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.     

Interaction between Mitochondrial Cytochromes and Linoleic Acid Hydroperoxide: POSSIBLE CONFUSION WITH LIPOXYGENASE AND ALTERNATIVE PATHWAY

O₂ uptake by tissue extracts in the presence of linoleic acid is generally ascribed to lipoxygenase. Such an O₂ uptake can be observed not only with mitochondria of Solanum tuberosum L. and Arum maculatum L. and pure lipoxygenase but also with cytochrome c. However, the rate of oxidation is highly dependent on the procedure used to prepare the solutions of linoleic acid. Unless special care is taken to prevent contact between linoleic acid and O₂, it appears that linoleic acid hydroperoxide is readily formed. This derivative can be readily oxidized by mitochondria or cytochrome c. On the other hand, the use of a rapid and specific enzymic procedure to estimate the disappearance of linoleic acid demonstrates that linoleic acid itself is not consumed at any appreciable rate by mitochondria or cytochrome c, the true substrate being linoleic acid hydroperoxide. During the reaction, the heme nucleus of added cytochrome c or of mitochondrial cytochromes undergoes deep alterations. Therefore, caution should be exerted when equating an O₂ uptake observed in the presence of linoleic acid to a lipoxygenase activity. The same holds true for the similarity of reaction towards specific inhibitors between lipoxygenase and the cyanide-insensitive pathway oxidase.     

Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b₅, cytochrome c₁ and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, and 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c₁ and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.     

Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase

The inhibition by superoxide dismutase of cytochrome c reduction by a range of semiquinone radicals has been studied. The semiquinones were produced from the parent quinones by reduction with xanthine and xanthine oxidase. Most of the quinones studied were favored over O₂ as the enzyme substrate, and in air as well as N₂, semiquinone radicals rather than superoxide were produced and they caused the cytochrome c reduction. With all but one of the quinones (benzoquinone), cytochrome c reduction in air was inhibited by superoxide dismutase, but the amount of enzyme required for inhibition was up to 100 times greater than that required to inhibit reduction by superoxide. It was highest for the quinones with the highest redox potential. These results demonstrate how superoxide dismutase can inhibit cytochrome c reduction by species other than superoxide. They can be explained by the dismutase displacing the equilibrium: semiquinone + O₂ ? quinone + O₂^? to the right, thereby allowing the forward reaction to out-compete other reactions of the semiquinone. The implication from these findings that superoxide dismutase-inhibitable reduction of cytochrome c may not be a specific test for superoxide production is discussed.     

Oxidative Half-reaction of Arabidopsis thaliana Sulfite Oxidase: GENERATION OF SUPEROXIDE BY A PEROXISOMAL ENZYME*

Vertebrate forms of the molybdenum-containing enzyme sulfite oxidase possess a b-type cytochrome prosthetic group that accepts reducing equivalents from the molybdenum center and passes them on to cytochrome c. The plant form of the enzyme, on the other hand, lacks a prosthetic group other than its molybdenum center and utilizes molecular oxygen as the physiological oxidant. Hydrogen peroxide is the ultimate product of the reaction. Here, we present data demonstrating that superoxide is produced essentially quantitatively both in the course of the reaction of reduced enzyme with O₂ and during steady-state turnover and only subsequently decays (presumably noncatalytically) to form hydrogen peroxide. Rapid-reaction kinetic studies directly following the reoxidation of reduced enzyme demonstrate a linear dependence of the rate constant for the reaction on [O₂] with a second-order rate constant of k_ox = 8.7 × 10⁴ ± 0.5 × 10⁴ m⁻¹s⁻¹. When the reaction is carried out in the presence of cytochrome c to follow superoxide generation, biphasic time courses are observed, indicating that a first equivalent of superoxide is generated in the oxidation of the fully reduced Mo(IV) state of the enzyme to Mo(V), followed by a slower oxidation of the Mo(V) state to Mo(VI). The physiological implications of plant sulfite oxidase as a copious generator of superoxide are discussed.     

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.     

Effect of crosslinking cytochrome c oxidase

Bovine heart cytochrome c oxidase and rat liver mitochondria were crosslinked in the presence and absence of cytochrome c. Biimidate treatment of purified cytochrome oxidase, which results in the crosslinkage of all of the oxidase protomers except subunit I when ? 20% of the free amines are modified, inhibits ascorbate-N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity. Intermolecular crosslinking of cytochrome oxidase molecules, which results in the formation of large enzyme aggregates displaying rotational correlation times ? 1 ms, does not affect oxidase activity. Crosslinking of mitochondria covalently binds the cytochrome bc₁ and aa₃ complexes to cytochrome c, and inhibits steady-state oxidase activity. Addition of cytochrome c to purified cytochrome oxidase or to cytochrome c-depleted mitoplasts increases this inhibition slightly. Cytochrome c oligomers act as competitive inhibitors of native cytochrome c; however, crosslinking of cytochrome c to cytochrome c-depleted mitoplasts or purified cytochrome oxidase results in a catalytically inactive complex. These experiments indicate that cytochrome c oxidase subunit interactions are required for activity, and that cytochrome c mobility may be essential for electron transport between cytochrome c reductase and oxidase.     

