BBA - Bioenergetics (v.1655, #C)

Introduction by Peter Brzezinski; Anders Ehrenberg; Cecilia Tommos (1).

Redox-driven proton pumps, radical initiation and propagation in biology, and small-molecule activation processes all involve the coupling of electron transfer to proton transport. A mechanistic framework in which to interpret these processes is being developed by examining proton-coupled electron transfer (PCET) in model and natural systems. Specifically, PCET investigations are underway on the following three fronts: (1) the elucidation of the PCET reaction mechanism by time-resolved laser spectroscopy of electron donors and acceptors juxtaposed by a proton transfer interface; (2) the role of amino acid radicals in biological catalysis with the radical initiation and transport processes of E. coli ribonucleotide reductase (RNR) as a focal point; and (3) the application of PCET towards small–molecule activation with emphasis on biologically relevant bond-breaking and bond-making processes involving oxygen and water. A review of recent developments in each of these areas is discussed.
Keywords: Proton-coupled electron transfer; Tyrosyl radical; Oxygen and water activation; Cytochrome c oxidase; Photosystem II; Ribonucleotide reductase; Monooxygenase; Catalase; Pacman; Hangman; Porphyrin; Hydrogen-bonding; Heme water channel; Dagwoods;

Theoretical studies of proton-coupled electron transfer reactions by Sharon Hammes-Schiffer; Nedialka Iordanova (29-36).
A theoretical formulation for proton-coupled electron transfer (PCET) is described. This theory allows the calculation of rates and kinetic isotope effects and provides insight into the underlying fundamental principles of PCET reactions. Applications of this theory to PCET reactions in iron bi-imidazoline complexes, oxoruthenium polypyridyl complexes, osmium–benzoquinone systems, amidinium–carboxylate salt bridges, DNA–acrylamide complexes, and ruthenium polypyridyl–tyrosine systems are summarized. The mechanistic insight gained from theoretical calculations on these model systems is relevant to PCET in more complex biological processes such as photosynthesis and respiration.
Keywords: Proton-coupled electron transfer; Hydrogen; Kinetic isotope effect;

A theory of proton coupled electron transfer (PCET) is reviewed with application to charge transfer steps in the photosystem II oxygen-evolving complex (PSII/OEC). The relation between PCET when it is a concerted electron proton transfer (ETPT) process and hydrogen-atom transfer (HAT) reactions is discussed. Signatures expected for HAT reactions in terms of the size of the kinetic isotope effect and overall magnitude of the rate constant are discussed in the context of PSII/OEC. The formal similarity of ETPT to proton transfer and translocation is used to introduce a combined quantum mechanical (for the transferring protons) and molecular dynamics for the heavy-atom degrees of freedom approach. The method is used to examine double proton transfer in cytochrome c oxidase where two waters and a glutamate (Glu286) that is implicated in the proton translocation mechanism form a cyclic hydrogen bonded structure. Protonation of the glutamate is found to occur in agreement with experimental results.
Keywords: Proton and electron transfer; Proton translocation; Photosystem II oxygen evolving complex; Cytcohrome c oxidase;

Important roles of tyrosines in Photosystem II and cytochrome oxidase by Per E.M. Siegbahn; Margareta R.A. Blomberg (45-50).
Theoretical studies (B3LYP) on models of the active sites in Photosystem II (PSII) and cytochrome oxidase are discussed. The role of a tyrosyl radical in the OO bond formation in PSII is investigated, as well as the tyrosyl radical formation. In cytochrome oxidase, mechanisms for OO bond cleavage involving tyrosyl radical formation are investigated, together with possible roles for the tyrosine in the proton translocation.
Keywords: Theory; B3LYP; Mechanism; Photosystem II; Cytochrome oxidase; Tyrosyl radical;

Reactions that involve transfer of an electron and a proton can proceed by stepwise pathways involving initial electron transfer (ET) or initial proton transfer (PT), or by a concerted pathway without an intermediate. The concerted mechanism is termed proton-coupled electron transfer (PCET). Understanding such reactions requires knowledge of the thermodynamics of the possible ET, PT, and PCET steps. Many reactions have a large thermochemical bias favoring the PCET pathway. This bias is often sufficient to rule out stepwise mechanisms. The ΔG° for ET, PT, or PCET has a strong influence on the rate of that step. Using the terminology of Marcus theory, PT and PCET reactions at CH bonds have higher intrinsic barriers than such reactions at OH or NH bonds. The intrinsic barriers to ET and PCET are often similar when there is a small intrinsic barrier to PT. Reactions with a thermochemical bias toward PCET and with similar intrinsic barriers for all the pathways are most likely to occur by concerted PCET.
Keywords: Proton-coupled electron transfer; Hydrogen atom transfer; Electron transfer; Proton transfer; Marcus theory; Iron;

Electron tunneling in rhenium-modified Pseudomonas aeruginosa azurins by Jeremiah E. Miller; Angel J. Di Bilio; William A. Wehbi; Michael T. Green; A.Katrine Museth; John R. Richards; Jay R. Winkler; Harry B. Gray (59-63).
Laser flash-quench methods have been used to generate tyrosine and tryptophan radicals in structurally characterized rhenium-modified Pseudomonas aeruginosa azurins. Cu(I) to “Re(II)” electron tunneling in Re(H107) azurin occurs in the microsecond range. This reaction is much faster than that studied previously for Cu(I) to Ru(III) tunneling in Ru(H107) azurin, suggesting that a multistep (“hopping”) mechanism might be involved. Although a Y108 radical can be generated by flash-quenching a Re(H107)M(II) (M=Cu, Zn) protein, the evidence suggests that it is not an active intermediate in the enhanced Cu(I) oxidation. Rather, the likely explanation is rapid conversion of Re(II)(H107) to deprotonated Re(I)(H107 radical), followed by electron tunneling from Cu(I) to the hole in the imidazole ligand.
Keywords: Electron tunneling; Rhenium complex; Amino acid radical; Blue copper; Azurin;

Intraprotein electron transfer and proton dynamics during photoactivation of DNA photolyase from E. coli: review and new insights from an “inverse” deuterium isotope effect by Martin Byrdin; Valérie Sartor; André P.M. Eker; Marten H. Vos; Corinne Aubert; Klaus Brettel; Paul Mathis (64-70).
We review our work on electron transfer and proton dynamics during photoactivation in DNA photolyase from E. coli and discuss a recent theoretical study on this issue. In addition, we present unpublished data on the charge recombination between the fully reduced FADH and the neutral (deprotonated) radical of the solvent exposed tryptophan W306. We found a pronounced acceleration with decreasing pH and an inverse deuterium isotope effect (k H/k D=0.35 at pL 6.5) and interpret it in a model of a fast protonation equilibrium for the W306 radical. Due to this fast equilibrium, two parallel recombination channels contribute differently at different pH values: one where reprotonation of the W306 radical is followed by electron transfer from FADH (electron transfer time constant τ et in the order of 10–50 μs), and one where electron transfer from FADH (τ et=25 ms) is followed by reprotonation of the W306 anion.
Keywords: Photolyase; Electron transfer; Proton transfer; Tryptophan radical; Deuterium isotope effect; Transient absorption spectroscopy;

