Encyclopedia of Life Sciences || Cytochrome c Oxidase

Cytochrome c OxidaseMarten Wikstrom, University of Helsinki, Helsinki, Finland

Cytochrome c oxidase is the key enzyme of cell respiration

in all eukaryotes and many prokaryotes. The cytochrome c

oxidases belong to the haem–copper superfamily of

structurally and functionally related enzymes; though

related in structure, some bacterial variants lack amino

acid residues that are known to be obligatory for the

function of the members of the main family. All haem–

copper oxidases have a unique bimetallic active site

catalysing reduction of dioxygen (O2) to water and an

adjacent second haem group that donates electrons to

this site. Here, the mechanism of O2 reduction is reviewed.

The membrane-bound enzyme couples this reaction to

translocation of protons across the membrane, and thus

functions as a primary energy transducer that contributes

to the formation of ATP (adenosine triphosphate) in aer-

obic life. The most recent knowledge of the function of

this ‘proton pump’ is discussed. It is concluded that cyto-

chrome c oxidase is an electrostatic energy-transducing

machine with high efficiency.

Introduction

Cytochrome coxidase is the key enzymeof cell respiration inall eukaryotes and many prokaryotes. The cytochrome coxidases actually belong to a superfamily of structurally andfunctionally related enzymes (the haem–copper oxidases),which also includes the quinol oxidases and cytochromecbb3-type oxidases found only in prokaryotes. In bacteria,they reside in the cell membrane, whereas in eukaryotic cellsthey are located in the inner mitochondrial membrane. The

genes for hundreds of members of this family have beensequenced (Hemp and Gennis, 2008). These enzymes typi-cally constitute the terminusof the ‘respiratory chain’which,as a whole, catalyses the flux of reducing equivalents fromfoodstuffs that have been oxidised in earlier stages ofcatabolism (e.g. the citric acid cycle) to molecular oxygen,reducing it to water. The haem–copper oxidases areresponsible for the final chemistry of this chain of events, vizthe binding, activation and reduction of the oxygen mol-ecule, as well as for the primary conservation of the freeenergy released in this reaction. See also: Mitochondria:Origin; Oxidative PhosphorylationCatabolism is designed to extract and conserve free

energy from the oxidation of foodstuffs, which is the keyfunction of the respiratory chains. Free energy from theoxidoreduction reactions in the chain is eventually con-served in the form of adenosine triphosphate (ATP). Thisfunction of respiratory chain protein complexes, includingcytochrome c oxidase, is intimately linked to their commonproperty of being membrane proteins. The respiratorychain complexes have the unique ability to convert theenergy released in the redox reactions into a transmem-brane electrochemical proton gradient DmHþð Þ, which cansubsequently be utilised as the driving force for ATP syn-thesis from adenosine diphosphate (ADP) and inorganicphosphate by another protein complex in the same mem-brane – the ATP synthase. The linkage of oxidoreductionreactions and ATP synthesis via an intermediary electro-chemical proton gradient is the essence of the che-miosmotic theory of Peter Mitchell (1920–1992; Mitchell,1979), which earned him the Nobel Prize in chemistry in1978. See also: Adenosine Triphosphate; Cell Biophysics;Cellular Thermodynamics; Mitchell, Peter DennisEvolution of cytochrome c oxidase and the other haem–

copper oxidases enables catabolism to utilise dioxygen (O2)as the terminal oxidant of foodstuffs. By this, biology cameto utilise a thermodynamically highly efficient oxidant,which owing to the vastly increased efficiency in the com-bustion of food led to more competitive and efficient aer-obic organisms. This is assumed to have occurred � 2500million years ago when the oxygen pressure of the Earth’satmosphere rose dramatically. However, there are someindications that prototypes of haem–copper oxidasesmay, in fact, have evolved even earlier – perhaps in more

Advanced article

Article Contents

. Introduction

. Structure of a Transmembrane Protein

. Electron Transfer and Binuclear Centres

. Reduction of Molecular Oxygen

. Binding and Kinetic Trapping of Dioxygen

. Coupled Proton Translocation

Online posting date: 19th May 2010

ELS subject area: Biochemistry

How to cite:Wikstrom, Marten (May 2010) Cytochrome c Oxidase. In: Encyclopediaof Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester.