A Biochemical Approach to Study the Role of the Terminal Oxidases in Aerobic Respiration in Shewanella oneidensis MR-1

The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb ₃-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb ₃-type oxidase is the major terminal oxidase under aerobic conditions while both cbb ₃-type and bd-type oxidases are involved in respiration at low-O₂ tensions. On the contrary, the low O₂-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa ₃-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa ₃-type oxidase in S. oneidensis MR-1 are discussed.     

Internal charge transfer in cytochrome c oxidase at a limited proton supply: Proton pumping ceases at high pH

Background

In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O₂ is linked to proton uptake from solution to form H₂O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane.

Methods

In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O₂. These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles.

Results

The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O₂ reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH.

Conclusions

The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the “wrong” side of the membrane) and that at this pH the net proton-uptake stoichiometry is ∼ 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme.

General significance

These results provide new insights into the function of cytochrome c oxidase.     

The solid state physical theory of cytochrome oxidase kinetics. Inhibition of second order rate constant,and second to first order kinetic shift with increasing oxygen,predicted from electron injection and trapping

Conduction of electrons through the solid protein cytochrome oxidase particle in accord with Ohm's law, driven by the difference in electrode potentials of two substrates which exchange electrons with the two sides of the enzyme particle, was previously shown to explain the inhibitory effect of cytochromec on the first order rate constant, and to predict the low semiconduction activation energy of dried cytochrome oxidase. If the solid conduction path in the cytochrome oxidase particle shows electron injection from sites of electron exchange with substrate, and shows trapping of conduction electrons by reversible O₂ complexes, then one may also predict that the first order kinetics observed as high O₂ concentrations will change to second order kinetics at lower O₂ concentrations, as observed by Gibson and Wharton. One may also predict quantitatively the inhibitory effect of increasing O₂ concentrations on the second order rate constant as observed by Gibson and Wharton. The same concept of electron trapping by O₂ complexes provides a possible reason for the unusually low semiconduction activation energy of cytochrome oxidase.     

Sulfide-Resistant Respiration in Leaves of Elodea canadensis Michx: Comparison with Cyanide-Resistant Respiration

The rate of dark O₂ uptake of Elodea canadensis leaves was titrated with either cyanide or sulfide in the presence and in the absence of 5 millimolar salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase. The inhibition of O₂ uptake by SHAM alone was very small (3-6%), suggesting that actual respiration mainly occurred through the cytochrome pathway. O₂ uptake was slightly stimulated by cyanide at concentrations of 50 micromolar or higher, but in the presence of SHAM respiration was strongly suppressed. The effects of sulfide on O₂ uptake were similar to those of cyanide, except that the percent stimulation of O₂ uptake by sulfide alone was somewhat higher than that of cyanide. However, the estimates of the capacity of the alternative pathway were similar with both inhibitors. Another difference is that maximal inhibition of respiration in the presence of SHAM was observed with lower concentrations of sulfide (50 micromolar) than cyanide (250 micromolar). The results suggest that sulfide can be used as a suitable inhibitor of cytochrome c oxidase in studies with intact plant tissues, and that sulfide does not apparently inhibit the alternative oxidase.     

Regulation of Cytochrome c- and Quinol Oxidases,and Piezotolerance of Their Activities in the Deep-Sea Piezophile Shewanella violacea DSS12 in Response to Growth Conditions

The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O₂ concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O₂ concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O₂ concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments.     

Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii

A membrane-bound cytochrome oxidase from Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using an ascorbate-TMPD oxidation assay. The oxidase was ‘solubilized’ from a sonic-type electron-transport particle (R₃ fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27–70% (NH₄)₂SO₄. The highly purified cytochrome oxidase has a V of 60–78 μgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN₃ and NH₂OH; NaNO₂ (but not NaNO₃) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c₄ and cytochrome o; cytochromes a₁ and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c₄?o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a₃ oxidase of mammalian mitochondria.     

