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Introduction

Cytochrome oxidase is one of a superfamily of proteins which act as the terminal enzymes of respiratory chains. The two main classes are cytochrome c oxidases, and quinol oxidases. The common features are:

Some of the variations among the heme-Cu oxidase superfamily are shown in this scheme. Cytochrome c oxidases (left) of mitochondria, of respiratory bacteria from the purple bacterial branch of the Eubacteria, and one of the cytochrome c oxidases of Rhodobacter sphaeroides, are of the aa₃-type. Others (ba₃-type) have a heme b in place of heme a. Not shown are cytochrome c oxidases which have cytochrome b attached to subunit II, and cytochrome oxidases (cbb₃-type) which have two membrane-bound cytochrome c molecules in place of subunit II. Quinol oxidases (right) come in several flavors; in the bo₃-type, a heme type-o replaces heme a at the binuclear center.

An excellent summary of the liganding of the metal centers in cytochrome oxidase is provided by the PROMISE site cytochrome oxidase pages.

For cytochrome oxidase, the overall reaction is:

Since cytochrome c is in the P-phase, 8 charges are transfered from N- to P-phase per oxygen consumed.

Structure of cytochrome oxidase from Paracoccus denitrificans

The following pictures are from Iwata et al. (1995) Cytochrome Oxidase from Paracoccus denitrificans. Nature 376 660-669; a more complete set can be found on the cytochrome oxidase home page

Click on thumbnails for larger image

The beef heart mitochondrial cytochrome oxidase

The structure of the beef heart mitochondrial complex has also been solved at a similar resolution, and shows essentially the same structure, but with the many additional small subunits forming membrane helical spans around the periphery of the main catalytic subunits. The cordinates for this structure are now available in the Brookhaven Protein Data Bank (entry 1occ.pdb)

Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-ltoh, K., Nakashima, R., Yaono, R. & Yoshikawa, S. (1996) The Whole Structure of the 13-Subunit Oxidized Cytochrome c Oxidase at 2.5 A. Science 272, 1136-1144.

Chime

Summary
The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution with an R value of 19.9 percent reveals 13 subunits, each different from the other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A and three copper, one magnesium, and one zinc. Of 3606 amino acid residues in the dimer, 3560 have been converged to a reasonable structure by refinement. A hydrogen-bonded system, including a propionate of a heme A (heme a), part of peptide backbone, and an imidazole ligand of Cu_A, could provide an electron transfer pathway between Cu_A and heme a. Two possible proton pathways for pumping, each spanning from the matrix to the cytosolic surfaces, were identified, including hydrogen bonds, internal cavities likely to contain water molecules, and structures that could form hydrogen bonds with small possible conformational change of amino acid side chains. Possible channels for chemical protons to produce H₂O, for removing the produced water, and for O₂, respectively, were identified.

Mechanism of cytochrome oxidase

Electron transfer from cytochrome c occurs by electrostatic binding of cyt c to subunit II, followed by electron transfer through Cu_A center to heme a. Although the docking site and orientation are not known with certainty, the interaction involves a conserved surface of positively charged residue around the heme crevice of cytochrome c, and a complementary surface of acidic residues on subunit II. The electrostatic effect is thought to provided an orienting force sufficient to line up the cytochrome c in the correct orientation during the approach to the docking site. Binding affinity is strongly dependent on ionic stength.

A possible pathway for electron transfer from Cu_A to heme a has been identified in the structure of the mitochndrial enzyme (see summary above).

Delivery of electrons to the binuclear center is thought to occur to heme a₃ via heme a, but the distance from the Cu_A center to heme a₃ is not much greater than the distance from heme a.

The main interest lies in the mechanism of O₂-reduction through the binuclear center. The diagrams below shows the dioxygen cycle proposed by Michel (left) or by Wikström (right) for turnover of the binuclear center.

From: (left) H. Michel, J. Behr, A. Harrenga, and A. Kannt (1998) Annu. Rev. Biophys. Biomol. Struct. 27, 329-56; (right) Wikström, M. (2000) Biochemistry, 239, 3515-3519

The binuclear center is represented by its metals, Fe^+III/Fe^+II for heme a₃, Cu^+II/Cu^+I for Cu_B.

• Starting from the oxidized state of the binuclear center (O), electrons are delivered by two successive tansfers via heme a, to form the half- (E) and fully (2-e^-) reduced center (R).
• Oxygen binds to form the oxy compound (A), and electron transfer within the center occurs to generate the peroxy intermediate (P), now thought to represent several states.
  • If the P state is indeed a peroxy intermediate (as indicated by P_M and P_R), internal electron transfer would regenerate the the oxidized centers.
  • However, the spectroscopy suggests that the heme a₃ Fe is present in the Fe^+IV state, suggesting a possible ferryl state, as indicated by the boxed path. Formation of the ferryl state would require reduction by four electrons, only three of which can be accounted for by the metal centers. It is suggested that a fourth electron must come from elsewhere, most plausibly from a tyrosine coordinated to one of the Cu_B histidine ligands.
• Transfer of an electron (the third), uptake of 2H⁺, and release of H₂O result in the formation of the classical ferryl intermediate, but the point of entry of the protons, and release of water, depend on the nature of the P state.
• Transfer of another electron (the fourth), and uptake of two more H⁺ leads via the H state to generation and release of a second H₂O molecule, and regeneration of the oxidized center.