DOI: 10.1002/9780470015902.a0000649.pub2

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http://dx.doi.org/10.1038/npg.els.0001381







isolated spots on this planet with a higher local oxygenconcentration.

The structure, function and evolution of cytochrome coxidase provide several biological issues of general interest,many of which will be reviewed here.

Structure of a Transmembrane Protein

Membrane proteins have turned out to be notoriouslydifficult to crystallise, probably owing mainly to the scar-city of extramembranous aqueous domains tomake crystalcontacts. This proved to be the case for cytochrome c oxi-dase from the soil bacterium Paracoccus denitrificans,which was eventually co-crystallised with the aid of aconformation-specific Fv antibody fragment directed at asubunit domain on the membrane surface, and the crystalstructure was solved at atomic resolution (Iwata et al.,1995).At about the same time,Tsukihara et al. (1995, 1996)succeeded in crystallising and solving the structure of thecytochrome c oxidase from bovine heart mitochondria.In this case, no extra ‘aid’ was required, except for thenecessity to use a detergent with a precise alkyl chainlength. Owing to the large number of nuclear-coded ‘extra’subunits, the mitochondrial enzyme has much largerhydrophilic domains than the simpler bacterial enzyme,which has only four subunits. Subsequently, the X-raystructures of cytochrome c oxidase from three other haem–copper oxidases have been published (Soulimane et al.,2000; Qin et al., 2006; Abramson et al., 2000). See also:Membrane Proteins

All haem–copper oxidases (except the least related cbb3enzymes) have in common anearly identical ‘catalytic core’consisting of three polypeptide subunits (I, II and III),which in the case of the mitochondrial enzyme are encodedin the mitochondrial deoxyribonucleic acid (mtDNA) andsynthesised on mitochondrial ribosomes. Subunits I and IIof the cytochrome coxidases contain all the redox cofactorsrequired for catalysis, whereas the role of subunit III is stillobscure (described in the following section). The functionof the fourth very small subunit of the bacterial enzymes isalso not known and shows no resemblance to subunits ofother oxidases. The mitochondrial cytochrome c oxidasestypically contain a large number of ‘extra’ subunits, 10 forthe mammalian enzymes that are all encoded in thenucleus, synthesised in the cytoplasm and then importedinto the mitochondrion. Isoforms of some of them havebeen described in different tissues, and they have beenimplicated as regulatory. See also: Coenzymes: Haem;Haem Structure and Function; Proteins: FundamentalChemical Properties

Subunit I

The largest subunit traverses the membrane 12 times bymeans of transmembrane helices that are connected to oneanother on either side of the membrane by hydrophilicloops. These helices are often tilted considerably from

perpendicularity with the membrane plane. Viewed fromoutside of the membrane (the extracellular face of thebacterial plasmamembrane or the extramitochondrial faceof the inner mitochondrial membrane), the helices arearranged (from N- to C-terminal of the polypeptide) incounterclockwise order as three semicircular arcs with fourhelices each, of which the last one (i.e. helices II, VI and X)has a key structural role in each case (Figure 1a). Thus, three‘channels’ are formed (A, B and C) perpendicular to themembrane and surrounded by helices III–VI, VII–X andXI–XII, respectively. The B and C channels are structur-ally blocked by the redox-active groups in this subunit, vizthe binuclear haem–copper centre and the low-spin haem,respectively, whereas the C channel is open. However, thelong hydroxyethyl-farnesyl side-chain of haem A is bentout of channel B, allowing access to the binuclear site fromthe inner side of the membrane (Figure 1a). Subunit I is themost important structure of the enzyme, and provides thebinuclear haem–copper site at which the binding andreduction of oxygen occurs.

