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.     

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.     

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.     

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.     

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.     

Copper and manganese electron spin resonance studies of cytochrome c oxidase from Paracoccus denitrificans

The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms. However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II). The pure Mn(II) spectrum is observed at ?40°C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P. denitrificans cells that had been grown in a Mn(II)-depleted medium. This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart. Manganese is apparently not an essential component of P. denitrificans cytochrome c oxidase since it is present in substoichiometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c. However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase. In contrast, manganese added to the yeast oxidase or to the manganese-free P. denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme. This suggests that the manganese normally associated with P. denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme.     

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.     

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.     

Reaction of CO with cytochrome c oxidase. Titration of the reaction site with chemical oxidant and reductant

The number of reducing equivalents required to form the reduced cytochrome a₃-CO compound has been determined for suspensions of submitochondrial particles and for isolated cytochrome c oxidase. Anaerobic preparations were titrated reductively with NADH and oxidatively with O₂ in the presence of high concentrations of CO (0.4 to 0.8 mM) while monitoring reduction of cytochrome a and the formation of the reduced cytochrome a₃-CO compound by their characteristic absorbance changes. Analysis of the titration data show that 2.0±0.3 and 2.1±0.2 reducing equivalents per mol of cytochrome oxidase (per cytochrome a) are required for formation of the reduced cytochrome a₃-CO compound in submitochondrial particles and isolated cytochrome c oxidase, respectively. In each case, the formation of the CO compound is proportional to the number of equivalents accepted by the preparation, indicating that the two equivalents are equal and the effective n value for the reaction is 2.0. Potentiometric titrations of cytochrome c oxidase using the cobalt orthophenanthrolene complex (E_{m, 7.0} = 0.37 V) as mediator give the same half-reduction potential values for cytochrome a and a₃ as those obtained using the ferro-ferricyanide couple. The formation of the reduced cytochrome a₃-CO compound at pH 7.0, in the presence of 0.6 mM CO and with CO-orthophenanthrolene as mediator occurs with a half-reduction potential of 0.45 V and requires two electrons. These data confirm and extend the observation of Lindsay et al. (Arch. Biochim. Biophys. (1975) 169, 492–505) that both the “invisible” copper and cytochrome a₃ must be reduced in order for CO to bind with high affinity.     

Formamide probes a role for water in the catalytic cycle of cytochrome c oxidase

Formamide is a slow-onset inhibitor of mitochondrial cytochrome c oxidase that is proposed to act by blocking water movement through the protein. In the presence of formamide the redox level of mitochondrial cytochrome c oxidase evolves over the steady state as the apparent electron transfer rate from cytochrome a to cytochrome a₃ slows. At maximal inhibition cytochrome a and cytochrome c are fully reduced, whereas cytochrome a₃ and Cu_B remain fully oxidized consistent with the idea that formamide interferes with electron transfer between cytochrome a and the oxygen reaction site. However, transient kinetic studies show that intrinsic rates of electron transfer are unchanged in the formamide-inhibited enzyme. Formamide inhibition is demonstrated for another member of the heme-oxidase family, cytochrome c oxidase from Bacillus subtilis, but the onset of inhibition is much quicker than for mitochondrial oxidase. If formamide inhibition arises from a steric blockade of water exchange during catalysis then water exchange in the smaller bacterial oxidase is more open. Subunit III removal from the mitochondrial oxidase hastens the onset of formamide inhibition suggesting a role for subunit III in controlling water exchange during the cytochrome c oxidase reaction.     

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.     

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.     

Cyanobacterial cytochrome cM: Probing its role as electron donor for CuA of cytochrome c oxidase

It is well known that efficient functioning of photosynthetic (PET) and respiratory electron transport (RET) in cyanobacteria requires the presence of either cytochrome c₆ (Cytc₆) or plastocyanin (PC). By contrast, the interaction of an additional redox carrier, cytochrome c_M (Cytc_M), with either PET or RET is still under discussion. Here, we focus on the (putative) role of Cytc_M in cyanobacterial respiration. It is demonstrated that genes encoding the main terminal oxidase (cytochrome c oxidase, COX) and cytochrome c_M are found in all 44 totally or partially sequenced cyanobacteria (except one strain). In order to check whether Cytc_M can act as electron donor to COX, we investigated the intermolecular electron transfer kinetics between Cytc_M and the soluble Cu_A domain (i.e. the donor binding and electron entry site) of subunit II of COX. Both proteins from Synechocystis PCC6803 were expressed heterologously in E. coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (2.4 ± 0.1) × 10⁵ M^− 1 s^− 1 and (9.6 ± 0.4) × 10³ M^− 1 s^− 1 (5 mM phosphate buffer, pH 7, 50 mM KCl). A comparative analysis with Cytc₆ and PC demonstrates that Cytc_M functions as electron donor to Cu_A as efficiently as Cytc₆ but more efficient than PC. Furthermore, we demonstrate the association of Cytc_M with the cytoplasmic and thylakoid membrane fractions by immunobloting and discuss the potential role of Cytc_M as electron donor for COX under stress conditions.     

The inside pH determines rates of electron and proton transfer in vesicle-reconstituted cytochrome c oxidase

Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane. The results show that the pH on the inside of the membrane fully determines the kinetics of internal electron transfers that are linked to proton pumping. Thus, even though proton release is rate limiting for these reaction steps (Salomonsson et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 17624), the transition kinetics is insensitive to the outside pH (in the range 6-9.5).     

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⁺.