Novel cyanide inhibition at cytochrome c 1 of Rhodobacter capsulatus cytochrome bc 1 by Artur Osyczka; Christopher C. Moser; P.Leslie Dutton (71-76).
Oxidized cytochrome c 1 in photosynthetic bacterium Rhodobacter capsulatus cytochrome bc 1 reversibly binds cyanide with surprisingly high, micromolar affinity. The binding dramatically lowers the redox midpoint potential of heme c 1 and inhibits steady-state turnover activity of the enzyme. As cytochrome c 1, an auxiliary redox center of the high-potential chain of cytochrome bc 1, does not interact directly with the catalytic quinone/quinol binding sites Qo and Qi, cyanide introduces a novel, Q-site independent locus of inhibition. This is the first report of a reversible inhibitor that manipulates the energetics and electron transfers of the high-potential redox chain of cytochrome bc 1, while maintaining quinone substrate catalytic sites in an intact form.
Keywords: Cytochrome bc 1; Cyanide inhibition; Heme; Electron transfer; Rhodobacter capsulatus;

The rate-limiting reaction of the bc 1 complex from Rhodobacter sphaeroides is transfer of the first electron from ubihydroquinone (quinol, QH2) to the [2Fe–2S] cluster of the Rieske iron–sulfur protein (ISP) at the Qo-site. Formation of the ES-complex requires participation of two substrates (S), QH2 and ISPox. From the variation of rate with [S], the binding constants for both substrates involved in formation of the complex can be estimated. The configuration of the ES-complex likely involves the dissociated form of the oxidized ISP (ISPox) docked at the b-interface on cyt b, in a complex in which Nε of His-161 (bovine sequence) forms a H-bond with the quinol OH. A coupled proton and electron transfer occurs along this H-bond. This brief review discusses the information available on the nature of this reaction from kinetic, structural and mutagenesis studies. The rate is much slower than expected from the distance involved, likely because it is controlled by the low probability of finding the proton in the configuration required for electron transfer. A simplified treatment of the activation barrier is developed in terms of a probability function determined by the Brønsted relationship, and a Marcus treatment of the electron transfer step. Incorporation of this relationship into a computer model allows exploration of the energy landscape. A set of parameters including reasonable values for activation energy, reorganization energy, distances between reactants, and driving forces, all consistent with experimental data, explains why the rate is slow, and accounts for the altered kinetics in mutant strains in which the driving force and energy profile are modified by changes in E m and/or pK of ISP or heme b L.
Keywords: Control of electron transfer; Proton transfer; bc 1 complex; Qo-site; Marcus theory; ES-complex;

The redox midpoint potential (E m) of QA, the primary quinone of bacterial reaction centers, is substantially modulated by the protein environment. Quite subtle mutations in the QA binding site, e.g., at residues M218, M252 and M265, cause significant increases in the equilibrium constant for electron transfer to QB, which indicate relative lowering of the E m of QA. However, reports of functional linkage between the QA and QB sites make it difficult to partition such effects between QA and QB from purely relative changes. We report here measurements on the yield of delayed fluorescence emission from the primary donor (P) accompanying the thermally activated charge recombination of P+QA to form the excited singlet state of the primary donor, P*. The results show that for mutations of the QA site residues, MetM218 and IleM265, essentially all the substantial thermodynamic effect is localized at QA, with no evidence for a significant effect of these residues on the properties of QB or the mutual influence (linkage) of QA and QB. We also report a significant lowering of the E m of QA by the native lipid, cardiolipin, which brings the E m in isolated reaction centers more in line with that seen in native membrane vesicles (chromatophores). Possible origins of this effect are discussed in the context of the QA binding site structure.
Keywords: Photosynthetic reaction center; Electron transfer; Quinone; Cardiolipin; Midpoint redox potential; Delayed fluorescence;

Surface-mediated proton-transfer reactions in membrane-bound proteins by Pia Ädelroth; Peter Brzezinski (102-115).
As outlined by Peter Mitchell in the chemiosmotic theory, an intermediate in energy conversion in biological systems is a proton electrochemical potential difference (“proton gradient”) across a membrane, generated by membrane-bound protein complexes. These protein complexes accommodate proton-transfer pathways through which protons are conducted. In this review, we focus specifically on the role of the protein–membrane surface and the surface–bulk water interface in the dynamics of proton delivery to these proton-transfer pathways. The general mechanisms are illustrated by experimental results from studies of bacterial photosynthetic reaction centres (RCs) and cytochrome c oxidase (CcO).
Keywords: Proton pathway; Cytochrome c oxidase; Reaction centre; Photosynthesis; Respiration; Electron transfer; Kinetics;

The function and characteristics of tyrosyl radical cofactors by Curtis W. Hoganson; Cecilia Tommos (116-122).
Amino-acid radicals are involved in the catalytic cycles of a number of enzymes. The main focus of this mini-review is to discuss the function and properties of tyrosyl radical cofactors. We start by briefly summarizing the experimental studies that led to the detection and identification of the two redox-active tyrosines, denoted YZ and YD, found in the water-oxidizing photosystem II (PSII) enzyme. More recent work that shows that the histidine-cross-linked tyrosine located in the active site of cytochrome c oxidase forms a radical during the catalytic oxygen–oxygen bond-cleavage process is also described.Advanced spectroscopic and structural studies have been performed to investigate the spin-density distribution, the protonation state and the hydrogen bonding of redox-active tyrosines. These studies have shown that the radical spin-density distribution is highly insensitive to the environment and that it is typical of a deprotonated species. In contrast, the hydrogen bonding and the nature of the proton acceptor or network of acceptors vary substantially in different systems. This is important for the function of the tyrosyl radical, as will be emphasized in a detailed discussion on the proposed function of YZ as a proton coupled electron-transfer cofactor in photosynthetic water oxidation.Amino-acid radical enzymes are typically large complexes containing multiple subunits, chromophores and redox cofactors. The structural and mechanistic complexity of these systems has hampered the detailed characterization of their radical cofactors. In the final section of this mini-review, we will describe a project aimed at investigating how the protein controls the thermodynamic and kinetic redox properties of aromatic residues by using de novo protein design. Two model proteins of different size have been constructed. The smaller protein is a 67-residue three-helix bundle containing either a single buried tryptophan or tyrosine residue. The high-resolution NMR structure of the tryptophan-containing protein, denoted α3W, shows that the aromatic side chain is involved in a π-cation interaction with a nearby lysine. The effects of this interaction on the tryptophan reduction potential were investigated by electrochemical and quantum mechanical methods. The calculations predict that the π-cation interaction increases the potential, which is consistent with the electrochemical characterization of α3W. A larger 117-residue four-helix bundle, α4W, has more recently been constructed to complement the work on the three-helix-bundles and expand the family of model radical proteins.
Keywords: Amino-acid radical; Tyrosyl radical; Oxygen evolution; Water oxidation; De novo protein; Protein design;

