Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with K_i values of 4.5, 91, 200 and 330 μM, respectively.2. The inhibition constant K_i of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme.3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k₁ of 33 M^?1 · s^?1 and dissociation constant K_d of 3.9 mM.4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M^?1 · s^?1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes.5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome a₃-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

The effect of inhibitors on the oxygen kinetics of cytochrome c oxidase.

1. The oxygen kinetics of purified beef heart cytochrome c oxidase were investigated. 2. The effect of addition of various fixed concentrations of the inhibitors CO, HN3, HCOOH, HCN and H2S on the double reciprocal plot of respiration rate against oxygen concentration was studied. 3. CO is strictly competitive, azide and formate are uncompetitive, and cyanide and sulfide are non-competitive inhibitors towards oxygen. 4. Binding constants for the various inhibitors from secondary plots of the oxygen kinetics at pH 7.4 are: CO: Ki = 0.32 micronM, azide: Ki = 33 micronM; formate: Ki = 15 mM; cyanide: Ki = 0.2 micronM and sulfide: Ki = 0.2 micronM. 5. The possible significance of these results in the elucidation of the reaction mechanism is discussed.     

Reduction of oxygen-pulsed cytochrome c oxidase by cytochrome c and other electron donors

1. Stopped-flow experiments were performed in which solutions containing dithionite were mixed with air-saturated buffer. Cytochrome c oxidase present in the dithionite-containing syringe is fully oxidized within the mixing time and the oxygen-pulsed form of the oxidase is produced.2. The reduction of this form by dithionite, by dithionite plus cytochrome c and by dithionite plus methyl viologen or benzyl viologen was followed and compared with the corresponding reduction reactions of the ‘resting’ oxidized enzyme. Reduction by dithionite is relatively slow, but the rate of reduction is greatly increased by addition of cytochrome c or the viologens, which are even more effective than cytochrome c on a molar basis.3. Profound differences between the transient kinetics of the reduction of the two oxidized oxidase derivatives were observed. The results are consistent with a direct reduction of cytochrome a followed by an intramolecular electron transfer to cytochrome a₃ (k^obs₁ = 7.5 s^?1 for the oxygen-pulsed oxidase).4. The spectrum of the oxygen-pulsed oxidase formed within 5 ms of the mixing closely resembles that of the ‘oxygenated’ compound, but there were small differences between the two spectra.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrateferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the K_m (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the ‘association’ rate but also by increasing the ‘dissociation’ rate for bound cytochrome c converting the ‘primary’ (T) site at high salt concentrations into a site similar kinetically to the ‘secondary’ (L) site at low ionic strength. A finite K_m of 170 μM at very high ionic strength indicates a ratio of $\text{K}{}^{\mathrm{\infty }}{}_{\mathrm{<mtext>M</mtext>}}\text{K}{}^0{}_{\mathrm{<mtext>M</mtext>}}$ of about 5000. It is proposed that anions either modify the E¹₀ of cytochrome c bound at the primary (T) site or that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O₂) conditions. The system measures the concentrations of O₂ and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O₂ concentration and electron turnover of the enzyme. At a high O₂ concentration (70 μM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O₂. At low O₂ (15 μM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O₂ consumption. At both high and low O₂ concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.     

The role of protein dynamics and thermal fluctuations in regulating cytochrome c/cytochrome c oxidase electron transfer

In this overview we present recent combined electrochemical, spectroelectrochemical, spectroscopic and computational studies from our group on the electron transfer reactions of cytochrome c and of the primary electron acceptor of cytochrome c oxidase, the Cu_A site, in biomimetic complexes. Based on these results, we discuss how protein dynamics and thermal fluctuations may impact on protein ET reactions, comment on the possible physiological relevance of these results, and finally propose a regulatory mechanism that may operate in the Cyt/CcO electron transfer reaction in vivo. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Transmembrane proton translocation by cytochrome c oxidase

Respiratory heme-copper oxidases are integral membrane proteins that catalyze the reduction of molecular oxygen to water using electrons donated by either quinol (quinol oxidases) or cytochrome c (cytochrome c oxidases, CcOs). Even though the X-ray crystal structures of several heme-copper oxidases and results from functional studies have provided significant insights into the mechanisms of O₂-reduction and, electron and proton transfer, the design of the proton-pumping machinery is not known. Here, we summarize the current knowledge on the identity of the structural elements involved in proton transfer in CcO. Furthermore, we discuss the order and timing of electron-transfer reactions in CcO during O₂ reduction and how these reactions might be energetically coupled to proton pumping across the membrane.     

