Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. The coxII/coxIII operon codes for structural and assembly proteins homologous to those in yeast.

The coxII/coxIII operon of Rhodobacter sphaeroides cytochrome c oxidase has been sequenced and characterized by insertional inactivation/complementation analysis. The organization of the genes in this locus (coxII.orf1.orf3.coxIII) is the same as that of the equivalent operon of Paracoccus denitrificans (ctaC.ctaB.ctaG.ctaE), but unlike that of other bacteria whose cytochrome oxidase genes have been characterized so far. The predicted amino acid sequence homology with eukaryotic oxidases is also higher for Rb. sphaeroides (and P. denitrificans) than for other bacterial versions of the enzyme. The inactivation of coxII results in loss of the characteristic cytochrome oxidase spectrum from membranes of the mutant strain. Full recovery requires introduction into the bacterium of the complete operon containing coxII.orf1.orf3.coxIII; partial complementation yielding a spectrally altered enzyme is achieved with a plasmid containing coxII or coxII.orf1.orf3. These results indicate that the peptides ORF1, ORF3, and COXIII are all required for assembly of native cytochrome c oxidase, suggesting an oxidase-specific assembly or chaperonin function for the ORFs in Rb. sphaeroides similar to that observed for the homologous gene products in yeast, COX10 and COX11.     

Alternative initial proton acceptors for the D pathway of Rhodobacter sphaeroides cytochrome c oxidase

To characterize protein structures that control proton uptake, we assayed forms of cytochrome c oxidase (CcO) containing a carboxyl or a thiol group in line with the initial, internal waters of the D pathway for proton transfer in the presence and absence of subunit III. Subunit III provides approximately half of the protein surrounding the entry region of the D pathway. The N139D/D132N mutant contains a carboxyl group 6 ? within the D pathway and lacks the normal, surface-exposed proton acceptor, Asp-132. With subunit III, the steady-state activity of this mutant is slow, but once subunit III is removed, its activity is the same as that of wild-type CcO lacking subunit III (～1800 H+/s). Thus, a carboxyl group～25% within the pathway enhances proton uptake even though the carboxyl has no direct contact with bulk solvent. Protons from solvent apparently move to internal Asp-139 through a short file of waters, normally blocked by subunit III. Cys-139 also supports rapid steady-state proton uptake, demonstrating that an anion other than a carboxyl can attract and transfer protons into the D pathway. When both Asp-132 and Asp/Cys-139 are present, the removal of subunit III increases CcO activity to rates greater than that of normal CcO because of simultaneous proton uptake by two initial acceptors. The results show how the environment of the initial proton acceptor for the D pathway in these CcO forms dictates the pH range of CcO activity, with implications for the function of Asp-132, the normal proton acceptor.     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. I. Biochemical, spectral, and kinetic characterization of surface mutants in subunit ii of Rhodobacter sphaeroides cytochrome aa(3)

To determine the interaction site for cytochrome c (Cc) on cytochrome c oxidase (CcO), a number of conserved carboxyl residues in subunit II of Rhodobacter sphaeroides CcO were mutated to neutral forms. A highly conserved tryptophan, Trp(143), was also mutated to phenylalanine and alanine. Spectroscopic and metal analyses of the surface carboxyl mutants revealed no overall structural changes. The double mutants D188Q/E189N and D151Q/E152N exhibit similar steady-state kinetic behavior as wild-type oxidase with horse Cc and R. sphaeroides Cc(2), showing that these residues are not involved in Cc binding. The single mutants E148Q, E157Q, D195N, and D214N have decreased activities and increased K(m) values, indicating they contribute to the Cc:CcO interface. However, their reactions with horse and R. sphaeroides Cc are different, as expected from the different distribution of surface lysines on these cytochromes c. Mutations at Trp(143) severely inhibit activity without changing the K(m) for Cc or disturbing the adjacent Cu(A) center. From these data, we identify a Cc binding area on CcO with Trp(143) and Asp(214) close to the site of electron transfer and Glu(148), Glu(157), and Asp(195) providing electrostatic guidance. The results are completely consistent with time-resolved kinetic measurements (Wang, K., Zhen, Y., Sadoski, R., Grinnell, S., Geren, L., Ferguson-Miller, S., Durham, B., and Millett, F. (1999) J. Biol. Chem. 274, 38042-38050) and computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).     

