The chemical biology of copper

286

The chemical biology of copper Bo G Malmstr6m* and Johan Leckneri Major progress was made in 1997 in the understanding of the biological transport of copper. Blue copper and Cu A sites have very low electron transfer reorganization energies. The mechanisms of copper-containing oxygenases and oxidases have been clarified by recent crystal structure determinations. Protein folding has been shown to tune the reduction potentials of blue copper proteins by hydrophobic encapsulation of the active sites and strict control of the axial ligation.

Addresses G6teborg University, Department of Chemistry, Biochemistry and Biophysics, PO Box 462, SE-405 30 G6teborg, Sweden *e-mail: [email protected] te-mail: [email protected] Current Opinion in Chemical Biology 1998, 2:286-292 http://biomednet.com/elecref/1367593100200286 © Current Biology Ltd ISSN 1367-5931

Abbreviation CCO cytochrome c oxidase

Introduction T h e biological functions of copper are intimately related to its properties as a transition metal (see [1] for a general description). Transition metal elements are characterized by having partially filled d orbitals in many of their compounds, and they generally have more than one stable valence state, for example, Co + and Cue+, or even Cu 3+, in tile case of cop.per. Consequently, they have the ability to mediate electron transfer by valence-shuttle mechanisms.

Some transition metals in their low valence state can bind dioxygen reversibly, as in hemocyanin, whose active site in the deoxy form is a dinuclear Cu+-Cu + complex [2]. An actb'e site, when reduced and oxygenated, c a n also activate dioxygen in copper-containing oxygenases (in which one or two atoms of dioxygen are incorporated into organic molecules) or in oxidascs (which reduce the dioxygen molecule to H 2 0 e or to two molecules of HeO) [31. T h e s e dioxygen-activating active sites are often dinuclear, as in tyrosinase [3,4"], or trinuclear, as in ascorbate oxidase and ceruloplasmin [4"',5",6]. Copper is one of tile most prevalent biological transition metals, second only to iron. Its concentration in biological habitats, such as sea water, is low, however, so that its accumulation in living cells requires active transport. Free Cu z+ is toxic, even in relatively low concentrations, and hence transport within organisms occurs in only complexes, usually in proteins.

This review will discuss recent papers, mostly from 1996 and 1997, which we deem particularly important and interesting. Emphasis will be on structure-function relationships so that, with a few exceptions, only proteins whose three-dimensional structures arc known will be discussed. T h e stress on publications from 1996 or later also has the consequence that proteins whose structures were determined a few or more years ago may not receive mention unless some novel mechanistic knowledge has emerged. Furthermore, enzymes that contain other cofactors in addition to copper, such as superoxide dismutase [3], cytochrome a3-Cu B in cytochrome oxidase [4"] and amine oxidases [7], will not be considered. Copper transport and storage Until recently the mechanism by which copper crosses the cell membrane and gets transported to the vesicular systems, where it is incorporated into functional proteins, has been largely unknown. During the past few years, however, a detailed picture of copper trafficking in yeast has emerged from investigations combining genetic, chemical and spectroscopic characterization of the proteins involved [8"]. Two pathways for copper transport to the final copper protein products are illustrated in Figure 1 [9°]. One leads to the formation of Fet3 (a P-type ATPase) in post-Golgi vesicles. This is a copper protein displaying sequence homology with laccase (a type 1 copper-containing oxidase), in particular [4"']. It possesses ferroxidase activity and is necessary for iron transport [8"], just like ceruloplasmin in vertebrates [10,11]. After entering the cell as Cu(I) via Ctrl, the metal is transported into the cytosol by the soluble Cu(I) receptor Atxl, which takes it to the transmembrane transport protein Ccc2 in the post-Golgi vesicle ([8"']; see Figure 1). Ccc2 is a cation transport ATPase of the P-type. An analogous transport chain, involving the proteins Ctrl, Coxl7 and an unknown analogue to Ccc2, exists for taking copper to cytochrome c oxidase (CCO) in the inner mitochondrial membrane.