Photosystem II (PSII), the multisubunit pigment–protein complex localised in the thylakoid membranes of oxygenic photosynthetic organisms, uses light energy to drive a series of remarkable reactions leading to the oxidation of water. The products of this oxidation are dioxygen, which is released to the atmosphere, and reducing equivalents destined to reduce carbon dioxide to organic molecules. The water oxidation occurs at catalytic sites composed of four manganese atoms (Mn4-cluster) and powered by the redox potential of an oxidised chlorophyll a molecule (P680 •+). Gerald T (Jerry) Babcock and colleagues showed that electron/proton transfer processes from substrate water to P680 •+ involved a tyrosine residue (YZ) and proposed an attractive reaction mechanism for the direct involvement of YZ in the chemistry of water oxidation. The ‘hydrogen-atom abstract/metalloradical’ mechanism he formulated is an expression of his genius and a highlight of his many other outstanding contributions to photosynthesis research. A structural basis for Jerry's model is now being revealed by X-ray crystallography.
Keywords: Photosynthesis; Photosystem II; Water oxidation; Hydrogen abstraction model; Babcock;

The evolutionary development of the protein complement of Photosystem 2 by Jason Raymond; Robert E. Blankenship (133-139).
During the transition from anoxygenic to oxygenic photosynthesis, the Type 2 reaction center underwent many changes, none so dramatic as the remarkable increase in complexity at the protein level, from only three or four subunits in the anoxygenic reaction center to possibly more than 25 in Photosystem 2 (PS2). The evolutionary source of most of these proteins is enigmatic, as they have no apparent homology to any other proteins in existing databases. However, some of the proteins in PS2 have apparent homologies to each other, suggesting ancient gene duplications have played an important role in the development of the complex. These homologies include the well-known examples of the D1 and D2 reaction center core proteins and the CP43 and CP47 core antenna proteins. In addition, PsbE and PsbF, the two subunits comprising cytochrome b-559, show homology to each other, suggesting that a homodimeric cytochrome preceded the heterodimeric one. Other potential homologies that appear to be statistically significant include PsbV with the N-terminal part of D1 and PsbT with PsbI. Most of the proteins that make up the photosynthetic apparatus bear no relation to any other proteins from any source. This suggests that a period of remarkable evolutionary innovation took place when the ability to make oxygen was invented. This was probably a response to the production of highly toxic oxygen and these new proteins served to protect and repair the photosynthetic apparatus from the harmful effects of oxygen.
Keywords: Photosystem 2; Evolution; Oxygenic photosynthesis;

The water-oxidation complex in photosynthesis by Kenneth Sauer; Vittal K. Yachandra (140-148).
Studies of the photosynthetic water-oxidation complex of photosystem II (PS II) using spectroscopic techniques have characterized not only important structural features, but also changes that occur in oxidation state of the Mn4 cluster and in its internal organization during the accumulation of oxidizing equivalents leading to O2 formation. Combining this spectroscopic information with that from the recently published relatively low-resolution X-ray diffraction studies, we have succeeded in limiting the range of likely cluster arrangements. This evidence strongly supports several options proposed earlier by DeRose et al. [J. Am. Chem. Soc. 116 (1994) 5239] and these can be further narrowed using compatibility with electron paramagnetic resonance (EPR) data.
Keywords: Photosystem II; Oxygen-evolving complex; EXAFS; XANES; EPR; S-state; Oxygen evolution;

The first spectroscopic model for the S1 state multiline signal of the OEC by Wen-Yuan Hsieh; Kristy A Campbell; Wolfgang Gregor; R David Britt; Derek W Yoder; James E Penner-Hahn; Vincent L Pecoraro (149-157).
The parallel-mode electron paramagnetic resonance (EPR) spectrum of the S1 state of the oxygen-evolving complex (OEC) shows a multiline signal centered around g=12, indicating an integer spin system. The series of [Mn2(2-OHsalpn)2] complexes were structurally characterized in four oxidation levels (MnII 2, MnIIMnIII, MnIII 2, and MnIIIMnIV). By using bulk electrolysis, the [MnIIIMnIV(2-OHsalpn)2(OH)] is oxidized to a species that contains MnIV oxidation state as detected by X-ray absorption near edge spectroscopy (XANES) and that can be formulated as MnIV 4 tetramer. The parallel-mode EPR spectrum of this multinuclear MnIV 4 complex shows 18 well-resolved hyperfine lines center around g=11 with an average hyperfine splitting of 36 G. This EPR spectrum is very similar to that found in the S1 state of the OEC. This is the first synthetic manganese model complex that shows an S1-like multiline spectrum in parallel-mode EPR.
Keywords: S1 state; Electron paramagnetic resonance (EPR); X-ray absorption near edge spectroscopy (XANES); Manganese complex;

Recent pulsed EPR studies of the Photosystem II oxygen-evolving complex: implications as to water oxidation mechanisms by R.David Britt; Kristy A Campbell; Jeffrey M Peloquin; M.Lane Gilchrist; Constantino P Aznar; Michelle M Dicus; John Robblee; Johannes Messinger (158-171).
The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S0-state are presented and interpreted. These pulsed EPR results are used in an evaluation of several recently proposed mechanisms for PSII water oxidation. We strongly favor mechanistic models where the substrate waters bind within the OEC early in the S-state cycle. Models in which the OO bond is formed by a nucleophilic attack by a Ca2+-bound water on a strong S4-state electrophile provide a good match to the pulsed EPR data.
Keywords: ENDOR; ESEEM; Multiline EPR signal; S0-state; Substrate water binding;

Water oxidation in PSII—H atom abstraction revisited by Ron J. Pace; Karin A. Åhrling (172-178).
A model for the water oxidation reaction in Photosystem II (PSII) is presented, based on an H atom abstraction mechanism. The model rationalises the S-state dependence of observed substrate water exchange kinetics [Biochim. Biophys. Acta 1503 (2001) 197] and assumes that H transfer occurs to an oxidised μ-oxo bridge oxygen on the S3→S4→S0 transition. The model requires that only one Mn-pair and a Ca ion be directly involved in the substrate binding and catalytic function. The multiline signal observed in the S0 state is shown to plausibly arise from such a system. A detailed molecular model of the three-metal site, assuming ligation by those residues identified by mutagenesis as Ca/Mn ligands is presented. This bears a resemblance to the dinuclear Mn site in Mn catalase and is generally consistent with the electron density map of cyanobacterial PSII recently presented [Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 98].
Keywords: Photosystem II; Mn cluster; Water oxidising complex; H atom abstraction;