Subunit II

This polypeptide has only two transmembrane helicesnear its C-terminal, and the rest forms a large ‘cap’ ofb-structure on the outer side of themembrane on top of thedomain of subunit I that contains the haem groups (Figure

1b). In the cytochrome c oxidases, the b domain of subunitII contains a unique copper centre, called CuA, where twocopper ions are ligated by two cysteine sulfurs (Cys196 andCys200), two histidine nitrogens (His161 and His204)and at a somewhat longer distance, a methionine sulfur(Met207) and by the backbone oxygen of a glutamic acidresidue (Glu198), respectively. (All amino acid residues arenumbered here on the basis of the numbering for cyto-chrome c oxidase from bovine heart mitochondria. Thecommon three-letter code is used for the amino acids.) Thecrystal structure of this CuA-binding domain has beensolved separately at high resolution. The related quinoloxidases have a homologous subunit II but the CuA centreand the copper ligands are all missing (Abramson et al.,2000) See also: Coenzymes and Cofactors

Subunit III

In the cytochrome c oxidases, this highly hydrophobicsubunit consists of seven transmembrane helices that arearranged as a ‘V’ with two helices in one and five in theother leg (Figure 1c and d). The ‘V’ widens towards the outerside of the membrane and the headgroup of at least onephospholipid molecule is tightly associated to its tip nearthe membrane interface on the inner side. The function ofsubunit III is not known, but a role as a store of O2 orconstituting part of an O2 import pathway from thehydrophobic core of the membrane into the enzyme’sactive site has been proposed. Part of subunit III contrib-utes to the entrance of the proton-conducting D channel

Cytochrome c Oxidase

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(Gilderson et al., 2003; described in the following section).See also: Hydrophobic Effect; Hydrophobicity Plots

Electron Transfer and BinuclearCentres

In solution, the chemical reaction catalysed by cytochromec oxidase may be written as in [I].

4cyt � c2þ þ 4Hþ þO2 ! 4cyt � c3þ þ 2H2O ½I�

This is a highly exergonic reaction with a free energychange (DG) of �2192 kJmol21 (246 kcalmol21), muchofwhich is conserved as an electrochemical protongradientDmHþð Þ across the membrane (described in the followingtext). It is especially noteworthy that the chemical reaction[I] requires transfer of both electrons and protons to O2 tomake possible its reduction to two water molecules, and

that in the membrane they are transferred from oppositesides. This arrangement of the chemistry (Figure 2a) willtherefore result in the translocation of four electricalcharge equivalents across the membrane per O2 moleculereduced. See also: Electron Carriers: Proteins and Cofac-tors in Oxidative Phosphorylation; Thermodynamics inBiochemistryThe electron donor, cytochrome c, is a peripheral water-

soluble haemoprotein located on the outer surface of themembrane and binds to a specific site in the b domain ofsubunit II. Cytochrome c donates one electron at a time tothe CuA centre, which is the primary electron acceptor of theenzyme. Despite its binuclear nature, the CuA centrecan only accept one electron at a time, shuttling betweenCu1+–Cu1+(reduced) andCu1.5+–Cu1.5+ (oxidised) states.Apparently, the energy of the fully oxidised Cu2+–Cu2+

state is too high to allow its formation. See also: Oxidation–Reduction ReactionsBoth haem groups, a and a3 (of type A) are located at

about the same depth in the membrane, approximately

Figure 1 (a) Stereo view of the transmembrane helix backbone arrangement of subunit I from the outside, perpendicular to the membrane.

Transmembrane helices are numbered I–XII, and they form ‘pores’ denoted A, B and C. Haems a and a3 are shown in pores C and B, respectively. (b) Stereo

view of the backbone of subunit II in the membrane plane, showing the binuclear CuA site and its relationship to the two haem groups in subunit I. (c) Top

view similar to that in (a) of the backbone of subunit III (highlighted) relative to subunit I with the two haem groups. (d) Side view of C along the membrane

plane. Notice the V shape with two transmembrane helices in one leg (left) and five in the other (right). These pictures are based on the crystal structure of

cytochrome c oxidase from bovine heart mitochondria (Tsukihara et al., 1996; Brookhaven protein data bank, accession number 1OCC).