The scheme is based on a large body of research in which the spectra of the intermediate forms have been characterized, and the kinetics measured using spectrophotometry, and a variety of techniques for rapid mixing and photochemical production of the oxidized form by flash photolysis of CO from the CO-liganded cytochrome oxidase. The scheme shows the 4 scalar protons, but not the protons which are pumped across the membrane; the points of entry of these latter are still controversial. Earlier experiments from Wikstrom had suggested that the work for these extra protons was provided by the reactions in which P is converted to O.

Proton pumping

The thermodynamics of the coupling of electron transfer to proton pumping have been determined by experiments from Mårten Wikström's group using intact mitochondria, in which the mitochondrial enzyme was driven in reverse by the proton gradient generated by ATP-hydrolysis.

The mitochondria were treated with rotenone, antimycin and myxothiazol to inhibit electron transfer from the respiratory chain, and ferricyanide was added to poise the metal centers in the oxidized (or partly oxidized) state. In A, ferricyanide was present to oxidize all the metal centers. Under these conditions, formation of the P intermediate from O can be driven by the reverse reaction, with the Cu_A/heme a pair as electron acceptor. In B, a mixture of ferri- and ferrocyanide was present to partly reduce the Cu_A/heme a pair, and thus limit the availablity of electron acceptors for the reverse reaction. Under these conditions, the reverse reaction forms the F intermediate. By varying the ATP/ADP ratio, the driving force available from ATP-hydrolysis (DG'_ATP) could be varied, and the extent of the reaction measured under both conditions. The figure shows how the ratio of reactants for the two redox half-cells varied as a function of the driving force. Since formation of ATP from ADP and phosphate requires 4H⁺ in intact mitochondria, hydrolysis of ATP pumps 4H⁺/ATP hydrolysed. In experiment A, the slope of 1 therefore indicates that the transition from P to F involves the transfer of 4H⁺ across the membrane; the slope of 0.75 in experiment B indicates that the transition from F to O involves the transfer of 3H⁺ across the membrane. In each case, two of the protons are those involved in reduction of oxygen. Recently, quantitative aspects of this work have been questioned, because the pattern of H⁺ uptake suggests that some uptake occurs in the reductive phase (see below).

Mechanism of proton pumping

The solution of the structure of cytochrome oxidase has led to much discussion of the mechanism of proton pumping. The figures below are taken from the paper by Iwata et al. (see above), which should be consulted for details. The groups thought to be involved in proton pumping are shown in the figure below. They have been identified by site-directed mutagenesis, mainly in the Gennis lab.

A tentative scheme for proton pumping involving these groups is shown below. Detail aspects of the mechanism are at present controversial; both labs involved in crystallographic studies have demonstrated that the histidine ligand to Cu_B (H333), which appeared to be mobile in the Iwata structure, is in the liganding position observed in the Yoshikawa structure in both oxidized and reduced enzyme in the absence of azide. It therefore seems unlikely that movement of this group through the sequence of states shown in the scheme below occurs. However, other variants of this scheme with movement linked to other transitions, are not excluded.

Michel has recently updated these ideas, and challenged Mårten Wikström's earlier conclusions about the points of entry of protons. A somewhat complicated scheme, taken from ref. 2, can be seen below.

FIGURE 3: Mechanistic model for the catalytic cycle of COX and its coupling to proton pumping by electrostatic repulsion. The left heme Fe symbol in each state stands for the heme a Fe atom with +III or +II as its formal oxidation number. A^- stands for a proton-accepting site at the heme a propionates. The Fe symbol more to the right stands for the heme a₃ iron. A water molecule (H₂O) is bound to it in the O-state. An OH^- bound to Cu_B is shown. B^- stands for a common protonation site near the heme a3-Cu_B binuclear site. For each state the overall net charge at the heme a Fe site (without A) is indicated within the left-hand circle, and the net charge in the immediate vicinity of the binuclear site (including HY, without B) is shown within the right-hand circle. All changes in the boxes are electroneutral. The reductions of heme a during the E -> R, F' -> P_R, and F -> O transitions are charge-compensated by proton uptake via the D pathway; these protons are stored in the proton-accepting site A. The resulting states are called arE, arF', and arF, respectively, where ar stands for heme a reduced. The proposal of a pumping step between arE and R is highly speculative; it might be achieved by electrostatic repulsion of the proton in the A-site by a proton entering the B-site via the D-pathway, if proton transfer from the A-site to the B-site is interrupted. e^- stands for electrons transferred from Cu_A to heme a. Protons taken up via the K-pathway are shown as H_K⁺, those taken up via the D-pathway as H_D⁺and those taken up via the D- or the K-pathway as H_D,K⁺. Proton pumping is indicated by a double arrow. Y^· stands for the neutral radical of the H27-H280 cross-link, and Y^- for the tyrosinate. For more details see text.

The controversy between Wikstrom and Michel was been played out in recent papers from both groups, which should be consulted for further details:

Reviews:

• Zaslavsky, D. and Gennis, R.B. (2000) Proton pumping by cytochrome oxidase: progress, problems and postulates. Biochim. Biophys. Acta 1458, 164-179
• Mills, D.A., Florens, L., Hiser, C., Qian, J. and Ferguson-Miller, S. (2000) Where is 'outside' in cytochrome c oxidase and how and when do protons get there? Biochim. Biophys. Acta 1458, 180-187

1. Michel, H. (1998) The mechanism of proton pumping by cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 95, 12819-12824
2. Michel, H. (1999) Cytochrome c Oxidase: Catalytic Cycle and Mechanisms of Proton Pumping-A Discussion. Biochemistry, 38, 15129-15140
3. Wikström, M. (2000) Proton Translocation by Cytochrome c Oxidase: A Rejoinder to Recent Criticism. Biochemistry, 239, 3515-3519