Biochemical and genetic studies [8°°,11,12,13°,14 "°] have shown that human homologs exist to some of the "yeast proteins as well as to some related bacterial ATPases [15]. In particular, Menkes and Wilson disease proteins are P-type ATPases similar to Ccc2 [12,13°,14"']. Thus, in Wilson disease copper accumulates in the cytosol of liver cells [11,13",14"], with resulting toxic effects and neurological damage. A c o m m o n Cu-binding motif, Met-X-Cys-X-X-Cys (where X can be any amino acid), has been found in Atxl, its human counterpart Hahl and Ccc2, as well as in the Wilson and Menkes proteins, and Cu(I)-S interactions with two- or three-coordination have been demonstrated by extended X-ray absorption fine structure (EXAFS) [8"°].

The chemical biology of copper Malmstr6m and Leckner

Figure 1

Plasma membrane

.......

L

~Atxl

(/~) Cox17

~.~Atxl

~ Cox17

Copper transport in yeast. Cu(I) is transported across the plasma membrane by Ctrl and then transferred to intracellular vesicles by soluble Cu chaperones (such as Atxl or Cox1 7). In the vesicles Cu is incorporated into specific proteins, Fet3 (a P-type ATPase) and cytochrome c oxidase (CCO).

Small blue copper proteins and Cu A domains High-resolution crystal structures have now been reported for two phytocyanins blue proteins in plants), stellacyanin [16] and cucumber basic protein [17]. T h e weak axial methionine ligand found in most blue proteins is replaced in stellacyanin by glutamine, whose e-oxygen, an oxygen atom of which forms a strong bond with copper (bond length is 2.21/~). This, together with the solvent exposure of the copper site, may explain the observation that stellacyanin has the lowest reduction potential of all blue proteins. In the azurin Metl21--+Glu mutant, a strong C u - O bond is also formed, and the reduction potential is low [18]. Site-directed mutagenesis has continued to give valuable information about small blue proteins, but major advances have been few. T h e inner-sphere reorganization energy for electron transfer to the copper site in azurin (blue copper protein found in some bacteria) has been determined to be very small (-0.2V) [19]. Studies of mutant forms of azurin [20,21] have established the crucial importance of the ligand His117 for electron transfer, and also that the second histidine ligand, His46, is required [22], even if it is not involved directly in the electron transfer. Several lines of evidence indicate that the role of Metl21 is to prevent the binding of strong axial ligands, either from the protein itself or from external ligands [23,24,25"]. Introduction of a second tryptophan into the electron-transfer pathway

287

in azurin has demonstrated that interacting rc orbitals of aromatic rings may enhance intramolecular electron transfer in proteins in general [26"1. The biological role of plastocyanin is to mediate electron transfer from cytochrome f to photosystem 1 in photosynthesis, and its interaction with the natural partners has also been characterized by site-directed mutagenesis [27,28]. T h e mutagenesis results demonstrate that both electrostatic and hydrophobic interactions are important for the reaction between plastocyanin and photosystem 1. Studies of ruthenium (Ru)-labelled plastocyanin suggest a strong electronic coupling along a Cu--+Cys-+His--+Ru pathway [29], which mimics the Cu--+Cu pathway in nitrite reductase and ascorbate oxidase. All studies of the Cu A site in cytochrome oxidase, which can be considered a dimer of two blue-protein sites, have recently been reviewed by Beinert [30"], who discovered this redox center in 1959. T h e expression of soluble domains containing the CUA site allowed, for the first time, studies of its spectroscopic properties, free from interference from the heme components of the oxidase. A domain from Thermus thermophilus cytochrome ba 3 is particularly useful for biophysical investigations because of its extreme stability [31 ]. Electron paramagnetic resonance (EPR) studies [32"] and paramagnetic N M R [33-35,36"] studies have established that the Cu A site is a highly delocalized, mixed-valence C u I ' S - C u 1"5 complex. It has a reduction potential of 0.24 V and cannot be oxidized to the Cue+-Cu e+ state without falling apart [37]. T h e entropy contribution to the reduction potential can be related to changes in solvation [38"], and it is smaller for the Cu A site [37] than for any blue copper protein [38"]; this suggests a low reorganization energy for the CUA site. T h e unique structure of the CUA site is important for electron transfer from cytochrome c to cytochrome oxidase, as demonstrated, for example, by its conversion to an inactive mononuclear center in the Cys216--+Ser mutant [39]. The Cu ions are held together by two bridging cysteine sulfurs, which is consistent with spectroscopic (resonance-Raman) measurements [40] and electronic structure calculations [41"]. These Cu ions are, however, so close (2.3~) that there is direct Cu-Cu bonding [42",43], as also found in model complexes [42",43,44]. There is little change in the bond length upon reduction of the sulfurs; this accounts for the low reorganization energy of the Cu A site.