one-third of the membrane thickness from the outermembrane surface. (Haem A is characterised by a longhydroxyethyl-farnesyl side-chain in position 2 and a formylgroup in position 8 of the haem ring. The two haem Agroups in the enzyme are termed haems a and a3, respect-ively, to distinguish their different modes of binding to theprotein, and for historical reasons.) The haem a group isligated axially by two histidines, one from helix II (His61)and the other from helix X (His378), and therefore haslow-spin character. The plane of haem a3 is orientatedalmost perpendicular to that of haem a and the haem edgesare almost at van der Waals distance from one another.The planes of both the haem groups are orientated al-most exactly perpendicularly to the membrane plane (seeFigure 1b). The haem a3 iron is ligated axially on the prox-imal side by a histidine from helix X (His376). The distalligation site, which will bind O2 and inhibitory ligands,such as carbon monoxide (CO), cyanide (CN2) andazide (N3

2), is either open or occupied by a water molecule(Figure 2b). Haem a3 forms a binuclear site of O2 reductiontogether with a close-lying copper ion, called CuB. Thecopper resides � 4–5 A from the haem iron on the distal

side and is ligated by three histidines, two adjacent ones inhelix VII (His290 andHis291) and one in helix VI (His240).A tyrosine residue (Tyr244) close to the CuB site is cova-lently bonded to one of the CuB histidine ligands (His240),an unusual posttranslational modification that appearsto be of great importance as it is conserved even in themost distant members of the haem–copper oxidases(Rauhamaki et al., 2006; Hemp et al., 2006). The cupricCuB has a fourth ligand, either water or an OH2 anion. Inthe oxidised enzyme, the high-spin haemFe(III) andCuB(II)metals of the O2-reduction site are tightly coupled mag-netically so that neither gives rise to electron paramagneticresonance (EPR) signals (van Gelder and Beinert, 1969).Upon reduction of the site at equilibrium conditions, CuBwill receive an electron first, by which this coupling is lostand a high-spin EPR signal becomes apparent from theferric haem a3.See also: Electron Paramagnetic Resonance(EPR) and Spin-labelling; Haem ProteinsElectron transfer from CuA to haem a is transmem-

branous owing to their respective locations relative to themembrane, and hence contributes to generation of electricmembrane potential (Figure 2a). The electron transfer

Distal histidineligandHis376

Haem a3

CuB

His291

His290

His240

Binding of O2

(b)

cyt.c

e–

CuA

H+

Output

Haem a

Haem a3

CuB

O214

H2O12

Input

Membranedomain

H+ H+(a)

Figure 2 (a) Schematic view of the functioning of cytochrome c oxidase as a generator of DmHþ . Protons consumed in the reduction of dioxygen to water

are shown in blue and protons translocated in red. Electron transfer is shown in light blue. (b) The binuclear O2-binding centre. The fourth water or

OH2 ligand of CuB, which has been identified by EXAFS and ENDOR spectroscopy, is not shown.





http://dx.doi.org/10.1002/9780470015902.a0003054

sequence is specific and, for example, precludes the transferof an electron fromCuA directly to haem a3. Subsequently,haem a transfers an electron to the haem a3–CuB centre.Electron transfer between the two haem groups has beenintensively discussed. The recent observation that the rateof this electron transfer is of the order of 1 ns21 (Pilet et al.,2004) is in excellent agreement with the rate predicted bythe empirical Moser–Dutton ‘ruler’ (Page et al., 1999).See also: Cell Membranes: Intracellular pH and Electro-chemical Potential; Membrane Potential

The kinetics of pure electron transfer between the twohaem groups and between the haems and the CuA centrecan be studied in the so-called mixed-valence enzymeblocked by CO. CO binds in place of O2 at the ferrous ironof haem a3, and the enzyme may be half-reduced by twoelectrons residing at haem a3 and CuB, respectively. TheFe–CO bond is photosensitive and may be broken by aflash of light. This leads to an increase of the energy (low-ering of midpoint potential) of the electrons in the binuc-lear site and hence to electron redistribution in the enzyme.In a first fast phase (t� 1 ns at room temperature; t is thefirst-order reaction time constant, equal to 1/k), electronsare redistributed from haem a3 to haem a, followed by asecond phase with t� 3 ms. In a third phase (t� 30ms)electrons in the two haem groups equilibrate with the CuAcentre. Such studies are rendered possible because thephotolysed COwill return only slowly to the haem iron. Infact, on photolysis it will first bind to CuB in a very fastreaction (t51 ps) and will then dissociate from the coppersite into the solution with t� 3ms, which is presumably thereaction that limits the rate of the second electron transferphase. The technique utilising CO can also be used suc-cessfully for studying the kinetics of O2 reduction by theenzyme, as will be explored further in the following section.