Biochemical and biophysical studies on cytochrome c oxidase. XVIII. Potentiometric titrations of cytochrome c oxidase followed by circular dichroism

1. Potentiometric circular dichroism titrations of cytochrome c oxidase, carried out in the absence of cytochrome c, confirm the potentiometric equivalence of the two heme a groups of cytochrome c oxidase. In the presence of cytochrome c, two different midpoint potentials are found for the two heme a groups of cytochrome c oxidase.2. Circular dichroism difference spectra (reduced minus oxidized) of the two heme a components of cytochrome c oxidase have been obtained by means of this potentiometric titration. On reduction of the first heme a group a circular dichroism difference spectrum is obtained with peaks at 425, 442 and 602.5 nm; the second heme a group shows difference peaks at 434, 447 and 608 nm. Whereas both heme a groups contribute about equally to the absorbance difference spectrum, the second heme a group reduced contributes about twice as much to the circular dichroism difference spectrum as does the first heme a group.3. From these spectral and circular dichroism differences it is concluded that, on reduction of or ligand binding to cytochrome c oxidase, conformational changes occur which affect the symmetry of the environments of the heme a groups.     

Biochemical and biophysical studies on cytochrome c oxidase XVI. Circular dichroic study of cytochrome c oxidase and its ligand complexes

1. CD spectra of cytochrome c oxidase have been determined both in the absence and presence of the extrinsic ligands CO, NO, cyanide and azide.2. CO and NO affect the CD spectrum of cytochrome c oxidase in a similar way.3. Cyanide and azide also affect the CD spectrum of cytochrome c oxidase in a similar way, but distinctly different from CO and NO.4. From the CD spectra of the oxidized and reduced enzyme, in the presence and absence of extrinsic ligands, CD difference spectra (reduced minus oxidized) are calculated for the so-called cytochrome a and cytochrome a₃ moieties of the enzyme.5. These spectra are largely dependent on the extrinsic ligand used. It is therefore concluded that these spectra do not represent independent cytochrome a and cytochrome a₃ difference spectra, but that heme-heme interactions occur within the cytochrome c oxidase molecule, in such a way that binding of a ligand to one of the heme a groups of cytochrome c oxidase affects the spectral properties of the other heme a group.6. As a consequence, ligand-binding studies cannot give information as to the pre-existence of separate cytochrome a and cytochrome a₃ moieties in the absence of extrinsic ligands.     

Inhibition of cytochrome c oxidase function by dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) reacted with beef heart cytochrome c oxidase to inhibit the proton-pumping function of this enzyme and to a lesser extent to inhibit electron transfer. The modification of cytochrome c oxidase in detergent dispersion or in vesicular membranes was in subunits II–IV. Labelling followed by fragmentation studies showed that there is one major site of modification in subunit III. DCCD was also incorporated into several sites in subunit II and at least one site in subunit IV. The major site in subunit III has a specificity for DCCD at least one order of magnitude greater than that of other sites (in subunits II and IV). Its modification could account for all of the observed effects of the reagent, at least for low concentrations of DCCD. Labelling of subunit II by DCCD was blocked by prior covalent attachment of arylazidocytochrome c, a cytochrome c derivative which binds to the high-affinity binding site for the substrate. The major site of DCCD binding in subunit III was sequenced. The label was found in glutamic acid 90 which is in a sequence of eight amino acids remarkably similar to the DCCD-binding site within the proteolipid protein of the mitochondrial ATP synthetase.     

Kinetics of cytochrome c oxidase from yeast. Membrane-facilitated electrostatic binding of cytochrome c showing a specific interaction with cytochrome c oxidase and inhibition by ATP (With an appendix on the occurrence of two-dimensional diffusion of cytochrome c on the membrane surface)

The steady-state kinetics of purified yeast cytochrome c oxidase were investigated at low ionic strength where the electrostatic interaction with cytochrome c is maximized. In 10 mM cacodylate/Tris (pH 6.5) the oxidation kinetics of yeast iso-1-cytochrome c were sigmoidal with a Hill coefficient of 2.35, suggesting cooperative binding. The half-saturation point was 1.14 μM. Horse cytochrome c exhibited Michaelis-Menten kinetics with a higher affinity (K_m = 0.35 μM) and a 100% higher maximal velocity.In 67 mM phosphate the Hill coefficient for yeast cytochrome c decreased to 1.42, and the species differences in Hill coefficients were lessened. Under the latter conditions, a yeast enzyme preparation partially depleted of phospholipids was activated on addition of diphosphatidylglycerol liposomes. When the enzyme was incorporated into sonicated yeast promitochondrial particles the apparent K_m for horse cytochrome c was considerably lower than the value for the isolated enzyme.ATP was found to inhibit both the isolated oxidase and the membrane-bound enzyme. With the isolated enzyme in 10 mM cacodylate/Tris, 3 mM ATP increased the half-saturation point with yeast cytochrome c 3-fold, without altering the maximal velocity or the Hill coefficient. 67 mM phosphate abolished the inhibition of the isolated oxidase by ATP.The increase in affinity for cytochrome c produced by binding the oxidase to the membrane was not observed in the presence of 3 mM ATP, with the result that the membrane-bound enzyme was more sensitive to inhibition by ATP. ADP was a less effective inhibitor than ATP, and did not prevent the inhibition by ATP.It is proposed that non-specific electrostatic binding of cytochrome c to phospholipid membranes, followed by rapid lateral diffusion, is responsible for the dependence of the affinity on the amount and nature of the phospholipids and on the ionic strength.ATP may interfere with the membrane-facilitated binding of cytochrome c by a specific electrostatic interaction with the membrane or by binding to cytochrome c.     