Oxygenases and oxidases T h e most studied copper-containing monooxygenase is undoubtedly dopamine [3-monooxygenase (see [7]), because of the important role this enzyme plays in controlling the level of neurotransmitters. T h e structure of a closely related enzyme, peptidylglycine ot-hydroxylating monooxygenase (PHM), has recently been determined [45"]. It consists of two domains, each having eight antiparallel

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[3 strands. T h e two Cu atoms, CuA and Cub, are bound in different strands 11 .~ apart. Dioxygen binds to reduced Cub, with the peptide substrate binding nearby. Each Ct, aton then donates one electron to dioxygen, and the hydroperoxide formed undergoes heterolytic cleavage, one O atom being reduced to H 2 0 and the other one being incorporated in the substrate.

Figure 2 (a)

1.1

[

reSY j:jox

0,9

~

0.7 0.5

._~ 0.3

Galactose oxidase, a mononuclear copper-containing oxidase, belongs to the growing family of tyrosyl radical enzymes (see [46 ° ] for a review). T h e Cu ion is pentacoordinated, with Tyr272, His581, His496 and a water molecule or an acetate ion in a plane, the apical ligand being Tyr495 (see [7]). Several models for the oxidized form of the galactose oxidasc active site have been described recently [47-511. In this form, Cu(I[) is strongly antiferromagnetically coupled to a Tyr272 radical [52]. T h e role of this radical in the catalytic mechanism is strongly supported by its inactivation using radical-probing 13-haloethanols as substrates [53]. In one mutant form, "l}'r495-->Phe, the catalytic efficiency of galactose oxidase is reduced 1000-tbld, which supports the idea that Tyr495 functions in a proton-abstraction step of the catalytic mechanism [54]. T h e main progress in our understanding of blue, coppercontaining oxidases in recent years has been the determination of high-resolution strucn, rcs for ascorbatc oxidase and ceruloplasmin, which has been repeatedly reviewed [5",6,55°]. As already mentioned, the strt, ctural work on ceruloplasmin has strengthened the view that this protein acts as a ferroxidase [10,55"]. T h e structures have established that the dioxygen-reducing site in these oxidases is a trinuclear complex consisting of the type 2 and type 3 Cu ions [4°*,5°].

T h e effect of protein folding on c o p p e r - c o n t a i n i n g active sites

t£ 0,1 i

-0.1

(b)

75 .=. 5 ~ 25

g

0

~

0

-25 ~ -50

0

1

2 3 [GuHCI] (M)

4

5

CurrentOpinionin ChemicalBiology Unfolding of oxidized (ox) and reduced (red) azurin. (a) The fraction unfolded is plotted as a function of the concentration of guanidine hydrochloride (GuHCI), whereas (b) gives the folding free energy (&G); extrapolation to zero concentration of guanidine hydrochloride yields the free energy in water. Data were obtained from [57].

however, shown that there is no strain on the metal ion in an oxidized blue site. This is qualitatively most easily understood from the orbital diagram for a blue site, shown in Figure 3. T h e shape of the sulfur p orbital is such that its two lobes can form bonds with two of the lobes of the Cu 2+ dx,__y, orbital. Together with the two ~ bonds from Cu to nitrogen atoms of the ligand histidine residues, the Cu 2+ ion thus gets an apparent square-planar coordination. Conseqt, ently, the trigonal core formed by thc cysteine sulfur and the histidine nitrogen atoms is a stable ligand arrangement in an oxidized blue site.

