Review article: structural and functional properties of cytochrome aa3 from bacteria

The aa3 oxidases from bacteria form a group of related enzymes that resemble the far more complex mitochondrial cytochrome c oxidase, both functionally and structurally. These enzymes catalyze electron transfer from ferrocytochrome c to oxygen to produce water. This transfer is coupled to proton translocation. Several oxidases of this type have been purified from cytoplasmic membranes of bacteria. This review summarizes the present knowledge on purified bacterial aa3 oxidases and correlates these findings with data available for the eukaryotic cytochrome c-oxidases.     

Elementary steps of proton translocation in the catalytic cycle of cytochrome oxidase

Proton translocation in the catalytic cycle of cytochrome c oxidase (CcO) proceeds sequentially in a four-stroke manner. Every electron donated by cytochrome c drives the enzyme from one of four relatively stable intermediates to another, and each of these transitions is coupled to proton translocation across the membrane, and to uptake of another proton for production of water in the catalytic site. Using cytochrome c oxidase from Paracoccus denitrificans we have studied the kinetics of electron transfer and electric potential generation during several such transitions, two of which are reported here. The extent of electric potential generation during initial electron equilibration between CuA and heme a confirms that this reaction is not kinetically linked to vectorial proton transfer, whereas oxidation of heme a is kinetically coupled to the main proton translocation events during functioning of the proton pump. We find that the rates and amplitudes in multiphase heme a oxidation are different in the OH-->EH and PM-->F steps of the catalytic cycle, and that this is reflected in the kinetics of electric potential generation. We discuss this difference in terms of different driving forces and relate our results, and data from the literature, to proposed mechanisms of proton pumping in cytochrome c oxidase.     

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase.

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa3) is coupled to translocation of H+ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa3 is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational "strain" in the cytochrome aa3 complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.     

Cytochrome c oxidase in prokaryotes

Abstract Several heme aa ₃-type cytochrome c oxidases, purified from the cytoplasmic membranes of bacteria, are able to catalyze the same reactions as the structurally far more complex eukaryotic enzyme, i.e., electron transport from cytochrome c to oxygen coupled to proton translocation. However, these oxidases show a very simple subunit pattern, and moreover, individual polypeptides even have homologous amino-acid sequences. This review summarizes the present data on purified bacterial cytochrome c oxidases and relates these findings to results obtained with the mitochondrial enzymes.     

Transmembrane charge separation during the ferryl-oxo -> oxidized transition in a nonpumping mutant of cytochrome c oxidase

The N139D mutant of cytochrome c oxidase from Rhodobacter sphaeroides retains full steady state oxidase activity but completely lacks proton translocation coupled to turnover in reconstituted liposomes (Pawate, A. S., Morgan, J., Namslauer, A., Mills, D., Brzezinski, P., Ferguson-Miller, S., and Gennis, R. B. (2002) Biochemistry 41, 13417-13423). Here, time-resolved electron transfer and vectorial charge translocation in the ferryl-oxo --> oxidized transition (transfer of the 4th electron in the catalytic cycle) have been studied with the N139D mutant using ruthenium(II)-tris-bipyridyl complex as a photoactive single-electron donor. With the wild type oxidase, the flash-induced generation of Deltaphi in the ferryl-oxo --> oxidized transition begins with rapid vectorial electron transfer from CuA to heme a (tau approximately 15 micros), followed by two protonic phases, referred to as the intermediate (0.4 ms) and slow electrogenic phases (1.5 ms). In the N139D mutant, only a single protonic phase (tau approximately 0.6 ms) is observed, which was associated with electron transfer from heme a to the heme a3/CuB site and decelerates approximately 4-fold in D2O. With the wild type oxidase, such a high H2O/D2O solvent isotope effect is characteristic of only the slow (1.5 ms) phase. Presumably, the 0.6-ms electrogenic phase in the N139D mutant reports proton transfer from the inner aqueous phase to Glu-286, replacing the "chemical" proton transferred from Glu-286 to the heme a3/CuB site. The transfer occurs through the D-channel, because it is observed also in the N139D/K362M double mutant in which the K-channel is blocked. It is concluded that the intermediate electrogenic phase observed in the wild type enzyme is missing in the N139D mutant and is because of translocation of the "pumped" proton from Glu-286 to the D-ring propionate of heme a3 or to release of this proton to the outer aqueous phase. Significantly, with the wild type oxidase, the protonic electrogenic phase associated with proton pumping (approximately 0.4 ms) precedes the electrogenic phase associated with the oxygen chemistry (approximately 1.5 ms).     