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.     

Product-controlled steady-state kinetics between cytochrome aa3 from Rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach

Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c^2 +) as the electron donor. In this study, the oxidation of horse cyt c^2 + by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c^2 + and cyt c^3 +. This allowed an analysis of the effects of cyt c^3 + on the steady-state kinetics between CcO and cyt c^2 +. The results show that cyt c^3 + exhibits product inhibition by two mechanisms: competition with cyt c^2 + at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c^2 + at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6 × His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

G204D, a mutation that blocks the proton-conducting D-channel of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides

Cytochrome c oxidase uses the free energy of oxygen reduction to establish a transmembrane proton gradient. The proton-conducting D-channel in this enzyme is the major input pathway for protons which go to the binuclear center for water formation ("chemical protons") and likely the only input pathway for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel starts at an acidic residue near the protein surface (D132, Rhodobacter sphaeroides numbering) and leads to another acidic residue near the binuclear center. Recent studies have shown that mutants that introduce an additional acidic residue in the channel (N139D) have the remarkable effect of accelerating steady-state oxidase activity but completely eliminating proton pumping. In this work, an aspartic acid was introduced at the position of glycine 204, G204D, which is also within the D-channel, and the effects were examined. In contrast to N139D, the G204D mutation results in a dramatic decrease of the steady-state oxygen reductase activity (<2% of wild type) [Aagaard, A., and Brzezinski, P. (2001) FEBS Lett. 494, 157-160]. The residual activity is not coupled to the proton pump, and furthermore, in reconstituted vesicles the mutant enzyme exhibits a reverse respiration control ratio; i.e., the mutant oxidase activity is stimulated rather than inhibited when working against a protonmotive force. Hence, the mutant behaves very much like the D132N, which blocks proton uptake through the D-channel. Single-turnover experiments show that the rate-limiting step in the reaction of O2 with the fully reduced G204D mutant is the F --> O transition, similar to the D132N mutant. The block of the D-channel in the D132N mutant can be partly bypassed by biochemically removing subunit III from the enzyme, indicating that removal of the subunit reveals an alternate entrance for protons to the channel. However, this is not observed with the G204D mutant. This suggests that the cryptic entrance to the D-channel that is revealed by the removal of subunit III is between the levels of G204 and D132.     

Flash-photolysis of fully reduced and mixed-valence CO-bound Rhodobacter sphaeroides cytochrome c oxidase: heme spectral shifts

Conformational changes, internal electron transfer, and CO rebinding processes in cytochrome c oxidase from Rhodobacter sphaeroides reduced to different degrees were investigated. The reactions were followed using a gated optical spectrometric multichannel analyzer. Light-induced difference spectra, recorded in the 350-700 nm region over the 100 ns to 1 s time interval, were analyzed by singular value decomposition and global exponential fitting. The photolyzed fully reduced enzyme showed two relaxations, approximately 1 and 190 mus, prior to the 20 ms CO rebinding process. Intramolecular electron transfer was monitored following photolysis of the mixed-valence CO-bound enzyme. The analysis revealed 1.1 micros, 2.4 micros, 31 micros, 68 ms, and 240 ms apparent lifetimes, the first three of which are attributed to electron transfer from heme a3 to heme a with contribution from a relaxation process at the heme a3 site. Spectral changes associated with the microsecond processes are consistent with 75% electron transfer from heme a3 to heme a. A comparison of the experimental spectra and model difference spectra for the intramolecular electron transfer indicated approximately 3 nm blue shift in the absolute spectra of both the oxidized heme a3 and reduced heme a generated in the process. The 68 and 240 ms lifetimes are due to CO recombination to heme a3 and are attributed to the presence of two conformers, the slower rate corresponding to the conformer in higher abundance. The dependency of the apparent rate of CO rebinding on the intensity of the probe beam in single-wavelength experiments is explained.     