T h e protein fold determines tire structure of the metalbinding site in metalloproteins [56**]. Since the site has an affinity for its intrinsic metal ion, binding of this ion will increase the folding free energy. In the case of redox-active metals, the degree of stabilization will depend on the redox state, as illustrated with .data for azurin [57] in Figure 2. It can be seen that, unlike heine proteins [58,59], the oxidized form of azurin is more stable against unfolding by guanidinium chloride than the reduced protein.

One of the many puzzles in blue copper protein research has been the question of how the reduction potentials can vary from about 200mV (stellacyanin) to almost 1V (fungal laccase) whereas the spectroscopic properties of the oxidized proteins are relatively constant. Spectroscopic experiments and electronic structure calculations [62 ° ] have suggested that the strength of the axial bond between Cu and a methionine sulfur is a key feature; lengthening of the bond results in an increased reduction potential. We have developed this idea further, as illustrated in Figure 4.

\Ve will discuss here how the protein fold controls the thermodynamics and kinetics of electron transfer in redox proteins, with blue copper proteins as examples. T h e finding that oxidized azurin is its most stable form [57] contradicts a long entertained idea about blue copper proteins: Cu 2÷ prefers a square-planar or tetragonal ligand arrangement [1], so it was believed to be strained by the protein fold in the lower symmetry of a blue copper site (sec [56"°]). Electronic structure calculations [60°,61] have,

Recent work has shown that unfolded azurin, as well as an unfolded CUA domain, have a reduction potential of about 450 mV [63]. We ascribe this relatively high potential to the strong electron-donating tendency of cysteine sulfur and the high affinity of Cu A for the strong ligands. T h e exposure of the Cu A site to 17120, which stabilizes the Cu 2+ form, prevents the potential from going even higher. Thus, the first effect of the protein fold is hydrophobic encapsulation, which increases the potential by excluding

The chemical biology of copper Malmstr6m and Leckner

Figure 3

289

Figure 4

Folded protein N "Cu --S

Fungallaccase

rl

N N/-'Cu--S

\S--

N/

N ~(~u-- S N

680mY

Rusticyanin

__C\\/ 0 H20 H20 H20 H20 N--Ou --S H20 H20 H20 Unfoldedprotein Hydrophobic encapsulation

450mV Plastocyanin

380mV

\S-N..I N/Cu-s

\S--

N

N;Cu__ s

N CurrentOpinionin ChemicalBiology

Molecular orbital diagram for a blue copper site. The Cu dx2 y2 orbital interacts with the two lobes of sulfur p orbital, and two histidine nitrogen atoms form a bonds.

N'', O

O 310mV

//

__C /

-% Stellacyanin

\

%

180mY

N "~u_ s N/,, O

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H 2 0 and by enclosing the metal ion in a medium of low dielectric constant (proteins can be regarded as the organic solvents of biology). T h e magnitude of this increase from the initial value of 450mV can be estimated, from the effect of heine encapsulation in cytochrome c, to be about 350mV [58], which brings the potential to the value for fungal laccase (800mV, Figure 4). In proteins with a lower reduction potential, we propose that the protein fold decreases the potential to different extents by varying the strength of axial ligation. This effect is strongest in stellacyanin, where the ligand methionine found in most blue proteins is replaced by a glutamine residue [16]; this protein has the lowest potential of copper-containing proteins studied. T h e azurin potential is higher because of the weak methionine sulfur bond, and the potential is raised further in plastocyanin, in which the bond to a carbonyl oxygen has been weakened. Finally, rusticyanin has a potential of only =100mV below that of fungal laccase; this situation is ascribed to further lengthening of the bond made by the carbonyl oxygen atom and to a very hydrophobic environment surrounding the metal site [64,65].