Cytochrome c oxidase: Intermediates of the catalytic cycle and their energy-coupled interconversion

Several issues relevant to the current studies of cytochrome c oxidase catalytic mechanism are discussed. The following points are raised. (1) The terminology currently used to describe the catalytic cycle of cytochrome oxidase is outdated and rather confusing. Presumably, it would be revised so as to share nomenclature of the intermediates with other oxygen-reactive heme enzymes like P450 or peroxidases. (2) A "catalytic cycle" of cytochrome oxidase involving complete reduction of the enzyme by 4 electrons followed by oxidation by O(2) is a chimera composed artificially from two partial reactions, reductive and oxidative phases, that never operate together as a true multi-turnover catalytic cycle. The 4e(-) reduction-oxidation cycle would not serve a paradigm for oxygen reduction mechanism and protonmotive function of cytochrome oxidase. (3) The foremost role of the K-proton channel in the catalytic cycle may consist in securing faultless delivery of protons for heterolytic O-O bond cleavage in the oxygen-reducing site, minimizing the danger of homolytic scission reaction route. (4) Protonmotive mechanism of cytochrome oxidase may vary notably for the different single-electron steps in the catalytic cycle.     

The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site

Cell respiration is catalyzed by the heme-copper oxidase superfamily of enzymes, which comprises cytochrome c and ubiquinol oxidases. These membrane proteins utilize different electron donors through dissimilar access mechanisms. We report here the first structure of a ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The overall structure of the enzyme is similar to those of cytochrome c oxidases; however, the membrane-spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer that is not present in the cytochrome c oxidase. Mutagenesis studies on these residues strongly suggest that this region forms a quinone binding site. A sequence comparison of this region with known quinone binding sites in other membrane proteins shows remarkable similarities. In light of these findings we suggest specific roles for these polar residues in electron and proton transfer in ubiquinol oxidase.     

Cytochrome c oxidase: catalytic cycle and mechanisms of proton pumping--a discussion

Cytochrome c oxidase catalyzes the reduction of molecular oxygen to water, a process in which four electrons, four protons, and one molecule of oxygen are consumed. The reaction is coupled to the pumping of four additional protons across the membrane. According to the currently accepted concept, the pumping of all four protons occurs after the binding of oxygen to the reduced enzyme and is exclusively coupled to the last two electron transfer steps. A careful analysis of the existing data shows that there is no experimental evidence for this paradigm. It is more likely that only three protons are pumped during the second half of the catalytic cycle of cytochrome c oxidase after the reaction with oxygen. In this article a variant of a recent mechanistic model of proton pumping by electrostatic repulsion is discussed. It is based on the electroneutrality principle in a way that in the catalytic cycle each electron transfer to the membrane-embedded electron acceptors is charge-compensated by uptake of one proton. The mechanism takes into account the findings with mutant cytochrome c oxidases and explains the results of many recent experiments, including the effects of hydrogen peroxide.     

Resolution of electrogenic steps coupled to conversion of cytochrome c oxidase from the peroxy to the ferryl-oxo state