Product-controlled steady-state kinetics between cytochrome aa(3) from Rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach

Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c(2+)) as the electron donor. In this study, the oxidation of horse cyt c(2+) by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c(2+) and cyt c(3+). This allowed an analysis of the effects of cyt c(3+) on the steady-state kinetics between CcO and cyt c(2+). The results show that cyt c(3+) exhibits product inhibition by two mechanisms: competition with cyt c(2+) at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c(2+) at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6×His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Palmitate decreases proton pumping of liver-type cytochrome c oxidase.

The H+/e- stoichiometry of reconstituted cytochrome c oxidase from bovine kidney, containing subunit VIaL (liver type), is 0.5 under standard conditions but 1.0 on addition of 1% cardiolipin to the lipid mixture (asolectin). Low concentrations of palmitate (half-maximal effect at 0.5 microm), but not laurate, myristate, stearate, oleate, 1-hexadecanol, palmitoyl glycerol and palmitoyl CoA, decreased the H+/e- ratio in the presence of cardiolipin from 1.0 to 0.5, accompanied by an increase of coupled, but not of uncoupled respiration of proteoliposomes. Cardiolipin and palmitate did not influence the H+/e- stoichiometry and respiration of reconstituted cytochrome c oxidase from bovine heart, containing subunit VIaH (heart-type). The H+/e- stoichiometry of the heart enzyme, however, is decreased from 1.0 to 0.5 by 5 mm intraliposomal ATP (instead of 5 mm ADP). It is assumed that palmitate binds to subunit VIaL. The partial uncoupling of proton pumping in cytochrome c oxidase is suggested to participate in mammalian thermogenesis.     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).     

Aspartate-132 in cytochrome c oxidase from Rhodobacter sphaeroides is involved in a two-step proton transfer during oxo-ferryl formation.

The aspartate-132 in subunit I (D(I-132)) of cytochrome c oxidase from Rhodobacter sphaeroides is located on the cytoplasmic surface of the protein at the entry point of a proton-transfer pathway used for both substrate and pumped protons (D-pathway). Replacement of D(I-132) by its nonprotonatable analogue asparagine (DN(I-132)) has been shown to result in a reduced overall activity of the enzyme and impaired proton pumping. The results from this study show that during oxidation of the fully reduced enzyme the reaction was inhibited after formation of the oxo-ferryl (F) intermediate (tau congruent with 120 microseconds). In contrast to the wild-type enzyme, in the mutant enzyme formation of this intermediate was not associated with proton uptake from solution, which is the reason the DN(I-132) enzyme does not pump protons. The proton needed to form F was presumably taken from a protonatable group in the D-pathway (e.g., E(I-286)), which indicates that in the wild-type enzyme the proton transfer during F formation takes place in two steps: proton transfer from the group in the pathway is followed by faster reprotonation from the bulk solution, through D(I-132). Unlike the wild-type enzyme, in which F formation is coupled to internal electron transfer from CuA to heme a, in the DN(I-132) enzyme this electron transfer was uncoupled from formation of the F intermediate, which presumably is due to the impaired charge-compensating proton uptake from solution. In the presence of arachidonic acid which has been shown to stimulate the turnover activity of the DN(I-132) enzyme (Fetter et al. (1996) FEBS Lett. 393, 155), proton uptake with a time constant of approximately 2 ms was observed. However, no proton uptake associated with formation of F (tau congruent with 120 micros) was observed, which indicates that arachidonic acid can replace the role of D(I-132), but it cannot transfer protons as fast as the Asp. The results from this study show that D(I-132) is crucial for efficient transfer of protons into the enzyme and that in the DN(I-132) mutant enzyme there is a "kinetic barrier" for proton transfer into the D-pathway.     

