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.     

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.     

Isolation and properties of cytochrome c-553, cytochrome c-550, and cytochrome c-549, 554 from Nitrobacter agilis

Three c-type cytochromes isolated from Nitrobacter agilis were purified to apparent homogeneity: cytochrome c-553, cytochrome c-550 and cytochrome c-549, 554. Their amino acid composition and other properties were studied. Cytochrome c-553 was isolated as a partially reduced form and could not be oxidized by ferricyanide. The completely reduced form of the cytochrome had absorption maxima at 419, 524 and 553 nm. It had a molecular weight of 25 000 and dissociated into two polypeptides of equal size of 11 500 during SDS gel electrophoresis. The isoelectric point of cytochrome c-553 was pH 6.8. The ferricytochrome c-550 exhibited an absorption peak at 410 nm and the ferrocytochrome c showed peaks at 416, 521 and 550 nm. The molecular weight of the cytochrome estimated by gel filtration and by SDS gel electrophoresis was 12 500. It had an E_m(7) value of 0.27 V and isoelectric point pH 8.51. The N-terminal sequence of cytochrome c-550 showed a clear homology with the corresponding portions of the sequences of other c-type cytochromes. Cytochrome c-549, 554 possessed atypical absorption spectra with absorption peaks at 402 nm as oxidized form and at 419, 523, 549 and 554 nm when reduced with Na₂S₂O₄. Its molecular weight estimated by gel filtration and SDS polyacrylamide gel electrophoresis was 90 000 and 46 000, respectively. The cytochrome had an isoelectric point of pH 5.6. Cytochrome c-549, 554 was highly autoxidizable.     

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.     

Reaction of cytochrome c in the electron-transport chain of Paracoccus denitrificans

The reaction of the cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) of Paracoccus denitrificans cytoplasmic membranes with the endogenous cytochrome c of the membranes was studied, as well as its interaction with added exogenous cytochrome c from P. denitrificans or bovine heart. The polarographic method was employed, using N,N,N′,N′-tetramethyl-p-phenylenediamine plus ascorbate to reduce the cytochrome c. We found that overall electron transport can proceed maximally while the cytochrome c remains membrane bound; NADH or succinoxidase activities were not inhibited by the addition of substances which bind the P. denitrificans cytochrome c strongly. In contrast to our observations with the spectrophotometric method (Smith, L., Davies, H.C. and Nava, M.E. (1976) Biochemistry 15, 5827–5831), in the polarographic assays the membrane-bound oxidase reacts with about equal rapidity with exogenous bovine and P. denitrificans cytochromes c. The reaction of the oxidase with the endogenous cytochrome c proceeds at high rates and preferentially to that with exogenous cytochrome c; the reaction with the latter, but not the former is inhibited by positively charged poly(l-lysine). The cytochrome c and the oxidase appear to be very closely associated on the membrane.     

Nitrobacter agilis Cytochrome c-550: Isolation, Physicochemical and Enzymatic Properties, and Primary Structure

Nitrobacter agilis cytochrome c-550 was purified to an electrophoreticallyhomogeneous state, and some of its properties were determined.The cytochrome showed an absorption peak at 410 nm in the oxidizedform, and peaks at 416, 521 and 550 nm in the reduced form.Its isoelectric point was 8.1 at 5?C. Analysis of the aminoacid composition showed that the cytochrome molecule was composedof 108 amino acid residues, 16 of which were lysine residues. The cytochrome reacted rapidly with N. agilis cytochrome c oxidaseand yeast cytochrome c peroxidase and more slowly with Pseudomonasaeruginosa nitrite reductase and bovine cytochrome c oxidase.The reactivities with these redox enzymes suggested that thecytochrome might be an evolutionary stage between bacterialand eukaryotic cytochromes c. The primary structure of the cytochrome from the N-terminusto the 85th residue was determined. The N-terminal sequencewas homologous to the corresponding portion of the primary structureof horse cytochrome c. ¹ Present adress: Department of Chemistry, Faculty of Science,Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo,152, Japan. (Received December 3, 1981; Accepted January 28, 1982)     

Transient Binding of CO to CuB in Cytochrome c Oxidase Is Dynamically Linked to Structural Changes around a Carboxyl Group: A Time-Resolved Step-Scan Fourier Transform Infrared Investigation