Charge translocation across the membrane coupled to transfer of the third electron in the reaction cycle of bovine cytochrome c oxidase (COX) has been studied. Flash-induced reduction of the peroxy intermediate (P) to the ferryl-oxo state (F) by tris-bipyridyl complex of Ru(II) in liposome-reconstituted COX is coupled to several phases of membrane potential generation that have been time-resolved with the use of an electrometric technique applied earlier in the studies of the ferryl-oxo-to-oxidized (F --> O) transition of the enzyme [Zaslavsky, D., et al. (1993) FEBS Lett. 336, 389-393]. As in the case of the F --> O transition, the electric response associated with photoreduction of P to F includes a rapid KCN-insensitive electrogenic phase with a tau of 40-50 microseconds (reduction of heme a by CuA) and a multiphasic slower part; this part is cyanide-sensitive and is assigned to vectorial transfer of protons coupled to reduction of oxygen intermediate in the binuclear center. The net KCN-sensitive phase of the response is approximately 4-fold more electrogenic than the rapid phase, which is similar to the characteristics of the F --> O electrogenic transition and is consistent with net transmembrane translocation of two protons per electron, including vectorial movement of both "chemical" and "pumped" protons. The protonic part of the P --> F electric response is faster than in the F --> O transition and can be deconvoluted into three exponential phases with tau values varying for different samples in the range of 0.25-0.33, 1-1.5, and 6-7.5 ms at pH 8. Of these three phases, the 1-1.5 ms component is the major one contributing 50-60%. The P --> F conversion induced by single electron photoreduction of the peroxy state as studied in this work is several times slower than the P --> F transition resolved during oxidation of the fully reduced oxidase by molecular oxygen. The role of the CuB redox state in controlling the rate of P --> F conversion of heme a3 is discussed.     

Proton transfer in ba(3) cytochrome c oxidase from Thermus thermophilus

The respiratory heme-copper oxidases catalyze reduction of O(2) to H(2)O, linking this process to transmembrane proton pumping. These oxidases have been classified according to the architecture, location and number of proton pathways. Most structural and functional studies to date have been performed on the A-class oxidases, which includes those that are found in the inner mitochondrial membrane and bacteria such as Rhodobacter sphaeroides and Paracoccus denitrificans (aa(3)-type oxidases in these bacteria). These oxidases pump protons with a stoichiometry of one proton per electron transferred to the catalytic site. The bacterial A-class oxidases use two proton pathways (denoted by letters D and K, respectively), for the transfer of protons to the catalytic site, and protons that are pumped across the membrane. The B-type oxidases such as, for example, the ba(3) oxidase from Thermus thermophilus, pump protons with a lower stoichiometry of 0.5 H(+)/electron and use only one proton pathway for the transfer of all protons. This pathway overlaps in space with the K pathway in the A class oxidases without showing any sequence homology though. Here, we review the functional properties of the A- and the B-class ba(3) oxidases with a focus on mechanisms of proton transfer and pumping.     

Electrochemical, FT-IR and UV/VIS spectroscopic properties of the caa3 oxidase from T. thermophilus.

The caa3-oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS and Fourier-transform infrared (FT-IR) spectroscopic approach. In this oxidase the electron donor, cytochrome c, is covalently bound to subunit II of the cytochrome c oxidase. Oxidative electrochemical redox titrations in the visible spectral range yielded a midpoint potential of -0.01 +/- 0.01 V (vs. Ag/AgCl/3m KCl, 0.218 V vs. SHE') for the heme c. This potential differs for about 50 mV from the midpoint potential of isolated cytochrome c, indicating the possible shifts of the cytochrome c potential when bound to cytochrome c oxidase. For the signals where the hemes a and a3 contribute, three potentials, = -0.075 V +/- 0.01 V, Em2 = 0.04 V +/- 0.01 V and Em3 = 0.17 V +/- 0.02 V (0.133, 0.248 and 0.378 V vs. SHE', respectively) could be obtained. Potential titrations after addition of the inhibitor cyanide yielded a midpoint potential of -0.22 V +/- 0.01 V for heme a3-CN- and of Em2 = 0.00 V +/- 0.02 V and Em3 = 0.17 V +/- 0.02 V for heme a (-0.012 V, 0.208 V and 0.378 V vs. SHE', respectively). The three phases of the potential-dependent development of the difference signals can be attributed to the cooperativity between the hemes a, a3 and the CuB center, showing typical behavior for cytochrome c oxidases. A stronger cooperativity of CuB is discussed to reflect the modulation of the enzyme to the different key residues involved in proton pumping. We thus studied the FT-IR spectroscopic properties of this enzyme to identify alternative protonatable sites. The vibrational modes of a protonated aspartic or glutamic acid at 1714 cm-1 concomitant with the reduced form of the protein can be identified, a mode which is not present for other cytochrome c oxidases. Furthermore modes at positions characteristic for tyrosine vibrations have been identified. Electrochemically induced FT-IR difference spectra after inhibition of the sample with cyanide allows assigning the formyl signals upon characteristic shifts of the nu(C=O) modes, which reflect the high degree of similarity of heme a3 to other typical heme copper oxidases. A comparison with previously studied cytochrome c oxidases is presented and on this basis the contributions of the reorganization of the polypeptide backbone, of individual amino acids and of the hemes c, a and a3 upon electron transfer to/from the redox active centers discussed.     