Reconstitution of the photosystem II Ca2+ binding site by Kirk A. Vander Meulen; Amanda Hobson; Charles F. Yocum (179-183).
The roles of Ca2+ in H2O oxidation may be as a site of substrate binding, and as a structural component of the photosystem II O2-evolving complex. One indication of this dual role of the metal is revealed by probing the Mn cluster in the Ca2+ depleted O2 evolving complex that retains extrinsic 23- and 17-kDa polypeptides with reductants (NH2OH and hydroquinone) [Biochemistry 41 (2002) 958]. Calcium appears to bind to photosystem II at a site where it could bind substrate H2O. Equilibration of Ca2+ with this binding site is facilitated by increased ionic strength, and incubation of Ca2+ reconstitution mixtures at 22 °C accelerates equilibration of Ca2+ with the site. The Ca2+ reconstituted enzyme system regains properties of unperturbed photosystem II: Sensitivity to NH2OH inhibition is decreased, and Cl binding with increased affinity can be detected. The ability of ionic strength and temperature to facilitate rebinding of Ca2+ to the intact O2 evolving complex suggests that the structural environment of the oxidizing side of photosystem II may be flexible, rather than rigid.
Keywords: Photosystem II; Water oxidation; Calcium; Manganese; Chloride;

Time-resolved oxygen production by PSII: chasing chemical intermediates by Jürgen Clausen; Richard J. Debus; Wolfgang Junge (184-194).
Photosystem II (PSII) produces dioxygen from water in a four-stepped process, which is driven by four quanta of light and catalysed by a Mn-cluster and tyrosine Z. Oxygen is liberated during one step, coined S3⇒S0. Chemical intermediates on the way from reversibly bound water to dioxygen have not yet been tracked, however, a break in the Arrhenius plot of the oxygen-evolving step has been taken as evidence for its existence.We scrutinised the temperature dependence of (i) UV-absorption transients attributable to the reduction of the Mn-cluster and tyrosine Z by water, and (ii) polarographic transients attributable to the release of dioxygen. Using a centrifugatable and kinetically competent Pt-electrode, we observed no deviation from a linear Arrhenius plot of oxygen release in the temperature range from −2 to 32 °C, and hence no evidence, by this approach, for a sufficiently long-lived chemical intermediate. The half-rise times of oxygen release differed between Synechocystis WT* (at 20 °C: 1.35 ms) and a point mutant (D1–D61N: 13.1 ms), and the activation energies differed between species (Spinacia oleracea, 30 kJ/mol versus Synechocystis, 41 kJ/mol) and preparations (PSII membranes, 41 kJ/mol versus core complexes, 33 kJ/mol, Synechocystis).Correction for polarographic artefacts revealed, for the first time, a temperature-dependent lag-phase of the polarographic transient (duration at 20 °C: 0.45 ms, activation energy: 31 kJ/mol), which was indicative of a short-lived intermediate. It was, however, not apparent in the UV-transients. Thus the “intermediate” was probably newly formed and transiently bound oxygen.
Keywords: Photosynthesis; Water oxidation; Activation energy; S-state; Photosystem II;

This minireview addresses questions on the mechanism of oxidative water cleavage with special emphasis on the coupling of electron (ET) and proton transfer (PT) of each individual redox step of the reaction sequence and on the mode of OO bond formation. The following topics are discussed: (1) the multiphasic kinetics of YZ ox formation by P680+• originate from three different types of rate limitations: (i) nonadiabatic electron transfer for the “fast” ns reaction, (ii) local “dielectric” relaxation for the “slow” ns reaction, and (iii) “large-scale” proton shift for the μs kinetics; (2) the ET/PT-coupling mode of the individual redox transitions within the water oxidizing complex (WOC) driven by YZ ox is assumed to depend on the redox state S i : the oxidation steps of S0 and S1 comprise separate ET and PT pathways while those of S2 and S3 take place via proton-coupled electron transfer (PCET) analogous to Jerry Babcock's hydrogen atom abstractor model [Biochim. Biophys. Acta, 1458 (2000) 199]; (3) S3 is postulated to be a multistate redox level of the WOC with fast dynamic equilibria of both redox isomerism and proton tautomerism. The primary event in the essential OO bond formation is the population of a state S3(P) characterized by an electronic configuration and nuclear geometry that corresponds with a complexed hydrogen peroxide; (4) the peroxidic type S3(P) is the entatic state for formation of complexed molecular oxygen through S3 oxidation by YZ ox; and (5) the protein matrix itself is proposed to exert catalytic activity by functioning as “PCET director”. The WOC is envisaged as a supermolecule that is especially tailored for oxidative water cleavage and acts as a molecular machine.
Keywords: OO bond formation; Primary donor; Redox active tyrosine; Redox isomerism; Tautomerism; Water cleavage;

Tyrosyl radicals in Photosystem II by Idelisa Pujols-Ayala; Bridgette A. Barry (205-216).
In PSII, there are two redox-active tyrosines, D and Z, with different midpoint potentials and different reduction kinetics. The factors responsible for these functional differences have not yet been elucidated. Recent model compound studies of tyrosinate and of tyrosine-containing dipeptides have demonstrated that perturbations of the amino and amide/imide group occur when the tyrosyl aromatic ring is oxidized [J. Am. Chem. Soc. 124 (2002) 5496]. Accompanying density functional calculations suggested that this perturbation is due to spin density delocalization from the aromatic ring onto the amino nitrogen. The implication of this finding is that spin density delocalization may occur in redox-active, tyrosine-containing enzymes, like Photosystem II. In this paper, we review the supporting evidence for the hypothesis that tyrosyl radical spin density delocalizes into the peptide bond in a conformationally sensitive, sequence-dependent manner. Our experimental measurements on tyrosyl radicals in dipeptides have suggested that the magnitude of the putative spin migration may be sequence-dependent. Vibrational spectroscopic studies on the tyrosyl radicals in Photosystem II, which are consistent with spin migration, are reviewed. Migration of the unpaired spin may provide a mechanism for control of the direction and possibly the rate of electron transfer.
Keywords: Redox active; Electron transfer; EPR; FT-IR; Photosynthetic water oxidation; Isotope labeling;

Photosynthetic water oxidation: the role of tyrosine radicals by Jonathan H.A. Nugent; Richard J. Ball; Michael C.W. Evans (217-221).
This mini-review outlines the involvement of the tyrosine electron carriers, YD and YZ, in the mechanism of electron transfer from water to P680. We discuss our data and put forward our ideas on the role of YD and YZ.
Keywords: Photosynthesis; Oxygen evolution; Photosystem II; Water oxidation; Tyrosine; Manganese;