Reduction of Molecular Oxygen

The primary binding of O2 to a haem group (dasAtmungsferment) in cell respiration was postulated byOttoWarburg already early in the last century on the basisof the inhibitory effect of cyanide and the action spectrumof CO. In the 1930s, David Keilin identified this haemo-protein by optical spectroscopy and called it cytochromea3; subsequently Britton Chance showed that cytochromea3 is identical toAtmungsferment. The oxygen reaction wasthen explored kinetically by Gibson and Greenwood(1963), who introduced the so-called flow/flash technique,and by Chance et al. (1975), who first identified key oxygenintermediates by applying this technique to studies atcryogenic temperatures. In this approach, CO is first usedto bind ferrous haem a3 and thus to ‘protect’ the oxygen-binding site from O2. CO dissociates away from the haemiron only slowly in the absence of light, so that O2 can beadded using a flow apparatus. Next, the Fe–CO bond isphotolysed by an intense flash of light, which enables O2 tobind and the oxygen reduction reaction to start. Sub-sequently, the reaction can be followed by spectroscopic

techniques, of which optical and resonance Raman spec-troscopy have been the most widely applied and fromwhich much of our present knowledge originates. See also:ResonanceRaman Spectroscopy;Warburg, OttoHeinrich

Binding and Kinetic Trapping ofDioxygen

The ferrous/cuprous binuclear haem a3/CuB centre firstbinds a molecule of O2 (t� 8ms at room temperature and1mmolL21 O2), forming the ferrous haem-oxy inter-mediate (A) (Figure 3), which is structurally analogous tooxyhaemoglobin and which was first identified in low-temperature studies by Chance et al. (1975) and later atroom temperature by time-resolved resonance Raman andoptical spectroscopy. Interestingly, the dissociation con-stant of the oxy complex is quite high (KD� 0.25mmol L21

O2), so that O2 binding per se is weak, especially con-sidering the low O2 concentrations in many tissues. Thisstands in contrast to the very high operational affinity(KM,app) of cell respiration towards O2 (� 0.1 mmolL21).The reason for the high operational oxygen affinity, whichis of obvious importance for effective cell respiration, iskinetic trapping of the O2 molecule by very fast electrontransfer (keT) to the bound oxygen molecule subsequent toits binding (Chance et al., 1975; Verkhovsky et al., 1996).The trapping electron transfer is orders ofmagnitude fasterthan the overall turnover (TN) of the enzyme. The resultis an apparent Michaelis constant for O2 (KM,app) thatis offset from the O2 dissociation constant by the ratiobetween the turnover and the trapping electron transferrate (eqn [1]). See also: Haemoglobin: Cooperativity

Figure 3 States of the binuclear oxygen reduction site during the catalytic

cycle. The rectangle shows the active site with haem a3, CuB and the

cross-linked Tyr-244. The proximal histidine ligand of the haem and the

three histidine ligands of CuB are not shown for simplicity. Red arrows

indicate steps coupled to proton translocation. Black H+ indicates uptake of

a ‘substrate proton’ forming the equivalent of water.









in Protein–Ligand Interactions; Substrate Binding toEnzymes

KM;app�KD TN=keTð Þ ½1�

In thisway, cytochrome coxidase achieves the importanthigh operational affinity for O2 without an energy invest-ment by tight binding of the O2 molecule, which mayinstead be conserved for the cell’s energy-requiring pro-cesses (described in the following text).