Heme-copper oxidases with modified D- and K-pathways are yet efficient proton pumps

The cytochrome aa(3)-type quinol oxidase from the archaeon Acidianus ambivalens and the ba(3)-type cytochrome c oxidase from Thermus thermophilus are divergent members of the heme-copper oxidase superfamily of enzymes. In particular they lack most of the key residues involved in the proposed proton transfer pathways. The pumping capability of the A. ambivalens enzyme was investigated and found to occur with the same efficiency as the canonical enzymes. This is the first demonstration of pumping of 1 H(+)/electron in a heme-copper oxidase that lacks most residues of the K- and D-channels. Also, the structure of the ba(3) oxidase from T. thermophilus was simulated by mutating Phe274 to threonine and Glu278 to isoleucine in the D-pathway of the Paracoccus denitrificans cytochrome c oxidase. This modification resulted in full efficiency of proton translocation albeit with a substantially lowered turnover. Together, these findings show that multiple structural solutions for efficient proton conduction arose during evolution of the respiratory oxidases, and that very few residues remain invariant among these enzymes to function in a common proton-pumping mechanism.     

Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus

The kinetics of the oxidation of fully-reduced ba(3) cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa(3)-type oxidases. However the F intermediate in ba(3) oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P-->F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F-->O transition in ba(3) oxidase is close to that observed during the F-->O transition in the aa(3) oxidases. However, the P(R)-->F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.     

A cooperative model for proton pumping in cytochrome c oxidase

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 Cu(A) 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 Cu(A)-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 Cu(B)-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.     

Cytochrome c Oxidase (Heme aa 3) from Paracoccus denitrificans: Analysis of Mutations in Putative Proton Channels of Subunit I

One of the challenging features of energy-transducing terminal oxidases, like the aa ₃ cytochrome c oxidase of Paracoccus denitrificans, is the translocation of protons across the cytoplasmic membrane, which is coupled to the transfer of electrons to oxygen. As a prerequisite for a more advanced examination of the enzymatic properties, several amino acid residues, selected on the basis of recent three-dimensional structure determinations, were exchanged in subunit I of the Paracoccus enzyme by site-directed mutagenesis. The properties of the mutated oxidases were analyzed by different methods to elucidate whether they are involved in the coupled and coordinated transfer of protons via two different pathways either to the site of oxygen reduction or through the enzyme from the cytoplasm to the periplasmic side.     

Theory and simulation of proton-coupled electron transfer, hydrogen-atom transfer, and proton translocation in proteins

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.     

Proton translocation by cytochrome c oxidase in different phases of the catalytic cycle

Since mitochondrial cytochrome c oxidase was found to be a redox-linked proton pump, most enzymes of the haem-copper oxidase family have been shown to share this function. Here, the most recent knowledge of how the individual reactions of the enzyme's catalytic cycle are coupled to proton translocation is reviewed. Two protons each are pumped during the oxidative and reductive halves of the cycle, respectively. An apparent controversy that concerns proton translocation during the reductive half is resolved. If the oxidised enzyme is allowed to relax in the absence of reductant, the binuclear haem-copper centre attains a state that lies outside the main catalytic cycle. Reduction of this form of the enzyme is not linked to proton translocation, but is necessary for a return to the main cycle. This phenomenon might be related to the previously described "pulsed" vs. "resting" and "fast" vs."slow" forms of haem-copper oxidases.     