The stable tyrosyl radical in Photosystem II: why D? by A.William Rutherford; Alain Boussac; Peter Faller (222-230).
Two redox-active tyrosines are present in Photosysytem II, the water-oxidizing enzyme. While the tyrosine that is kinetically competent in electron transfer, TyrZ, may also have a role in the enzyme mechanism, the second tyrosine, TyrD, has a stable radical and is not directly involved in the redox chemistry associated with enzyme function. Nevertheless, reasonable mechanistic roles for TyrD have been postulated that satisfy desires to rationalise the presence of this cofactor, or, in English, we think we know what it does. First, the TyrD radical acts an oxidant of the Mn cluster in the lowest state of the redox accumulation cycle (i.e., S0), providing potential benefits in maintaining the cluster in the more stable higher valence states. This redox role may also be important during Mn assembly and indeed overreduced forms of the Mn cluster appear to be oxidised by TyrD. Second, the proton generated by the TyrD radical is thought to remain in its vicinity having an electrostatic influence on the location and potential of the chlorophyll cation, P+. This effect may be important for the kinetics of TyrZ oxidation and may provide a significant thermodynamic boost to the enzyme. In addition, through its electrostatic influence, TyrD(H+) may confine the highly oxidising cation P+ to the chlorophyll nearest to TyrZ, thereby accelerating TyrZ oxidation and restricting the potentially damaging redox chemistry to one side of the reaction centre: the disposable D1 side. This second role, evidence for which is beginning to emerge, constitutes a new role for a redox-active tyrosine in biology: as a positive charge generator in a hydrophobic environment. In this short review, we focus on work relevant to these two roles.
Keywords: Photosystem II; Tyrosine D; Redox reaction; Tyrosyl radical; G.T. Babcock;

Protein dynamics and reactions of Photosystem II by Anders Ehrenberg (231-234).
The kinetics of charge recombination by electron transfer from QA •− to P680 •+ on the reducing branch of PSII is likely to be strongly dependent on protein dynamics, in analogy with the kinetics of the corresponding reaction in the reaction center of purple bacteria [Biophys. J. 74 (1998) 2567]. On the oxidizing branch of PSII, the kinetics of electron hole transfer from P680 •+ to YZ is known to be multiexponential. This transfer is in the Babcock model of the reactions of the water-oxidizing complex coupled with proton transfer from YZ. The proton is via switching hydrogen bonds in the protein transferred to the thylakoid lumen. The demand for successive proton transfers requires rearrangement of the hydrogen bonds, which in turn requires a flexible protein making fluctuating excursions among all its conformations. In the equilibrated protein, only a fractional part of the molecules is in a conformation that is able to support the proton transfer from YZ. The kinetics of the rearrangement to this active conformation will be multiexponential and dependent on the distribution among all conformations, which is likely to be sensitive to various influences, in particular from changes in the protein coordination to the (Mn)4 cluster between the different S states.
Keywords: Photosystem II; Reaction kinetics; Protein dynamics; Protein conformation;

Keywords: G.T. (Jerry) Babcock; Cytochrome oxidase; Spectroscopy;

Since its discovery [Nature 266 (1977) 271] [1], the function of cytochrome c oxidase (and other haem-copper oxidases) as a redox-driven proton pump has been subject of both intense research and controversy, and is one of the key unsolved issues of bioenergetics and of biochemistry more generally. Despite the fact that the mechanism of proton translocation is not yet fully understood on the molecular level, many important details and principles have been learned. In the hope of accelerating progress, some of these will be reviewed here, together with a brief presentation of a novel proton pump mechanism, and of the emergence of a molecular basis for control of its efficiency.
Keywords: Haem-copper oxidase; Proton pump; Chemiosmotic theory;

The use of stable isotopes and spectroscopy to investigate the energy transducing function of cytochrome c oxidase by Bryan Schmidt; Warwick Hillier; John McCracken; Shelagh Ferguson-Miller (248-255).
We have used EPR and FTIR spectroscopy in combination with 17O and 15N stable isotopes to investigate the mechanism of cytochrome c oxidase (CcO). A high-spin state of heme a 3 was found in high yield by EPR, achieved upon turning over the enzyme until it was anaerobic, and shown to be a mixture of heme with a coordinated oxygen-based ligand and five-coordinate heme. Allowing the enzyme to consume 17O2 for a few milliseconds before freezing, we also showed that the product H2 17O exits toward the external side of the enzyme, binding to the nonredox active Mg/Mn site en route.Specific 15N labeling of histidine, in comparison with global 15N labeling and unlabeled samples, allowed us to more definitively assign heme and histidine peaks in the electrochemically induced FTIR difference spectrum. Additionally, the assignment of heme bands affords a reliable method of spectrum normalization between samples, providing a more accurate comparison of the spectral features of bovine with bacterial cytochrome oxidase and revealing multiple differences between the two species.
Keywords: Cytochrome aa 3; EPR; FTIR; Stable isotope; Water; Heme;

The cytochrome oxidase family of heme-copper oxidases has been the subject of intense kinetic and mechanistic enquiry. Much of this work has focussed on transient kinetic studies of the partial reactions of the enzyme with the goal being to build a kinetic model describing the catalytic cycle that the enzyme undergoes to direct the oxidation of substrate, reduction of oxygen and vectorial proton transfer. A key aspect of such a model is to define the structures of each of the intermediate forms the enzyme takes up as it traverses the catalytic cycle. One complication that has been prevalent with mitochondrial cytochrome c oxidase is the existence of structural variants of the enzyme, as isolated, that may not be participants in catalysis. Studies of structurally simpler procaryotic members of the family may offer new insight on the intermediates of catalysis. In this paper transient-state and steady-state kinetic studies of cytochrome aa 3-600 from Bacillus subtilis are integrated into a model of the catalytic cycle. This model specifies that the P intermediate accumulates in the steady-state and it is proposed that the step following its formation is limited by proton uptake.
Keywords: Cytochrome oxidase; Transient kinetics; Steady state kinetics; Oxygen reaction;

Time-resolved optical absorption studies of cytochrome oxidase dynamics by Ólöf Einarsdóttir; Istvan Szundi (263-273).
Time-resolved spectroscopic studies in our laboratory of bovine heart cytochrome c oxidase dynamics are summarized. Intramolecular electron transfer was investigated upon photolysis of CO from the mixed-valence enzyme, by pulse radiolysis, and upon light-induced electron injection into the cytochrome c/cytochrome oxidase complex from a novel photoactivatable dye. The reduction of dioxygen to water was monitored by a gated multichannel analyzer using the CO flow-flash method or a synthetic caged dioxygen carrier. The pH dependence of the intermediate spectra suggests a mechanism of dioxygen reduction more complex than the conventional unidirectional sequential scheme. A branched model is proposed, in which one branch produces the P form and the other branch the F form. The rate of exchange between the two branches is pH-dependent. A cross-linked histidine–phenol was synthesized and characterized to explore the role of the cross-linked His–Tyr cofactor in the function of the enzyme. Time-resolved optical absorption spectra, EPR and FTIR spectra of the compound generated after UV photolysis indicated the presence of a radical residing primarily on the phenoxyl ring. The relevance of these results to cytochrome oxidase function is discussed.
Keywords: Flow-flash; Branched mechanism; Oxygen carrier; Tyrosyl radical;