Reduction of the bound O2 molecule

The oxygen-bound active site now undergoes fast changes,the nature of which are fairly well understood today(Figure 3). If no electron is available in the CuA or haem acentres, the oxy intermediate (Fe(II)–O=O CuB(I)) decaysspontaneously andmore slowly (t� 200ms) to a state calledPM (Figure 3) or ‘CompoundC’ (Chance et al., 1975), whichis relatively stable. In contrast to an originally envisaged‘peroxy’ structure, resonance Raman data from thelaboratory of TeizoKitagawa (Kitagawa andOgura, 1997)as well as other evidence (Fabian et al., 1999) have shownthat theO–Obond is broken in this state, one of the oxygenatoms being bound to the haem, which attains the ferrylstructure Fe(IV)=O22, the CuB is cupric (although notdetectable by EPR in this state; described in the followingtext), with scission of the O–O bond. As suggested by GTBabcock and others (Babcock, 1999), the missing electrondonor is probably the conserved tyrosine residue nearby(Tyr244) that is covalently bonded to the CuB ligandHis240 (described in the preceding text), forming a neutralradical. This view is supported by recent FTIR data(Gorbikova et al., 2008) which indicate that Tyr244 alsodonates the proton involved in scission of the O–O bond.

However, if an electron is available in the CuA or haem acentres, it is transferred to the oxygen-bound binuclear site(t� 30 ms) that now instead of PM forms the PR inter-mediate where the haem is again in the ferryl state. TheCuBsite is cupric, as evidenced from its unique EPR signal(Morgan et al., 2001), carrying the other oxygen atom as ahydroxide ligand. Hence, O2 is fully reduced by four elec-trons already at the stage of the P intermediates. PR has aunique optical spectrum, identical to that of PM, and con-sistent with the identical redox states of haem a3 andCuB inthese intermediates.

PR decays to the ferryl or F intermediate (t� 100–200ms), which probably has the structure Fe(IV)=O2– CuB(II)–OH2, and this step is associated with the netuptake of one proton. Note that this structure is similar tothe proposed structure for PR. The very different spectro-scopic properties of these two intermediates may be due tothe additional proton in the site in the case of F.Finally, theF intermediate receives another electron from haem a, andthe binuclear site is converted, to the O state, with thebinuclear site structure Fe(III) Cu(II) (Figure 3). This step isalso associated with net uptake of one proton. However,the exact position of this proton is not known, and the O

state is indeed heterogeneous. It is apparent that O of theactively functioning enzyme (OH) is a metastable state,different from state O in the ‘as isolated’ enzyme, as firstdescribed byAntonini et al. (1977). Recently,much interesthas been centered on the chemical nature of the ‘active’ OH

state, but its structural definition is yet unclear (described inthe following text). Recently, it has been suggested on thebasis of X-ray data (Aoyama et al., 2009; Koepke et al.,2009) that there is a peroxide molecule bridging haem a3iron and CuB in the ‘relaxed’ O state, but this proposal stillremains somewhat controversial.

Re-reduction and protonation of the enzyme

The binuclear site must be reduced prior to the binding ofthe next O2 molecule, and this occurs in the second half ofthe catalytic cycle (Figure 3), which is the rate-limiting partfor enzyme turnover (Mason et al., 2009).CuB(II) is reducedbefore haem a3, and this is associated with breaking of thestrong magnetic interaction between these two metals (vanGelder and Beinert, 1969). Reduction of the binuclear siteis associated with uptake of two protons from themedium,one of which may be consumed in converting an OH2

ligand of CuB to water. Complete reduction of the enzymefrom its fully oxidised state is linked to uptake of � 2.4protons from the medium at pH 7 (Rich et al., 1996).