The redox-driven proton pump cytochrome c oxidase is that enzymatic machinery of the respiratory chain that transfers electrons from cytochrome c to molecular oxygen and thereby splits molecular oxygen to form water. To investigate the reaction mechanism of cytochrome c oxidase on the single vibrational level, we used time-resolved step-scan Fourier transform infrared spectroscopy and studied the dynamics of the reduced enzyme after photodissociation of bound carbon monoxide across the midinfrared range (2300-950 cm⁻¹). Difference spectra of the bovine complex were obtained at -20°C with 5 μs time resolution. The data demonstrate a dynamic link between the transient binding of CO to Cu_B and changes in hydrogen bonding at the functionally important residue E(I-286). Variation of the pH revealed that the pK_a of E(I-286) is >9.3 in the fully reduced CO-bound oxidase. Difference spectra of cytochrome c oxidase from beef heart are compared with those of the oxidase isolated from Rhodobacter sphaeroides. The bacterial enzyme does not show the environmental change in the vicinity of E(I-286) upon CO dissociation. The characteristic band shape appears, however, in redox-induced difference spectra of the bacterial enzyme but is absent in redox-induced difference spectra of mammalian enzyme. In conclusion, it is demonstrated that the dynamics of a large protein complex such as cytochrome c oxidase can be resolved on the single vibrational level with microsecond Fourier transform infrared spectroscopy. The applied methodology provides the basis for future investigations of the physiological reaction steps of this important enzyme.     

Turnover and vectorial properties of cytochrome c oxidase in reconstituted vesicles

1. Proteoliposomes containing cytochrome c oxidase and phospholipid have been made by sonication and by the cholate dialysis procedure. In both methods of preparation, only about 50% of the enzyme molecules are oriented in the membrane with their cytochrome c reaction sites exposed to the outside of the vesicle.2. The activity of cytochrome c oxidase in the reconstituted vesicles is not increased by incubation in 1% Tween 80. Experiments on reconstituted vesicles containing internal (entrapped) cytochrome c indicate that turnover of enzyme oxidising entrapped cytochrome c in the presence of N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine is at a very much lower rate than enzyme oxidising external ferrocytochrome c.3. Oxidation of ascorbate by externally added cytochrome c results in an electrogenic production of OH^? inside the vesicles, which can be monitored using entrapped phenol red. Polylysine inhibits, but does not abolish, the internal alkalinity change in reconstituted vesicles oxidising internal (entrapped) cytochrome c using externally added ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine. When 2,3,5,6-tetramethyl-p-phenylenediamine is used as the permeable redox mediator, an increase in internal acidity can be monitored under the same conditions.     

Cytochrome c-cytochrome oxidase interaction at subzero temperatures

Cytochrome oxidase forms two distinctive compounds with oxygen at ?105 and ?90°C, one appears to be oxycytochrome oxidase (Compound A) and the other peroxycytochrome oxidase (Compound B). The functional role of compound B in the oxidation of cytochrome c has been examined in a variety of mitochondrial preparations. The rate and the extent of the reaction have been found to be dependent upon the presence of a fluid phase in the vicinity of the site of the reaction of cytochrome c and cytochrome oxidase. The kinetics of cytochrome c oxidation and of the slowly reacting component of cytochrome oxidase are found to be linked to one another even in cytochrome c depleted preparations, but under appropriate conditions, especially low temperatures, the oxidation of cytochrome c precedes that of this component of cytochrome oxidase. Based upon the identification of the slowly reacting components of cytochrome oxidase with cytochrome c, various mechanisms are considered which allow cytochrome c to be oxidized without the intervention of cytochrome a at very low temperatures, and tunneling seems an appropriate mechanism.     

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.     

The role of the quinone pool in the cyclic electron-transfer chain of Rhodopseudomonas sphaeroides A modified Q-cycle mechanism

(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c₂ oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c₁ and c₂, the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a low-potential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0–3 mol ubiquinol/mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c₁ and c₂, and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycin-sensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 ix oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 became completely reduced following one flash in the presence of antimycin. The results are interpreted as showing that at potentials higher than 180 mV, ubiquinol stoichiometric with cytochrome b-561 reaches the complex from the reaction center. The increased extent of the carotenoid change, when one extra ubiquinol is available in the pool, is interpreted as showing that the ubiquinol oxidase site turns over twice, and the ubiquinone reductase sites turns over once, for a complete turnover of the ubiquinol:cytochrome c₂ oxidoreductase complex, and the net oxidation of one ubiquinol/complex. (7) The antimycin-sensitive reduction of cytochrome c₁ and c₂ is shown to reflect the second turnover of the ubiquinol oxidase site. (8) We suggest that, in the presence of antimycin, the ubiquinol oxidase site reaches a quasi equilibrium with ubiquinol from the pool and the high- and low-potential chains, and that the equilibrium constant of the reaction catalysed constrains the site to the single turnover under most conditions. (9) The results are discussed in the context of a detailed mechanism. The modified Q-cycle proposed is described by physicochemical parameters which account well for the results reported.     