Interaction of cytochrome c with cytochrome oxidase: two different docking scenarios by Oliver Maneg; Francesco Malatesta; Bernd Ludwig; Viktoria Drosou (274-281).
Cytochrome c is the specific and efficient electron transfer mediator between the two last redox complexes of the mitochondrial respiratory chain. Its interaction with both partner proteins, namely cytochrome c 1 (of complex III) and the hydrophilic CuA domain (of subunit II of oxidase), is transient, and known to be guided mainly by electrostatic interactions, with a set of acidic residues on the presumed docking site on the CuA domain surface and a complementary region of opposite charges exposed on cytochrome c. Information from recent structure determinations of oxidases from both mitochondria and bacteria, site-directed mutagenesis approaches, kinetic data obtained from the analysis of isolated soluble modules of interacting redox partners, and computational approaches have yielded new insights into the docking and electron transfer mechanisms. Here, we summarize and discuss recent results obtained from bacterial cytochrome c oxidases from both Paracoccus denitrificans, in which the primary electrostatic encounter most closely matches the mitochondrial situation, and the Thermus thermophilus ba 3 oxidase in which docking and electron transfer is predominantly based on hydrophobic interactions.
Keywords: Electrostatic interaction; Docking complex; Electron transfer; CuA center; Paracoccus denitrificans; Thermus thermophilus;

The UV properties of key oxygen intermediates of cytochrome c oxidase have been investigated by transient absorption spectroscopy. The temporal behavior of P m species upon aerobic incubation with CO or in the reaction with H2O2 is closely concurred by a new optical shift at 290/260 nm. In the acid-induced conversion of P m to F , it is replaced by another shift at 323/288 nm. The wavelength and intensity of the UV signal observed in F match closely the properties of model Trp• in agreement with results of ENDOR studies on this species. The UV spectrum of Tyr• gives the closest match with the 290/260 nm signal observed in P m . On the basis of analysis of possible UV chromophores in CcO and similarity to Tyr• , the 290/260 nm signal is proposed to originate from the H240-Y244 site. Possible effects of local environment on UV properties of this site are discussed.
Keywords: Cytochrome oxidase; Reactive intermediate; Tyrosine radical; Tryptophan radical; Optical absorption;

Reduced cytochrome c oxidase binds molecular oxygen, yielding an oxygenated intermediate first (Oxy) and then converts it to water via the reaction intermediates of P, F, and O in the order of appearance. We have determined the iron–oxygen stretching frequencies for all the intermediates by using time-resolved resonance Raman spectroscopy. The bound dioxygen in Oxy does not form a bridged structure with CuB and the rate of the reaction from Oxy to P (PR) is slower at higher pH in the pH range between 6.8 and 8.0. It was established that the P intermediate has an oxo-heme and definitely not the Fe a 3 –O–O–CuB peroxy bridged structure. The Fe a 3 O stretching (νFeO) frequency of the PR intermediate, 804/764 cm−1 for 16O/18O, is distinctly higher than that of F intermediate, 785/750 cm−1. The rate of reaction from P to F in D2O solution is evidently slower than that in H2O solution, implicating the coupling of the electron transfer with vector proton transfer in this process. The P intermediate (607-nm form) generated in the reaction of oxidized enzyme with H2O2 gave the νFeO band at 803/769 cm−1 for H2 16O2/H2 18O2 and the simultaneously measured absorption spectrum exhibited the difference peak at 607 nm. Reaction of the mixed valence CO adduct with O2 provided the P intermediate (PM) giving rise to an absorption peak at 607 nm and the νFeO bands at 804/768 cm−1. Thus, three kinds of P intermediates are considered to have the same oxo-heme a 3 structure. The ν4 and ν2 modes of heme a 3 of the P intermediate were identified at 1377 and 1591 cm−1, respectively. The Raman excitation profiles of the νFeO bands were different between P and F. These observations may mean the formation of a π cation radical of porphyrin macrocycle in P.
Keywords: Cytochrome c oxidase; Oxygen activation; P intermediate; Proton pump; Resonance Raman;

Implications of ligand binding studies for the catalytic mechanism of cytochrome c oxidase by Marian Fabian; Ludovit Skultety; Daniel Jancura; Graham Palmer (298-305).
The reaction of oxidized bovine heart cytochrome c oxidase (CcO) with one equivalent of hydrogen peroxide results in the formation of two spectrally distinct species. The yield of these two forms is controlled by the ionization of a group with a pK a of 6.6. At basic pH, where this group is deprotonated, an intermediate called P dominates (P, because it was initially believed to be a peroxy compound). At acidic pH where the group is protonated, a different species, called F (ferryl intermediate) is obtained. We previously proposed that the only difference between these two species is the presence of one proton in the catalytic center of F that is absent in P. It is now suggested that the catalytic center of this F form has the same redox and protonation state as a second ferryl intermediate produced at basic pH by two equivalents of hydrogen peroxide; the role of the second equivalent of H2O2 is that of a proton donor in the conversion of P to F.Two chloride-binding sites have been detected in oxidized CcO. One site is located at the binuclear center; the second site was identified from the sensitivity of g=3 signal of cytochrome a to chloride in the EPR spectra of oxidized CcO. Turnover of CcO releases chloride from the catalytic center into the medium probably by one of the hydrophobic channels, proposed for oxygen access, with an orientation parallel to the membrane plane.Chloride in the binuclear center is most likely not involved in CcO catalysis. The influence of the second chloride site upon several reactions of CcO has been assessed. No correlation was found between chloride binding to the second site and the reactions that were examined.
Keywords: Cytochrome c oxidase; Hydrogen peroxide; Chloride interaction;

Active site structure of the aa 3 quinol oxidase of Acidianus ambivalens by Tapan Kanti Das; Cláudio M. Gomes; Tiago M. Bandeiras; Manuela M. Pereira; Miguel Teixeira; Denis L. Rousseau (306-320).
The membrane bound aa 3-type quinol:oxygen oxidoreductase from the hyperthermophilic archaeon, Acidianus ambivalens, which thrives at a pH of 2.5 and a temperature of 80 °C, has several unique structural and functional features as compared to the other members of the heme–copper oxygen reductase superfamily, but shares the common redox-coupled, proton-pumping function. To better understand the properties of the heme a 3–CuB catalytic site, a resonance Raman spectroscopic study of the enzyme under a variety of conditions and in the presence of various ligands was carried out. Assignments of several heme vibrational modes as well as iron–ligand stretching modes are made to serve as a basis for comparing the structure of the enzyme to that of other oxygen reductases. The CO-bound oxidase has conformations that are similar to those of other oxygen reductases. However, the addition of CO to the resting enzyme does not generate a mixed valence species as in the bovine aa 3 enzyme. The cyanide complex of the oxidized enzyme of A. ambivalens does not display the high stability of its bovine counterpart, and a redox titration demonstrates that there is an extensive heme–heme interaction reflected in the midpoint potentials of the cyanide adduct. The A. ambivalens oxygen reductase is very stable under acidic conditions, but it undergoes an earlier alkaline transition than the bovine enzyme. The A. ambivalens enzyme exhibits a redox-linked reversible conformational transition in the heme a 3–CuB center. The pH dependence and H/D exchange demonstrate that the conformational transition is associated with proton movements involving a group or groups with a pK a of ∼3.8. The observed reversibility and involvement of protons in the redox-coupled conformational transition support the proton translocation model presented earlier. The implications of such conformational changes are discussed in relation to general redox-coupled proton pumping mechanisms in the heme–copper oxygen reductases.
Keywords: Raman spectroscopy; Bioenergetics; Midpoint potential; Heme proteins; Proton translocation;