Variations in the Alternative Oxidase in Chlamydomonas Grown in Air or High CO(2)

Chlamydomonas in the resting phase of growth has an equal capacity of about 15 micromole O₂ uptake per hour per milligram of chlorophyll for both the cytochrome c, CN-sensitive respiration, and for the alternative, salicylhydroxamic acid-sensitive respiration. Alternative respiration capacity was measured as salicylhydroxamic acid inhibited O₂ uptake in the presence of CN, and cytochrome c respiration capacity as CN inhibition of O₂ uptake in the presence of salicylhydroxamic acid. Measured total respiration was considerably less than the combined capacities for respiration. During the log phase of growth on high (2-5%) CO₂, the alternative respiration capacity decreased about 90% but returned as the culture entered the lag phase. When the alternative oxidase capacity was low, addition of salicylic acid or cyanide induced its reappearance. When cells were grown on low (air-level) CO₂, which induced a CO₂ concentrating mechanism, the alternative oxidase capacity did not decrease during the growth phase. Attempts to measure in vivo distribution of respiration between the two pathways with either CN or salicylhydroxamic acid alone were inconclusive.     

Light-induced electron transport pathways in membrane preparations from Rhodopseudomonas capsulata

The photosynthetic electron transport chain in Rhodopseudomonas capsulata cells was investigated by studying light-induced noncyclic electron transport from external donors to O₂. Two membrane preparations with opposite membrane polarity, heavy chromatophores and regular chromatophores, were used to characterize this electron transport. It was shown that with lipophylic electron donors such as dichloroindophenol, diaminobenzidine, and phenazine methosulfate the electron transport activities were similar in both types of chromatophores, whereas horse heart cytochrome c, K₄Fe(CN)₆, 3-sulfonic acid phenazine methosulfate, and ascorbate, which cannot penetrate the membrane, were more active in the heavy chromatophores than in the regular chromatophores. Partial depletion of cytochrome c₂ from the heavy chromatophores caused a decrease in the light-induced O₂ uptake from reduced dichloroindophenol or ascorbate. The activity could be restored with higher concentrations of dichloroindophenol or with purified cytochrome c₂ from Rps. capsulata. It is assumed that in the heavy chromatophores the artificial electron donors are oxidized on the cytochrome c₂ level which faces the outside medium. However, cytochrome c₂ is not exposed to the outside medium in the regular chromatophores. Therefore, only lipophylic donors would interact with cytochrome c₂ in this system, while hydrophylic donors would be oxidized by another component of the electron transport chain which is exposed to the external medium. Studies with inhibitors of photophosphorylation show that antimycin A enhances the light-dependent electron transport to O₂ whereas 1:10 phenanthroline inhibited the reaction, but dibromothymoquinone did not affect it. It is assumed that a nonheme iron protein is taking part in this electron transport but not a dibromothymoquinone-sensitive quinone. The terminal oxidase of the light-dependent pathway is different from the two oxidases of the respiratory chain. The ratio between electrons entering the system and molecules of O₂ consumed is 4, which means that the end product of O₂ reduction is H₂O.     

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.     

A Critical Role for the cccA Gene Product,Cytochrome c2, in Diverting Electrons from Aerobic Respiration to Denitrification in Neisseria gonorrhoeae

Neisseria gonorrhoeae is a microaerophile that, when oxygen availability is limited, supplements aerobic respiration with a truncated denitrification pathway, nitrite reduction to nitrous oxide. We demonstrate that the cccA gene of Neisseria gonorrhoeae strain F62 (accession number NG0292) is expressed, but the product, cytochrome c₂, accumulates to only low levels. Nevertheless, a cccA mutant reduced nitrite at about half the rate of the parent strain. We previously reported that cytochromes c₄ and c₅ transfer electrons to cytochrome oxidase cbb₃ by two independent pathways and that the CcoP subunit of cytochrome oxidase cbb₃ transfers electrons to nitrite. We show that mutants defective in either cytochrome c₄ or c₅ also reduce nitrite more slowly than the parent. By combining mutations in cccA (Δc₂), cycA (Δc₄), cycB (Δc₅), and ccoP (ccoP-C368A), we demonstrate that cytochrome c₂ is required for electron transfer from cytochrome c₄ via the third heme group of CcoP to the nitrite reductase, AniA, and that cytochrome c₅ transfers electrons to nitrite reductase by an independent pathway. We propose that cytochrome c₂ forms a complex with cytochrome oxidase. If so, the redox state of cytochrome c₂ might regulate electron transfer to nitrite or oxygen. However, our data are more consistent with a mechanism in which cytochrome c₂ and the CcoQ subunit of cytochrome oxidase form alternative complexes that preferentially catalyze nitrite and oxygen reduction, respectively. Comparison with the much simpler electron transfer pathway for nitrite reduction in the meningococcus provides fascinating insights into niche adaptation within the pathogenic neisseriae.     

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.