Coupled Proton Translocation

In addition to taking up four protons to reduce each O2

molecule to water, the enzyme translocates four protonsacross themembrane per O2 reduced (Wikstrom, 1977). Bythis means, energy conservation as DmHþ is doubled withtranslocation of a total of eight electrical charges across themembrane per O2 reduced. The total cytochrome c oxidasereaction should, therefore, be written as eqn [II], wheresubscripts i and o refer to the aqueous protonic input andoutput sides of the membrane, respectively, correspondingto thematrix and intermembrane spaces on each side of theinner mitochondrial membrane (see Figure 2a). See also:Mitochondria: Structure and Role in Respiration

4cyt � c2þ þ 8Hþi þO2 ! 4cyt � c3þ þ 4Hþo þ 2H2O ½II�

Linkage of proton translocation to thecatalytic cycle

Proton translocation was initially suggested to occur dur-ing the P!F and F!O transitions in the catalytic cycle(two protons translocated in each step; Figure 3) on thebasis of equilibrium titrations in isolated mitochondria(Wikstrom, 1989). These two reactions indeed appear to bethe most exergonic in the cycle, whereas reduction of CuB(in O!E) and haem a3 (in E!R) are known to occur atredox potentials of 5400mV in equilibrium titrations;thus, apparently providing far too little driving force for







proton translocation. However, subsequent kinetic workdemonstrated that only two protons are translocated dur-ing the P!F and F!O transitions (one in each), in add-ition to the uptake of one proton in each step for formationof the equivalent of water (Verkhovsky et al., 1999). Theremaining two protons are translocated during the O!Eand E!R reactions (one in each step; Bloch et al., 2004),which posed a thermodynamic dilemma, since these latterreactions do not seem to have a sufficient driving force(described in the preceding text). A partial solution to thisproblem came from the observations that the O stateoccupied during turnover has different properties from theanalogous state in the ‘as isolated’ enzyme. The structuralbasis for this difference is still not known, but CuB in thetransient metastable O state (OH) appears to have a muchhigher midpoint redox potential than in the stable O state,thus providing a higher driving force for theO!E reaction(Belevich et al., 2007).

Pathways of proton transfer

Uptake of protons into the enzyme, either to be consumedin O2 reduction to water at the binuclear haem a3–CuB

centre or translocated across the membrane, takes placefrom thematrix side of the innermitochondrialmembrane,or in bacteria from the cytosolic side of the cell mem-brane. Pathways of proton uptake have been identified bysite-directed mutagenesis experiments combined with thecrystal structures. From such experiments, it has beenconjectured that all protons taken up in the P!O sequenceof the catalytic cycle (Figure 3), whether they will be con-sumed or translocated, are transferred via the so-called Dchannel in subunit I (Figure 4). This ‘channel’ begins nearresidue Asp91 on the inside of themembrane, and containsseveral polar residues and bound water molecules. It endsnear the middle of the membrane domain in a nonpolarcavity that extends towards the binuclear site and containsthe well-conserved residue Glu242, to which protons fromthe D-channel may be transferred via bound water mol-ecules. Glu242 has indeed been shown to be involved in theproton translocation process, and itsmutation also leads tosevere inhibition of O2 reduction, suggesting that the pro-ton path to the O2 reduction centre is also blocked. Thefurther pathof the protons beyondGlu242 almost certainlyinvolves water molecules in the nonpolar cavity, althoughthey are not seen in theX-ray structures, presumably due to

Figure 4 Pathways of proton uptake. The backbones of subunits I (green) and II (silver) are shown in a phospholipid membrane. The P- and N-sides indicate

the positively and negatively charged sides, respectively. Subunit I includes the heme groups a (left) and a3 (right) drawn in purple, and a nonpolar cavity

(in yellow) above the residue Glu242 (E242). Two proton uptake pathways are indicated by red arrows. The D-pathway (left) starts with Asp91 (D91) and

ends at Glu242. The K-pathway is named after the conserved residue Lys319 (K319) and ends at Tyr244 (not shown) near heme a3.



their mobility (Figure 4). The path of the protons to bepumped across the membrane appears to transientlyinvolve theD-propionate group of haem a3 (Puustinen andWikstrom, 1999), which forms an ion pair with Arg438 inthe crystal structures (Figure 4). One key question is howproton transfer fromGlu242 either to the binuclear site forconsumption or across the membrane (pumping) is regu-lated. Arrangement of the water molecules in the nonpolarcavity to form distinct proton transfer paths for the twopurposes, regulated by the state of the electron transferprocess, has been suggested on the basis of moleculardynamics simulations (Wikstrom et al., 2003). Such simu-lations also suggest that the side-chain of Glu242 performs‘up/down’ dynamics (‘down’ in Figure 4), which may givethis residue a valve function (Kaila et al., 2008). See also:Bacterial Cytoplasmic Membrane