FTIR studies of internal proton transfer reactions linked to inter-heme electron transfer in bovine cytochrome c oxidase by Benjamin H. McMahon; Marian Fabian; Farol Tomson; Timothy P. Causgrove; James A. Bailey; Francisca N. Rein; R.Brian Dyer; Graham Palmer; Robert B. Gennis; William H. Woodruff (321-331).
FTIR difference spectroscopy is used to reveal changes in the internal structure and amino acid protonation states of bovine cytochrome c oxidase (CcO) that occur upon photolysis of the CO adduct of the two-electron reduced (mixed valence, MV) and four-electron reduced (fully reduced, FR) forms of the enzyme. FTIR difference spectra were obtained in D2O (pH 6–9.3) between the MV-CO adduct (heme a3 and CuB reduced; heme a and CuA oxidized) and a photostationary state in which the MV-CO enzyme is photodissociated under constant illumination. In the photostationary state, part of the enzyme population has heme a3 oxidized and heme a reduced. In MV-CO, the frequency of the stretch mode of CO bound to ferrous heme a3 decreases from 1965.3 cm−1 at pH* ≤7 to 1963.7 cm−1 at pH* 9.3. In the CO adduct of the fully reduced enzyme (FR-CO), the CO stretching frequency is observed at 1963.46±0.05 cm−1, independent of pH. This indicates that in MV-CO there is a group proximal to heme a that deprotonates with a pK a of about 8.3, but that remains protonated over the entire pH* range 6–9.3 in FR-CO. The pK a of this group is therefore strongly coupled to the redox state of heme a. Following photodissociation of CO from heme a3 in MV oxidases, the extent of electron transfer from heme a3 to heme a shows a pH-dependent phase between pH 7 and 9, and a pH-independent phase at all pH's. The FTIR difference spectrum resulting from photolysis of MV-CO exhibits vibrational features of the protein backbone and side chains associated with (1) the loss of CO by the a3 heme in the absence of electron transfer, (2) the pH-independent phase of the electron transfer, and (3) the pH-dependent phase of the electron transfer. Many infrared features change intensity or frequency during both electron transfer phases and thus appear as positive or negative features in the difference spectra. In particular, a negative band at 1735 cm−1 and a positive band at 1412 cm−1 are consistent with the deprotonation of the acidic residue E242. Positive features at 1552 and 1661 cm−1 are due to amide backbone modes. Other positive and negative features between 1600 and 1700 cm−1 are consistent with redox-induced shifts in heme formyl vibrations, and the redox-linked protonation of an arginine residue, accompanying electron transfer from heme a3 to heme a. An arginine could be the residue responsible for the pH-dependent shift in the carbonyl frequency of MV-CO. Specific possibilities as to the functional significance of these observations are discussed.
Keywords: FTIR; Cytochrome oxidase; Glutamate; Mixed-valence;

Although subunit III of cytochrome c oxidase is part of the catalytic core of the enzyme, its function has remained enigmatic. Comparison of the wild-type oxidase and forms lacking subunit III shows that the presence of subunit III maintains rapid proton uptake into the D pathway at the pH of the bacterial cytoplasm or mitochondrial matrix, apparently by contributing to the protein environment of D132, the initial proton acceptor of the D pathway. Subunit III also appears to contribute to the conformation of the normal proton exit pathway, allowing this pathway to take up protons from the outer surface of the oxidase in the presence of ΔΨ and ΔpH. Subunit III prevents turnover-induced inactivation of the oxidase (suicide inactivation) and the subsequent loss of CuB from the active site. This function of subunit III appears partly related to its ability to maintain rapid proton flow to the active site, thereby shortening the lifetime of reactive O2 reduction intermediates. Analysis of proton pumping by subunit III-depleted oxidase forms leads to the proposal that the trapping of two protons in the D pathway, one on E286 and one on D132, is required for efficient proton pumping.
Keywords: Cytochrome oxidase; Proton pathway; Proton pumping; Suicide inactivation;

Haem-copper oxygen reductases are the widest spread enzymes involved in aerobic respiratory chains, in Eukarya, Bacteria and Archaea. However, both the catalytic mechanism for oxygen reduction and its coupling to proton translocation remain to be fully understood. In this article we analyse the experimental data gathered in recent years for haem-copper reductases presenting features distinct from the mitochondrial-like enzymes. These features further support the classification of several families of haem-copper oxygen reductases based on their proton pathways and previously proposed by us [Biochim. Biophys. Acta 1505 (2001) 185], and allow to identify the minimal essential elements for these enzymes.
Keywords: Cytochrome oxidase; Proton channel; Binuclear site;

Time-resolved step-scan Fourier transform infrared investigation of heme-copper oxidases: implications for O2 input and H2O/H+ output channels by Constantinos Koutsoupakis; Eftychia Pinakoulaki; Stavros Stavrakis; Vangelis Daskalakis; Constantinos Varotsis (347-352).
We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS2-FTIR) spectroscopy to investigate the dynamics of the heme-CuB binuclear center and the protein dynamics of mammalian aa 3, Pseudomonas stutzeri cbb 3, and caa 3 and ba 3 from Thermus thermophilus cytochrome oxidases. The implications of these results with respect to (1) the molecular motions that are general to the photodynamics of the binuclear center in heme-copper oxidases, and (2) the proton pathways located in the ring A propionate of heme a 3-Asp372-H2O site that is conserved among all structurally known oxidases are discussed.

A cooperative model for proton pumping in cytochrome c oxidase by Sergio Papa; Nazzareno Capitanio; Giuseppe Capitanio (353-364).
In this paper, the mechanism of proton pumping in cytochrome c oxidase is examined. Data on cooperative linkage of vectorial proton translocation to oxido-reduction of CuA and heme a in the CO-inhibited, liposome-reconstituted bovine cytochrome c oxidase are reviewed. Results on proton translocation associated to single-turnover oxido-reduction of the four metal centers in the unliganded, membrane-reconstituted oxidase are also presented. On the basis of these results, X-ray crystallographic structures and spectrometric data for a proton pumping model in cytochrome c oxidase is proposed.This model, which is specifically derived from data available for the bovine cytochrome c oxidase, is intended to illustrate the essential features of cooperative coupling of proton translocation at the low potential redox site. Variants will have to be introduced for those members of the heme copper oxidase family which differ in the redox components of the low potential site and in the amino acid network connected to this site.The model we present describes in detail steps of cooperative coupling of proton pumping at the low potential CuA-heme a site in the bovine enzyme. It is then outlined how this cooperative proton transfer can be thermodynamically and kinetically coupled to the chemistry of oxygen reduction to water at the high potential CuB-heme a 3 center, so as to result in proton pumping, in the turning-over enzyme, against a transmembrane electrochemical proton gradient of some 250 mV.
Keywords: Cytochrome c oxidase; Proton pumping; Cooperativity; Mitochondria;