Conclusions

Biological transport of copper across membranes has been shown to involve P-type ATPases, examples being the Menkes and Wilson disease proteins in humans. Blue and purple (those containing a Cu A site) copper proteins have been found to be facile electron transfer agents because of the very low reorganization energies

Increasing axial interaction

CurrentOpinionin ChemicalBiology The effect of the protein fold on the reduction potentials of blue copper sites. In the unfolded protein, Cu is still bound to cysteine (shown as its sulfur atoms, S) and one histidine (shown as its nigrogen atom, N) and it is surrounded by water molecules, which results in an intermediate potential. Exclusion of water by hydrophobic encapsulation of the copper site raises the potential to almost 1 V. In proteins with lower reduction potentials such as plastocyanin and stellacyanin, these are down-tuned by varying strengths of axial ligation.

associated with electron transfer. T h e two Cu ions in peptidylglycine ct-hydroxylating monooxygenase, an enzyme closely related in function to dopamine ]3-monooxygenase, donate two electrons to dioxygen. Several model studies have advanced our understanding of the role of the interacting Cu2+ ion-tyrosyl radical pair in galactose oxidase. Folding studies on blue copper proteins have led to a detailed understanding of how the protein rack (the strain in metalloprotein active sites) controls the functional properties of active sites in metalloproteins. Future directions of copper protein research will undoubtedly include detailed analyses of electron-transfer pathways in proteins containing several copper atoms. In addition, experiments with electron-transfer-triggered folding of proteins will no longer be restricted to heine proteins but will include blue copper proteins as well.

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Bio-inorganic chemistry

Acknowledgements We wish to thank Harry B Gray and Pernilla Wittung-Stafshede for extremely valuable discussions, Bo G MalmstrSm acknowledges support from the Nobel Committee for Chemistry.

Copper transport and copper-containing enzymes in humans are discussed in detail (168 references) in this paper. Excellent figures summarize normal copper transport and its disruption in pathological states. 15.

Silver M, Phung LT: Bacterial heavy metal resistance: new surprises. Annu Rev Microbio11996, 50:753-789.

16.

Hart PJ, Nersissian AM, Herrmann RG, Nalbandyan RM, Valentine JS, Eisenberg D: A missing link in cupredoxins: crystal structure of cucumber stellacyanin at 1.6 A resolution. Protein Sci 1996, 5:2175-2183.

17.

Guss JM, Merritt EA, Phizackerley RP, Freeman HC: The structure of a phytocyanin, the basic blue protein from cucumber, refined at 1.8A resolution. J Mol Bio11996, 262:686-705.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest 00 of outstanding interest 1.

Shriver DF, Atkins PW, Langford CH: Inorganic Chemistry, edn 2. New York: WH Freeman and Co; 1994.

2.

Jameson GB, Ibers JA: Biological and synthetic dioxygen carriers. In Bioinorganic Chemistry. Edited by Bertini I, Gray HB, Lippard SJ, Valentine JS. Mill Valley: University Science Books: 1994:167-252.

18.

Karlsson BG, Tsai L-C, Nar H, Sanders-Loehr J, Bonander N, Langer V, Sj5lin L: X-ray structure determination and characterization of the Pseudomonas aeruginosa azurin mutant Met121Glu. Biochemistry 1997, 36:4089-4095.

Valentine JS: Dioxygen Reactions. In Bioinorganic Chemistry. Edited by Bertini I, Gray HB, Lippard SJ, Valentine JS. Mill Valley: University Science Books; 1994:253-313.

19.

Di Bill• AJ, Hill MG, Bonander N, Karlsson BG, Villahermosa RM, MalmstrOm BG, Winkler JR, Gray HB: Reorganization energy of blue copper: effects of temperature and driving force on the rates of electron transfer in ruthenium- and osmium-modified azurins. J Am Chem Soc 1997, 119:9921-9922.