Energy diagrams and mechanism for proton pumping in cytochrome c oxidase

The powerful technique of energy diagrams has been used to analyze the mechanism for proton pumping in cytochrome c oxidase. Energy levels and barriers are derived starting out from recent kinetic experiments for the O to E transition, and are then refined using general criteria and a few additional experimental facts. Both allowed and non-allowed pathways are obtained in this way. A useful requirement is that the forward and backward rate should approach each other for the full membrane gradient. A key finding is that an electron on heme a (or the binuclear center) must have a significant lowering effect on the barrier for proton uptake, in order to prevent backflow from the pump-site to the N-side. While there is no structural gating in the present mechanism, there is thus an electronic gating provided by the electron on heme a. A quantitative analysis of the energy levels in the diagrams, leads to Prop-A of heme a(3) as the most likely position for the pump-site, and the Glu278 region as the place for the transition state for proton uptake. Variations of key redox potentials and pK(a) values during the pumping process are derived for comparison to experiments.     

Electrostatic control of proton pumping in cytochrome c oxidase

As part of the mitochondrial respiratory chain, cytochrome c oxidase utilizes the energy produced by the reduction of O2 to water to fuel vectorial proton transport. The mechanism coupling proton pumping to redox chemistry is unknown. Recent advances have provided evidence that each of the four observable transitions in the complex catalytic cycle consists of a similar sequence of events. However, the physico-chemical basis underlying this recurring sequence has not been identified. We identify this recurring pattern based on a comprehensive model of the catalytic cycle derived from the analysis of oxygen chemistry and available experimental evidence. The catalytic cycle involves the periodic repetition of a sequence of three states differing in the spatial distribution of charge in the active site: [0|1], [1|0], and [1|1], where the total charge of heme a and the binuclear center appears on the left and on the right, respectively. This sequence recurs four times per turnover despite differences in the redox chemistry. This model leads to a simple, robust, and reproducible sequence of electron and proton transfer steps and rationalizes the pumping mechanism in terms of electrostatic coupling of proton translocation to redox chemistry. Continuum electrostatic calculations support the proposed mechanism and suggest an electrostatic origin for the decoupled and inactive phenotypes of ionic mutants in the principal proton-uptake pathway.     

The binding domain on horse cytochrome c and Rhodobacter sphaeroides cytochrome c2 for the Rhodobacter sphaeroides cytochrome bc1 complex

The interaction of the Rhodobacter sphaeroides cytochrome bc1 complex with Rb. sphaeroides cytochrome c2 and horse cytochrome c was studied by using specific lysine modification and ionic strength dependence methods. The rate of the reactions with both cytochrome c and cytochrome c2 decreased rapidly with increasing ionic strength above 0.2 M NaCl. The ionic strength dependence suggested that electrostatic interactions were equally important to the reactions of the two cytochromes, even though they have opposite net charges at pH 7.0. In order to define the interaction domain on horse cytochrome c, the reaction rates of derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This result indicates that lysines surrounding the heme crevice of horse cytochrome c are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. In order to define the reaction domain on cytochrome c2, a fraction consisting of a mixture of singly labeled 4-carboxy-2,6-dinitrophenylcytochrome c2 derivatives modified at lysine-35, -88, -95, -97, and -105 and several unidentified lysines was prepared. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The rate of reaction of this fraction was significantly smaller than that of native cytochrome c2, suggesting that the binding domain on cytochrome c2 is also located at the heme crevice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome a1 of Nitrosomonas europaea resembles aa3-type cytochrome c oxidase in many respects

