed that the restricted nature of the heme pocket limited the number of alternate ligands to heme a3 (Das et al., 1998). Thus Das et al. postulate that the CUB ligand His-284, if not cross-linked, would have the flexibility to coordinate to the Fe ion of a3 in the Y228F protein. Sodium dithionite is able to reduce Y288F heme a3, and reoxidization by oxygen was observed. This suggests that H284 can be at most only weakly coordinated to heme a3 and thus easily displaced. Interestingly, heme a3 could not be reduced by cytochrome c in the Y288F variant, which may reflect a significant change in the environment of the a3-CuB site. One possibility is that the binuclear site becomes more accessible to solvent in the absence of the cross-link. This may also rationalize the loss of copper from the binuclear site. The Fe-C m o d e of Fe-CO complex is observed in the resonance Raman spectrum of cytochrome c oxidase (Argade et al., 1984). The frequency of this vibration is d e p e n d e n t on the iron-copper distance and is sensitive to pH. In Y288E the Fe-C stretching frequency was found to be p H insensitive and equal to the frequency in the wild-type enzyme where the copper is sufficiently distant so as not to perturb Fe-CO. This structural rearrangement of the binuclear site in Y288F may facilitate the loss of the CuB from the site. In summary, the properties of the Y288F variant are clearly consistent with a role for the Tyr-His cross-link in maintaining the binuclear site architecture. The covalent link prevents His-284 from ligating heine a3 (with the subsequent loss of the CuB ion), preserving its availability as a copper ligand. Furthermore, it appears that the cross-link ensures the structural integrity of the binuclear site, modulating the redox potential of heme a3 and minimizing loss of CuB. The rigid nature of the binuclear site, originating with the cross-link, may prevent the generation of peroxide. This could occur if the tyrosine hydrogen atom were donated to the proximal oxygen b o u n d to heme a3 rather than the distal oxygen. Investigation of the crystal structure shows that the tyrosine hydroxyl group is positioned too far away to be an effective donor (Gennis, 1998; Yoshikawa et al., 1998; Proshlyakov et al., 1998). It may be possible that the cross-link maintains tyrosine sufficiently far away from the distal oxygen to prevent generation of peroxide. E Biogenesis of the Tyrosine-Histidine Cross-Link

As discussed elsewhere, the nonenzymatic formation of covalently or oxidatively modified amino acids has been d e m o n s t r a t e d in both amine oxidase (Dooley, 1999) and galactose oxidase (Rogers et al.,

POSTTRANSLATIONALLY MODIFIED TYROSINES

431

2000). It is possible that cytochrome c oxidase is a n o t h e r example of a self-processing enzyme. Buse et al. (1999) have suggested that the generation of an oxygen radical mechanism in the presence of the e l e c t r o n - d o n a t i n g metals of the p r o t e i n results in the Tyr-His crosslink (Buse et al., 1999). No proposals for the biogenesis of this cross-link have been f o r m u l a t e d at this time. Histidine and tyrosine have been reported to be sensitive to radical reactions (Davies et al., 1987), and Buse et al. are exploring the cross-linking reaction in a synthetic peptide in the presence of oxygen, copper, and iron. The cross-link reaction may occur during the first turnover (MacMillan et al., 1999) via a side reaction of the tyrosyl radical. Michel (1999) argues that the presence of the cross-link is itself evidence that a tyrosyl radical is f o r m e d during the catalytic cycle. The involvement of a c h a p e r o n e or enzyme to catalyze the formation of the cross-link must also be considered, although the catalytic center is buried in the protein and thus it is likely that histidine and tyrosine are inaccessible (Zaslavsky and Gennis, 2000). Precedent favors a self-processing reaction in the presence of oxygen and metal ion.

IV. FINAL COMMENTS

The study of posttranslationally modified redox-active amino acids is a new and continuing area of biochemistry, with many examples now recognized (Okeley and van der Donk, 2000). However, we are still at the early stages in our understanding of the reactivity., structure and function relationships, and "biogenesis" of these new cot;actors. The model studies on compounds representing the galactose oxidase and cytochrome c oxidase cofactors have demonstrated that substitution at the ortho position of the tyrosine side chain may modity the redox potential, the b o n d dissociation energy, and the pKa of the hydroxyl group. Behavior attributable to such perturbations are evident in studies on both enzymes. Yet the effect of the covalent modification may have different outcomes. In cytochrome c oxidase, the tyrosyl radical species in cytochrome c oxidase is proposed to arise as a resuh of donating a hydrogen atom (electron + proton) to b o u n d dioxygen to assure O - O bond cleavage. This contrasts with galactose oxidase where the stabilized, cross-linked tyrosyl radical abstracts a hydrogen atom from the activated (by coordination to copper) substrate. Finally we should include the possibility that the cross-link may also serve a protective role, perhaps controlling the reactivity of the tyrosyl radical, and preventing deleterious ligand radical coupling reactions.

432

MELANIES. ROGERSAND DAVIDM. DOOLEY

The capability for self processing, using available reagents such as metal ions and dioxygen, to generate new types of reactivity, might represent a key step in the evolution of enzymes. New redox functions can thus be created without the need for a complex biosynthesis machinery. It is currently unclear whether the formation of the novel cofactor might result from an independent biogenesis pathway or be collateral to the first turnover of the enzyme. Therefore, an in-depth understanding of the formation and function of such novel cofactors might facilitate their incorporation into modified or designed enzymes. Cross-linking amino acids certainly provides enzymes with new types of redox reactivity. Thus we expect that more proteins will be discovered with covalently modified tyrosine residues that display new functions.

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