20.

Faham S, Mizoguchi TJ, Adman ET, Gray HB, Richards JH, Rees DC: Role of the active site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the Cull(Cys112Asp) protein. J B/o/Inorg Chem 1997, 2:464-469.

21.

Gorren ACF, den Blaauwen T, Canters GW, Hopper DJ, Duine JA: The role of His117 in the redox reactions of azurin from Pseudomonas aeruginosa. FEBS Lett 1996, 381:140-142.

22.

Van Pouderoyen G, Andrew CR, Loehr TM, Sanders-Loehr J, Mazumdar S, Hill HA, Canters GW: Spectroscopic and mechanistic studies of type-1 and type-2 copper sites in Pseudomonas aeruginosa azurin as obtained by external ligands to mutant His46Gly. Biochemistry 1996, 35:1397-1407.

23.

Bonander N, Karlsson BG, V~nng&rd T: Environment of copper in Pseudomonas aeruginosa azurin probed by binding of exogenous ligands to Met121X (X=Gly, Ala, Val, Leu, or Asp) mutants. Biochemistry 1996, 35:2429-2436.

24.

Tsai L-C, Bonander N, Harata K, Karlsson G, V~.nng,~rdT, Langer V, Sj~lin L: Mutant Met121Ala of Pseudomonas aeruginosa azurin and its azide derivative: crystal structure and spectral properties. Acta Crystallogr D 1996, 52:950-958.

4. ;is

Solomon El, Sundaram UM, Machonkin TE: MuRicopper oxidases and oxygenases. Chem Rev 1996, 96:2563-2605. review supplements Solomon's spectroscopy article in the Messerschmidt book [5"] and contains outstanding presentations of the chemical and electronic structures as well as the reaction mechanisms of the enzymes involved, it appears in a special issue of Chemical Reviews, devoted to bioinorganic enzymology, which contains other valuable articles on electron transfer and oxygen metabolism (for example, [7]). 5. •

Messerschmidt A: Spatial structures of ascorbate oxidase, laccase and related proteins. In Multi-Copper Oxidases. Edited by Messerschmidt A. Singapore: World Scientific; 1997:23-79. This is a very useful book, which contains excellent chapters on the structure of ceruloplasmin, spectroscopy, electron transfer reactions and redox properties of multi-copper oxidases, among others. Messerschmidt A: Metal sites in small blue copper proteins, blue copper oxidases and vanadium-containing enzymes. Structure Bonding 1998, 90:37-68. Klinman JP: Mechanisms whereby mononuclear copper proteins functionalize organic substrates. Chem Rev 1996, 96:2541-2561. 8. 00

Pufahl RA, Singer CP, Peariso KL, Lin S-J, Schmidt PJ, Fahrni CJ, Cizewski Culotta V, Penner-Hahn JE, •'Hall•ran TV: Metal ion chaperone function of the soluble Cu(I) receptor Atxl. Science 1997, 278:853-856. This is a key paper concerning copper transport in yeast, and references to earlier contributions can be found in it. 9. Valentine JS, Gralla EB: Delivering copper inside yeast and • human cells. Science 1997, 278:817-818. This is just a short commentary on [8"'] but provides a very valuble summary.

10.

Lindley PF, Card G, Zaitseva I, Zaitsev V, Reinhammar B, SelinLindgren E, Yoshida K: An X-ray structural study of human ceruloplasmin in relation to ferroxidase activity. J Biol Inorg Chem 1997, 2:454-463.

11.

Harris ZL, Morita H, Gitlin JD: The biology of human ceruloplasmin. In Multi-Copper Oxidases. Edited by Messerschmidt A. Singapore: World Scientific; 1997:285-305.

12.

T(~mer Z, Horn N: Menkes disease: recent advances and new aspects. J Med Genet 1997, 34:265-274.