Another pathway, the so-calledK-channel, has alsobeenimplicated in proton transfer from both mutagenesisexperiments and the crystal structures. The key residue ofthis potential pathway is Lys319; a serine and a threonineresidue and two bound water molecules have also beendeemed important (Figure 4). It has been suggested that thispathway, which appears to lead to the invariant Tyr244beneath the binuclear site is used in the proton uptakeprocess that is coupled to the O!R steps in the catalyticcycle (Figure 3).

The exit path of the pumped protons has been proposedto involve the domain just above the D propionates ofthe two haem groups, which contains several polar andcharged residues and bound water molecules (Figure 4).

The proton pump mechanism

Cytochrome coxidase is a proton translocating device, thatis a molecular proton pump (Figure 2a and Figure 3). In thelight-induced proton pump bacteriorhodopsin of Halo-bacteria, a Schiff base between the cofactor retinal and alysine side-chain in themembrane protein forms the ‘pumpelement’ to which a proton originating from one side of themembrane is bound and from which the proton is releasedto the opposite side. By analogy, such a pump element isexpected to exist also in the redox-linked proton pumpcytochrome c oxidase. The isomeric structure of the ret-inal–Schiff base complex in bacteriorhodopsin is sensitiveto light, which drives proton translocation. By analogy, the‘pump element’ (or ‘proton-loading site’, PLS) in cyto-chrome c oxidase should be sensitive to the driving oxygenreduction chemistry at the binuclear site, and recent re-sults suggest that it may be the A-propionate of haema3 (Siegbahn and Blomberg, 2007; Wikstrom andVerkhovsky, 2007). Recent time-resolved experiments(Belevich et al., 2007) have gone some way to elucidate themechanism of proton pumping by cytochrome c oxidase,although there are still some unsolved questions. Theproton pump appears to function as a solid state electro-static device, where arrival of the electron at the ‘electron-queueing’ haem a leads to twokey effects, both electrostaticin nature:Theprotonaffinity (pKa) of thePLS is raised, and

the activation barrier for proton transfer from Glu242 tothe PLS is lowered. At the same time, the barrier for protontransfer from Glu242 to the binuclear centre is very high,and its pKa does not favour acceptance of the proton. Theproton of Glu242 is therefore transferred to the PLS forboth kinetic and thermodynamic reasons. Arrival of theproton at the PLS increases the affinity of the binuclear sitefor the electron (again for electrostatic reasons), whichmoves over by a fast tunneling process (Pilet et al., 2004).Reciprocally, the electron at the binuclear centre furtherstabilises the PLS proton, since the distance between elec-tron and proton is now shortened.However, the electron atthe binuclear site also increases the proton affinity of thissite. Meanwhile, Glu242 has become reprotonated via theD-pathway. Themovement of the electron to the binuclearcentre has raised the proton transfer barrier betweenGlu242 and the PLS, and lowered the corresponding bar-rier between Glu242 and the binuclear site. Control of theproton transfer barrier between Glu242 and the PLS bythe position of the electron is an essential feature of themechanism, which contributes to minimisation of protonleaks (Siegbahn and Blomberg, 2007; Wikstrom andVerkhovsky, 2007). The stage is now set for transfer of thesecond proton (per electron) from Glu242 to the binuclearsite, where it completes the oxygen reduction chemistrywith formation of the equivalent of water. Neutralisationof the electron charge at this site by the ‘substrate proton’lowers the proton affinity of the PLS, and the PLS proton isejected to the proton output side of the membrane byelectrostatic repulsion. Therefore, ‘the energy of bindingthe chemical protons is used to eject the pumped protons’(Wikstrom et al., 1994) that is a drive towards electro-neutrality that has been emphasised in this connection byPeter Rich (1995). See also: Halophiles

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