Properties and reconstitution of a cytochrome oxidase deficient in subunit III

Three different preparations of beef heart cytochrome oxidase (EC 1.9.3.1) were reconstituted into the membranes of artificial liposomes, and the electrical charge/electron ratios were determined for charge translocation coupled to enzymic activity. Our previously characterised subunit-III-deficient preparation, which apparently lacks H+ translocation capacity [Saraste et al. (1981) Eur. J. Biochem. 115, 261-268] has a decreased charge/electron ratio (0.9-1.0) as determined from the uptake of potassium in the presence of valinomycin, in contrast to the intact reconstituted cytochrome oxidase (1.9-2.0). A third preparation that was depleted of three minor polypeptides by trypsin treatment (these polypeptides are also removed together with subunit III using the present method), but which retains subunit III, had a K+/e- ratio of 1.5 but also a relatively low respiratory control index. The pH-dependence of the Em of cytochrome a determined in the presence of cyanide is abolished in the subunit-III-deficient enzyme. Electron transfer activities are nearly identical for the original and subunit-III-depleted enzymes at an infinite concentration of cytochrome c in a polarographic assay with supplemented phospholipids. The optical spectral properties are very similar for both preparations, but with a small shift to the blue of the alpha-peak in the modified enzyme. These results support the hypothesis that the removal of subunit III abolishes the H+-translocating function of cytochrome oxidase. This occurs by an intrinsic decoupling of H+ transport from electron transfer, and yields a preparation with only half-maximal efficiency of energy conservation.     

Deuterium isotope effect of proton pumping in cytochrome c oxidase

In mitochondria and many aerobic bacteria cytochrome c oxidase is the terminal enzyme of the respiratory chain where it catalyses the reduction of oxygen to water. The free energy released in this process is used to translocate (pump) protons across the membrane such that each electron transfer to the catalytic site is accompanied by proton pumping. To investigate the mechanism of electron-proton coupling in cytochrome c oxidase we have studied the pH-dependence of the kinetic deuterium isotope effect of specific reaction steps associated with proton transfer in wild-type and structural variants of cytochrome c oxidases in which amino-acid residues in proton-transfer pathways have been modified. In addition, we have solved the structure of one of these mutant enzymes, where a key component of the proton-transfer machinery, Glu286, was modified to an Asp. The results indicate that the P3-->F3 transition rate is determined by a direct proton-transfer event to the catalytic site. In contrast, the rate of the F3-->O4 transition, which involves simultaneous electron transfer to the catalytic site and is characteristic of any transition during CytcO turnover, is determined by two events with similar rates and different kinetic isotope effects. These reaction steps involve transfer of protons, that are pumped, via a segment of the protein including Glu286 and Arg481.     

Terminal oxidases in the trypanosomatid Trypanosoma cruzi

Titration of Trypanosoma cruzi respiration with cyanide, with results treated as Dixon plots, indicated the presence of several terminal oxidases. The inhibitions obtained at low cyanide concentrations (0-300 microM), taken together with cyanide effects on cytochrome aa3-deficient, dyskinetoplastic epimastigotes, supported cytochrome aa3 as T. cruzi main terminal oxidase. By increasing cyanide concentration to 1.0 mM, two alternative terminal oxidases could be detected. One of these was active in both kinetoplastic and dyskinetoplastic (cytochrome aa3-deficient) epimastigotes, and azide- and antimycin-insensitive. Complementary cytochrome studies with intact epimastigotes and mitochondrial membranes revealed the presence of cytochromes aa3, b, c558, o and possibly d, as components of the parasite electron transport system. Fractionation studies demonstrated that both o and d were bound to the mitochondrial membrane. Reduction by endogenous substrates and complex formation with cyanide supported cytochrome o as alternative terminal oxidase. EB-cultured, dyskinetoplastic epimastigotes showed the same respiration rate as the kinetoplastic cells, despite the significant decrease of cytochrome aa3, thus indicating adaptive mechanisms that determine the expression of alternative oxidases, whenever the main terminal activity is depressed.