13. Linder MC, Hazegh-Azam M: Copper biochemistry and T'his ravimclecularew biology. Am J C/in Nutr 1996, 63:797S-811S. provides a very readable account of copper biochemistry, includo ing nutrition and disease states. 14. =e

DiD•nat• M, Sarkar B: Copper transport and its alterations in Menkes and Wilson diseases. Biochim Biophys Acta 1997, 1360:3-16.

25. •

Bauer R, Danielsen El Hemmingsen L, Bjerrum MJ, Hansson C), Singh K: Interplay between oxidations state and coordination geometry of metal ions in azurin. J Am Chem Soc 1997, 119:157-162. This study employs a rather unusual method - perturbed angular correlation of y-rays spectroscopy. This is the only copper protein study available using this method. 26. •

FarverO, Skov LK, Young S, Bonander N, Karlsson BG, V~nngb.rdT, Pecht I: Aromatic residues may enhance intramolecular electron transfer in azurin. J Am Chem Soc 1997, 119:5453-5454. This paper describes direct evidence for a role of aromatic residues in electron transfer in proteins. 27.

Sigfridsson K, Young S, Hansson C): Electron transfer between spinach plastocyanin mutants and photosystem 1. Eur J Biochem 1997, 245:805-812.

28.

Young S, Sigfridsson K, Olesen K, Hansson (3: The involvement of two acidic patches of spinach plastocyanin in the reaction with photosystem 1. Biochim Biophys Acta 1997, 1322:106-114.

29.

Sigfridsson K, Sundahl M, Bjerrum MJ, Hansson (~: Intraprotein electron transfer in a ruthenium-modified Tyr83-His plastocyanin mutant: evidence for strong electronic coupling. J Biol/norg Chem 1996, 1:405-414.

The chemical biology of copper Malmstr6m and Leckner

30. -=

Beinert H: Copper A of cytochrome c oxidase, a novel, longembattled, biological electron-transfer site. Eur J Biochem 1997, 245:521-532. This review offers a fascinating account of the controversies surrounding research on Cu A and its dinuclear nature. Its dinuclear nature was resisted by some investigators as late as the spring of 1995, when, in the minds of most people in the field, it had already been established by analytical and spectroscopic data on soluble domains; it was not fully accepted until the crystal structures were published later in that year. 31.

Slutter C, Sanders D, Wittung P, Malmstr5m BG, Aasa R, Richards JH, Gray HB, Fee JA: Water-soluble, recombinant CuA-Domain of the cytochrome ba 3 subunit II from Thermus thermophilus. Biochemistry 1996, 35:3387-3395.

Karpefor'sM, Slutter C, Fee JA, Aasa R, K&llebring B, Larsson S, V~nng&rd T: Electron paramagnetic resonance studies of the soluble CUA protein from the cytochrome be 3 of Thermus thermophilus. Biophys J 1996, 71:2823-2829. In addition to its scientific significance, this paper describes a technically very elegant investigation, employing different Cu isotopes and several microwave frequencies as well electronic-structure calculations.

valence and fully reduced forms of the soluble CUA domains of cytochrome c oxidase, J Am Chem Soc 1997, 119:6135-6143. This study gives very accurate bond lengths in Cu A of cytochrome c oxidase, consistent with Cu-Cu bonding, and shows that there is very little change in these on reduction, accounting for the low reorganization energy. 43.

Williams KR, Gamelin DG, LaCroix LB, Houser RP, Tolman WB, Mulder TC, de Vries S, Hedman B, Hodgson KO, Solomon El: Influence of copper-sulfur covalency and copper-copper bonding on valence delocalization and electron transfer in the Cu A site of cytochrome c oxidase. J Am Chem Soc 1997, 119:613-614.

44.

Farrar JA, Grinter R, Neese F, Nelson J, Thomson AJ: The electronic structure of the mixed-valence copper dimer Cu 2 N(CH2CH2N==NCH2CH2)3N3+. J Chem Soc Dalton Trans 199?:4083-4087.

32. •

33.