Control of cytochrome c oxidase activity by nitric oxide by Maurizio Brunori; Alessandro Giuffrè; Elena Forte; Daniela Mastronicola; Maria Cecilia Barone; Paolo Sarti (365-371).
Over the past decade it was discovered that, over-and-above multiple regulatory functions, nitric oxide (NO) is responsible for the modulation of cell respiration by inhibiting cytochrome c oxidase (CcOX). As assessed at different integration levels (from the purified enzyme in detergent solution to intact cells), CcOX can react with NO following two alternative reaction pathways, both leading to an effective, fully reversible inhibition of respiration. A crucial finding is that the rate of electron flux through the respiratory chain controls the mechanism of inhibition by NO, leading to either a “nitrosyl” or a “nitrite” derivative. The two mechanisms can be discriminated on the basis of the differential photosensitivity of the inhibited state. Of relevance to cell pathophysiology, the pathway involving the nitrite derivative leads to oxidative degradation of NO, thereby protecting the cell from NO toxicity. The aim of this work is to review the information available on these two mechanisms of inhibition of respiration.
Keywords: Free radical; Signaling; Respiration; NO scavenging; Mitochondria; Hemeprotein;

It has been reported that different amino acid radicals are formed following the addition of hydrogen peroxide to cytochrome c oxidase (CcO) from bovine heart or from Paracoccus denitrificans. A broad unresolved signal in the electron paramagnetic resonance (EPR) spectra of bovine CcO has been assigned to a tryptophan radical, probably Trp126 [Rigby et al. Biochemistry 2000, 39, 5921–5928]. In the P. denitrificans enzyme, a similarly broad signal but with a well-resolved hyperfine structure was shown to originate from a tyrosyl radical and was tentatively assigned to the active site Tyr280 [MacMillan et al. Biochemistry 1999, 38, 9179–9184]. We confirm that the EPR signal from P. denitrificans CcO can be simulated using spectral parameters typical for known Tyr radicals in other systems. However, the rotational conformation of the phenolic ring of Tyr280 is inconsistent with our simulation. Instead, the simulation parameters we used correspond to the rotational conformation of ring that matches very accurately the conformation found in Tyr167, a residue that is close enough (∼10 Å) to the binuclear centre to readily donate an electron. The broad unresolved EPR signal in the bovine oxidase has been thought previously to be inconsistent with a tyrosyl radical. However, we have simulated a hypothetical EPR spectrum arising from a Tyr129 radical (the equivalent of Tyr167 in P. denitrificans CcO) and showed that it is similar to the observed broad signal. The possibility exists, therefore, that the homological tyrosine amino acid (Tyr167/Tyr129) is responsible for the EPR spectrum in both the Paraccoccus and the bovine enzyme. This correspondence between the two enzymes at least allows the possibility that this radical may have functional importance.
Keywords: Tyrosine; Tryptophan; Free radical; Electron transfer; Cytochrome c oxidase; Hydrogen peroxide; EPR spectroscopy;

In the aerobic steady state of the classical eukaryotic cytochrome c oxidase, three aa 3 redox metal centres (cytochrome a, CuA and CuB) are partially reduced while the fourth, cytochrome a 3, remains almost fully oxidized. Turnover depends primarily upon the rate of cytochrome a 3 reduction. When prokaryotic cytochrome c-552 oxidase (ba 3) of Thermus thermophilus turns over, three different metal centres (cytochromes b, a 3 and CuA) share the steady state electrons; it is the fourth, CuB, that apparently remains almost fully oxidized until anaerobiosis. Cytochrome a 3 stays partially reduced during turnover and a possible P/F state may also be populated. Cyanide traps the aerobic ba 3 CuB centre in the a 3 2+CNCuB2+ state; the corresponding eukaryotic cyanide trapped state is a 3 3+CNCuB+. Both states become the fully reduced a 3 2+CNCuB+ upon anaerobiosis.The different reactivities of the aa 3 and ba 3 binuclear centres may be correlated with the very different proximal histidine εN-Fe distances in the two enzymes (3.3 Å for ba 3 compared to 1.9 Å for aa 3) which may in turn relate to the functioning of thermophilic Thermus cytochrome ba 3 in vivo at a very elevated temperature. But the differences may also just exemplify how evolution can find surprisingly different solutions to the common problem of electron transfer to oxygen. Some of these alternatives were potentially enshrined in a model of the oxidase reaction already adopted by Gerry Babcock in the early 1990s.

The bacterial cytochrome cbb 3 oxidases by Robert S. Pitcher; Nicholas J. Watmough (388-399).
Cytochrome cbb 3 oxidases are found almost exclusively in Proteobacteria, and represent a distinctive class of proton-pumping respiratory heme-copper oxidases (HCO) that lack many of the key structural features that contribute to the reaction cycle of the intensely studied mitochondrial cytochrome c oxidase (CcO). Expression of cytochrome cbb 3 oxidase allows human pathogens to colonise anoxic tissues and agronomically important diazotrophs to sustain N2 fixation. We review recent progress in the biochemical characterisation of these distinctive oxidases that lays the foundation for understanding the basis of their proposed high affinity for oxygen, an apparent degeneracy in their electron input pathways and whether or not they acquired the ability to pump protons independently of other HCOs.
Keywords: Microaerobic metabolism; Cytochrome cbb 3 oxidase; Heme-copper oxidase; Pseudomonas;

The possible role of cytochrome c oxidase in stress-induced apoptosis and degenerative diseases by Bernhard Kadenbach; Susanne Arnold; Icksoo Lee; Maik Hüttemann (400-408).
Apoptotic cell death can occur by two different pathways. Type 1 is initiated by the activation of death receptors (Fas, TNF-receptor-family) on the plasma membrane followed by activation of caspase 8. Type 2 involves changes in mitochondrial integrity initiated by various effectors like Ca2+, reactive oxygen species (ROS), Bax, or ceramide, leading to the release of cytochrome c and activation of caspase 9. The release of cytochrome c is followed by a decrease of the mitochondrial membrane potential ΔΨ m. Recent publications have demonstrated, however, that induction of apoptosis by various effectors involves primarily a transient increase of ΔΨ m for unknown reason. Here we propose a new mechanism for the increased ΔΨ m based on experiments on the allosteric ATP-inhibition of cytochrome c oxidase at high matrix ATP/ADP ratios, which was concluded to maintain low levels of ΔΨ m in vivo under relaxed conditions. This regulatory mechanism is based on the potential-dependency of the ATP synthase, which has maximal activity at ΔΨ m=100–120 mV. The mechanism is turned off either through calcium-activated dephosphorylation of cytochrome c oxidase or by 3,5-diiodo-l-thyronine, palmitate, and probably other so far unknown effectors. Consequently, energy metabolism changes to an excited state. We propose that this change causes an increase in ΔΨ m, a condition for the formation of ROS and induction of apoptosis.
Keywords: Mitochondrial membrane potential hyperpolarization; Cytochrome c oxidase; Apoptosis; 3,5-diiodo-l-thyronine; Palmitate; Calcium-activated protein dephosphorylation; cAMP-dependent phosphorylation;