Functional and Structural Comparisons between Prokaryotic and Eukaryotic aa3-Type Cytochrome c Oxidases from an Evolutionary Point of View

The specificities for cytochrome c of the aa3-type cytochromec oxidase were studied with enzymes derived from Thiobacillusnovellas, Nitrobacter agilis, Paracoccus denitrificans and thecow in reaction with the cytochromes c from 5 prokaryotes and7 eukaryotes. The T. novellus enzyme reacted most rapidly withthe cytochromes c of Candida krusei, tuna and bonito as wellas T. novellus cytochrome c; the specificity for cytochromec of the N. agilis enzyme was similar to that of the T. novellusenzyme. The bovine enzyme reacted rapidly with all the eukaryoticcytochromes c tested. The P. denitrificans enzyme showed a specificitysimilar to that of the bovine enzyme, except that it reactedrapidly with P. denitrificans cytochrome c, while the bovineenzyme reacted with it very poorly. All four kinds of enzymesshowed an extremely limited reaction with Pseudomonas aeruginosacytochrome c. The amino acid composition of subunit I of the N. agilis enzymeresembled that of the bovine enzyme, while the compositionsof their subunits II were different. On the basis of these results,an evolutionary relationship between bacterial and eukaryoticenzymes was discussed. (Received May 21, 1981; Accepted August 20, 1981)     

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 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).     

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.     

Respiratory components and oxidase activities in Alcaligenes eutrophus

1. Cells of the hydrogen bacterium Alcaligenes eutrophus are broken by gentle lysis using lysozyme treatment in hypertonic sucrose followed by osmotic shock. By this method, 93% of the in vivo activity of the H₂ oxidase is recovered and the ATPase remains particle bound. In contrast, cell disruption in a French pressure cell diminishes the in vivo activity of the H₂ oxidase by 50% and solubilizes the bulk of the ATPase.2. The bacterium contains a periplasmic cytochrome c with bands at 418, 521 and 550 nm (difference spectrum). In addition to cytochrome aa₃, b-560, c-553 and o, low temperature difference spectra of membranes show the presence of two further cytochromes (shoulders at 551 and 553 nm).3. The unsupplemented membrane fraction catalyses the oxidation of hydrogen, NADH, NADPH, succinate, formate and endogenous substrate (NAD linked) at rates 2–3-fold higher than membranes obtained from cells disrupted in a French pressure cell. With the exception of the H₂ oxidase all oxidase activities in lysozyme membranes are sensitive to carbonylcyanide m-chlorophenylhydrazone (20–100% stimulation of oxygen uptake).4. The cytoplasmic fraction contains a B-type cytochrome with absorption maxima at 436 and 560 nm, capable of combining with CO; it contains non-covalently bound protohaem. In alkaline solutions a spectral transition to the haemochrome type with bands at 423, 526 and 556 nm occurs. The addition of NADH to an aerobic suspension of this cytochrome elicits new absorption maxima at 418, 545 and 577 nm (difference spectrum), which are believed to represent an oxygenated form of the reduced cytochrome.     

The terminal oxidase of Photobacterium phosphoreum. A novel cytochrome

The terminal oxidase of Photobacterium phosphoreum has been purified to the electrophoretically homogeneous state and some of its properties have been studied.The enzyme catalyses oxidation of ascorbate in the presence of phenazine methosulphate or N,N,N′,N′-tetramethyl-p-phenylenediamine. The reaction is inhibited by cyanide. Nitrite at comparatively high concentrations inhibits the enzyme, but the enzyme does not catalyse nitrite reduction with ascorbate plus the electron mediator as the electron donor.The enzyme shows the absorption peaks at 632, 565, 534 and 436 nm in the reduced form. It has two kinds of haems: protohaem and haem d. Namely, the enzyme is a ‘cytochrome bd’-type oxidase; a novel cytochrome.     

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.     

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.     

The photosynthetic electron transfer chain of Chromatium vinosum chromatophores flash-induced cytochrome b reduction

Reduction of a cytochrome b following excitation by a single, short, near-saturating light flash has been demonstrated in Chromatium vinosum chromatophores. The extent of reduction is increased by addition of antimycin. The cytochrome has an α-band maximum at 562 nm in the presence of antimycin.The cytochrome b reduction is most readily observed in the presence of antimycin at high redox potential when cytochrome c-555 is oxidised before excitation. Under these conditions the half-time for reduction is about 20 ms, and the extent is about 0.5 mol of cytochrome b reduced per mol of reaction center oxidised. This extent of reduction is observed on the first flash-excitation from the dark-adapted state, and there was no indication that the reaction center quinone acceptor complex acted as a two-electron accumulating system. With cytochrome c-555 reduced before excitation, the extent of cytochrome b reduction is approximately halved. The factors which result in substoichiometric cytochrome b reduction are not yet understood.Agents which appear to inhibit primary acceptor oxidation by the secondary acceptor (UHDBT, PHDBT, DDAQQ, HOQNO, o-phenanthroline), inhibit reduction of the cytochrome b. DBMIB inhibits cytochrome b reduction but does not appear to inhibit primary acceptor oxidation.These observations confirm that a cytochrome b receives electrons delivered from the primary acceptor complex, and indicate that the photoreduced cytochrome b is reoxidised via an antimycin-sensitive pathway.