Bertini I, Bren KL, Clemente A, Fee JA, Gray HB, Luchinat C, Malmstrbm BG, Richards JH, Sanders D, Slutter CE: The Cu A center of a soluble domain from Thermus cytochrome be3. An NMR investigation of the paramagnetic protein. J Am Chem Soc 1996, 118:11658-1 t 659.

34.

Dennison C, Berg A, de Vries S, Canters GW: 1H NMR studies of the paramagnetic Cu A center of cytochrome oxidase. FEBS Lett 1996, 394:340-344.

35.

Dennison C, Berg A, Canters GW: A 1H NMR study of the paramagneti¢ active site of the Cu A variant of amicyanin. Biochemistry 1997, 36:3262-3269.

36. •

Luchinat C, Soriano A, Diinovic-Carugo K, Saraste M, Malmstr0m BG, Bertini I: Electronic and geometric structure of the Cu A site studied by 1NMR in a soluble domain of cytochrome c oxidase from Paracoccus denitrificans. J Am Chem Soc 1997, 119:11023-11027. The paramagnetically shifted NMR lines are unusually narrow because of the short relaxation time of Cu A, and this paper analyzes effect this in terms of electronic structure. 37.

Immoos C, Hill MG, Sanders D, Fee JA, Slutter CE, Richards JH, Gray HB: Electrochemistry of the Cu A domain of Thermus thermophilus cytochrome ba 3. J Biol Inorg Chem 1996, 1:529531.

Battistuzzi G, Borsari M, Loschi L, Sola M: Redox thermodynamics, acid-base equilibria and salt-induced effects for the cucumber basic protein. General implications for bluecopper proteins. J Bio//norg Chem 1997, 2:350-359. This article gives a valuable discussion of the control of the reduction potentials of blue copper proteins in thermodynamic terms.

45. •e

Prigge ST, Kolhekar AS, Eipper EA, Mains RE, Amzel LM: Amidation of bioactive peptides: the structure of peptidylglycine c(-hydroxylating monooxygenase. Science 1997, 278:1300-1305. This paper describes the first structure determination of a member of this group of copper-containing oxygenases. The mechanistic knowledge extracted can be extrapolated to other members of the group, including dopamine ~-monooxygenase. 46. •

Babcock GT, Espe M, Hoganson C, Lydakis-Simantiris N, McCracken C, Shi W, Styring S, Tommos C, Warncke K: Tyrosyl radicals in enzyme catalysis: some properties and a focus on photosynthetic water oxidation. Acta Chem Scand 1997, 51:533-540. A description is given of enzymes that require a red•x-active amino acid for catalysis, Tyrosine-based radical enzymes, such as galactose oxidase and ribonucleotide reductase, are discussed; spectroscopic insights into the control of radical reactivity by the protein are discussed, in particular,. The major emphasis in the review is, however, not on copper proteins but on photosystem 2 in photosynthesis. 47.

Zurita D, Gautier-Luneau I, M~nage S, Pierre .I-L, Saint-Aman E: A first model for the oxidized active form of the active site in galactose oxidase: a free-radical copper complex. J Biol Inorg Chem 1997, 2:46-55.

48.

Zurita D, Scheer C, Pierre J-L, Saint-Aman E: Solution studies of copper(ll) complexes as models for the active site in galactose oxidase. J Chem Soc Dalton Trans 1996:4331-4336.

49.

Halfen JA, Jazdzewski BA, Mahapatra S, Berreau LM, Wilkinson EC, Clue L Jr, Tolman WB: Synthetic models of the inactive copper(ll)-tyrosinate and active copper(ll)-tyrosyl radical forms of galactose and glyoxal oxidases. J Am Chem Soc 1997, 119:8217-8227.

50.

Itch S, Takayama S, Arakawa R, Furuta A, Komatsu M, ]shida A, Takamuko S, Fukuzumi S: Active site models for galactose oxidase. Electronic effect of the thioether group in the novel organic cofactor. /norg Chem 199?, 36:1407-1416.

51.

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