The effect of pH on the oxygen kinetics of cytochrome c oxidase proteoliposomes

The effect of pH on the oxygen kinetics of cytochrome c oxidase incorporated into phospholipid vesicles is studied. The pH profiles of the oxygen kinetics of energized and deenergized oxidase vesicles are similar. An effect of pH on the slope of the reciprocal plot of rate against oxygen concentration is observed, and this may indicate that protons are involved in the rate limiting step of the reaction between oxygen and reduced oxidase. In contrast to the pH dependence of the oxygen kinetics, the binding of CO to the oxidase is not pH dependent.     

Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated.Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H⁺ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K⁺. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons.The proton ejection is the result of true translocation of H⁺ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system.Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions.It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H⁺.     

A 1H-NMR longitudinal relaxation study of the interaction between cytochrome c and cytochrome c oxidase

The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by ¹H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand.     

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.     

Cytochrome c reactivity in its complexes with mammalian cytochrome c oxidase and yeast peroxidase

1.

1. The ascorbate reducibility of cytochrome c (beef or horse heart) in its complexes with cytochrome c oxidase (beef heart) and cytochrome c peroxidase (yeast) has been studied.     

Water-gated mechanism of proton translocation by cytochrome c oxidase

Cytochrome c oxidase is essential for aerobic life as a membrane-bound energy transducer. O₂ reduction at the haem a₃-Cu_B centre consumes electrons transferred via haem a from cytochrome c outside the membrane. Protons are taken up from the inside, both to form water and to be pumped across the membrane (M.K.F. Wikström, Nature 266 (1977) 271 [1]; M. Wikström, K. Krab, M. Saraste, Cytochrome Oxidase, A Synthesis, Academic Press, London, 1981 [2]). The resulting electrochemical proton gradient drives ATP synthesis (P. Mitchell, Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin, UK, 1966 [3]). Here we present a molecular mechanism for proton pumping coupled to oxygen reduction that is based on the unique properties of water in hydrophobic cavities. An array of water molecules conducts protons from a conserved glutamic acid, either to the Δ-propionate of haem a₃ (pumping), or to haem a₃-Cu_B (water formation). Switching between these pathways is controlled by the redox-state-dependent electric field between haem a and haem a₃-Cu_B, which determines the water-dipole orientation, and therefore the proton transfer direction. Proton transfer via the propionate provides a gate to O₂ reduction. This pumping mechanism explains the unique arrangement of the metal cofactors in the structure. It is consistent with the large body of biochemical data, and is shown to be plausible by molecular dynamics simulations.     

Effect of cardiolipin on the enzymatic activity of Nitrobacter agilis cytochrome c oxidase

Effects of cardiolipin on the reaction rates of Nitrobacter agilis cytochrome c oxidase with cytochrome c were studied at various concentrations of phosphate buffer. Cardiolipin stimulated greatly the oxidation by the enzyme of horse and yeast ferrocytochromes c, especially at higher ionic strengths. However, the oxidation by the enzyme of N. agilis ferrocytochrome c-550, the physiological electron donor for the oxidase, was not accelerated by addition of cardiolipin. Analysis of the lipid compositions showed that neither the cell membranes of N. agilis nor the enzyme preparation contained cardiolipin. These results suggest that cardiolipin is not necessary for the reaction of N. agilis cytochrome c oxidase with N. agilis cytochrome c-550. On the basis of these results, the difference in the reactivity with cytochrome c of cytochrome c oxidase between the bacterial and mitochondrial enzymes is discussed.     

The effect of mitochondrial energization on cytochrome c oxidase kinetics as measured at low temperatures. I. The reaction with carbon monoxide

The kinetics of CO binding by the cytochrome c oxidase of pigeon heart mitochondria were studied as a function of membrane energization at temperatures from 180 to 280°K in an ethylene glycol/water medium. Samples energized by ATP showed acceleration of CO binding compared to those untreated or uncoupled by carbonylcyanide p-trifluoromethoxyphenylhydrazone but only at relatively low temperatures and high CO concentrations. Experiments using samples in a “mixed valency” (partially oxidized) state showed that the acceleration of ligand binding is not due to the formation of a partially oxidized state via reverse electron transport.It is concluded that in the deenergized state one CO molecule can be closely associated with the cytochrome a₃ heme site without actually being bound to the heme iron; in the energized state, two or more ligand molecules can occupy this intermediate position.The change in the apparent ligand capacity of a region near the heme iron in response to energization is evidence for an interaction between cytochrome oxidase and the ATPase system. Furthermore, these results suggest a control mechanism for O₂ binding.