Cytochrome c oxidase of Nitrosomonas europaea has been called cytochrome a₁ by Erickson et al. (Erickson, R.H., Hooper, A.B. and Terry, K.R. (1972) Biochim. Biophys. Acta 283, 155–166) because the reduced form of their preparation had the α peak at 595 nm. In the present studies, the enzyme was purified to an electrophoretically almost homogeneous state and some of its properties were studied. The enzyme much resembled cytochrome aa₃-type oxidase although its reduced form showed the α peak at 597 nm. (1) The absorption spectra of the CO compound of the reduced enzyme and CN⁻ compounds of the oxidized and reduced enzyme were similar to those of the respective compounds of cytochrome aa₃, as well as the absorption spectrum of the intact enzyme resembled that of the cytochrome. (2) The enzyme possessed two molecules of haem a and 1–2 atoms of copper in the molecule. (3) The enzyme molecule was composed of two kinds of subunits of M_r 50000 and 33000, respectively, as are other bacterial cytochromes aa₃. Although the enzyme resembled other bacterial cytochromes aa₃ in many properties, it differed greatly in two properties; its CO compound was easily dissociated into the oxidized enzyme and CO in air, and 50% inhibition of its activity by CN⁻ required approx. 100 μM of the reagent. The enzyme oxidized 0.57, 1.6 and 1.8 mol horse, Candida krusei and N. europaea ferrocytochromes c per s per mol haem a, respectively, in 10 mM phosphate buffer, pH 6.0. The turnover numbers with eukaryotic ferrocytochromes c were increased to 32 and 14, respectively, by addition of cardiolipin (14 μ · ml⁻¹).     

A role for subunit III in proton uptake into the D pathway and a possible proton exit pathway in Rhodobacter sphaeroides cytochrome c oxidase

Protons are transferred from the inner surface of cytochrome c oxidase to the active site by the D and K pathways, as well as from the D pathway to the outer surface by a largely undefined proton exit route. Alteration of the initial proton acceptor of the D pathway, D132, to alanine has previously been shown to greatly inhibit oxidase turnover and slow proton uptake into the D pathway. Here it is shown that the removal of subunit III restores a substantial rate of O(2) reduction to D132A. Presumably an alternative proton acceptor for the D pathway becomes active in the absence of subunit III and D132. Thus, in the absence of subunit III cytochrome oxidase shows greater flexibility in terms of proton entry into the D pathway. In the presence of DeltaPsi and DeltapH, turnover of the wild-type oxidase or D132A is slower in the absence of subunit III. Comparison of the turnover rates of subunit III-depleted wild-type oxidase to those of the zinc-inhibited wild-type oxidase containing subunit III, both reconstituted into vesicles, leads to the hypothesis that the absence of subunit III inhibits the ability of the normal proton exit pathway to take up protons from the outside in the presence of DeltaPsi and DeltapH. Thus, subunit III appears to affect the transfer of protons from both the inner and outer surfaces of cytochrome oxidase, perhaps accounting for the long-observed lower efficiency of proton pumping by the subunit III-depleted oxidase.     

Differential effects of glutamate-286 mutations in the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo(3) ubiquinol oxidase from Escherichia coli

Both the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcO(aa3)) and the closely related bo(3)-type ubiquinol oxidase from Escherichia coli (EcQO(bo3)) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcO(aa3) result in long range conformational changes that influence the protein interactions with both heme a and heme a(3). Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsCcO(aa3) to ~0.05% is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a(3)-Cu(B) active site. In contrast, the E286C mutation of EcQO(bo3) exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3%) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsCcO(aa3), the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQO(bo3) is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Ferrocyanide as electron donor to cytochrome aa3. Cytochrome c requirement for oxygen uptake.

1. In the absence of cytochrome c, ferrocyanide or ferrous sulphate reduces cytochrome c oxidase (EC 1.9.3.1), but no continuous oxygen uptake ensues, as it does with N,N,N',N'-tetramethyl-p-phenylenediamine or reduced phenazine methosulphate as reductants, unless a substoichiometric amount of cytochrome c or an excess of clupein is present. Cytochrome c cannot be replaced by porphyrin cytochrome c. 2. Cytochrome c, porphyrin cytochrome c and clupein all stimulate the reduction of cytochrome aa3 by ferrocyanide. 3. A model is proposed to explain these findings in which a high-affinity site for cytochrome c on the oxidase regulates the access of hydrophilic electron donors to a low-affinity site, and reduction via the high-affinity site is required for continuous oxygen uptake. 4. Furthermore, it is shown that upon reaction of oxidase with ferrocyanide, cyano-oxidase is formed.     

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.