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.     

13th International Congress of Biochemistry

Cytochrome caa₃ (cytochrome oxidase) from the thermophilic bacterium PS3 can exhibit full catalytic activity in the presence of ascorbate and TMPD or other electron donors and in the absence of added soluble c-type cytochromes. It appears to possess only a low-affinity and not a high-affinity site for the soluble cytochromes. Proteoliposomal cytochrome caa₃ develops an effective membrane potential in the presence of ascorbate and TMPD or PMS, in the absence of added soluble cytochrome c. Reduction of the a₃ centre is blocked in the presence of cyanide. During reductive titrations of the cyanide-inhibited enzyme, electrons initially equilibrate among three centres, the c haem, the a haem and one of the associated Cu atoms. During steady-state turnover, electrons probably enter the complex via the bound c haem; the a haem and perhaps an associated Cu_A atom are reduced next. It is concluded that, despite its size and hydrophobic association with the aa₃ complex, the haem c-containing subunit can behave in an analogous way to that of mammalian cytochrome c, bound at the high-affinity site of the eucaryotic enzyme.     

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.     

Control of respiration in proteoliposomes containing cytochrome aa3 II. Inhibition by carbon monoxide and azide

1. Carbon monoxide (CO) acts competitively towards oxygen when the latter is taken up in respiration by cytochrome aa₃-containing proteoliposomes, both in the presence of p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin (deenergized state) and in their absence (energized state). At high levels of CO, the double reciprocal plots ($\text{1}\text{v}$ vs. $\text{1}\text{[}\text{O}{}_2\text{]}$) in the energized and deenergized states are parallel, i.e. energization acts “anti-competitively” towards oxygen, and the “respiratory control ratio” decreases as the oxygen concentration decreases.2. Azide acts non-competitively towards cytochrome c when the latter is oxidized by cytochrome aa₃-containing proteoliposomes both in the energized and deenergized (plus p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin) conditions. At low azide concentrations the apparent K_i for azide is unaffected by energization, but at high azide levels the K_i increases in energized liposomes, i.e. the “respiratory control ratio” decreases as the azide concentration increases.3. It is concluded that the inhibitor experiments are consistent with but do not prove the concept that the oxidase molecules in a single vesicle are responding to a single “energization state” or set of electrochemical gradients. This and other models are discussed.     

Solubilization and purification of cytochrome a1 from Nitrosomonas

Cytochrome a₁ was solubilized with Triton X-100 from a membrane-envelope preparation of Nitrosomonas and partially purified by repeated fractionation with (NH₄)₂SO₄. The purified fraction of cytochrome a₁ was enriched over the crude extract by a factor of 16 and 300 with respect to protein and c-type cytochrome, respectively. The cytochrome was characterized as cytochrome a₁ on the basis of (a) reduced absorption maxima at 444 nm and 595 nm, (b) acid acetone extractibility and ether solubility of the heme and (c) absorption maximum of 587 nm of the ferro-hemochrome in alkaline pyridine. The α absorption band shifted from 600 nm to 595 nm upon solubilization of the cytochrome with Triton X-100. Spectral shifts were observed in the presence of cyanide and azide and the cytochrome changed with aging to a form with a reduced absorption band at 422 nm. Cytochrome a₁ was reduced anaerobically in the presence of reduced mammalian cytochrome c and was rapidly reoxidized in the presence of O₂. CO caused a shift in the soret peak of the reduced form but did not prevent reoxidation of cytochrome a₁ in the presence of CO-O₂ (95:5, v/v).     

The effect of sulphide on cytochromeaa3 Isosteric and allosteric shifts of the reduced α-peak

1. Sulphide, like cyanide, is a slow-binding inhibitor of cytochromeaa₃ with a high affinity (K_d < 0.1 μM).2. Unlike cyanide binding, the binding of sulphide is apparently independent of the redox state of components of the oxidase other than cytochromea₃and shows no anomalous kinetics during complex formation.3. Sulphide binding to cytochrome a₃³⁺ is accompanied by a blue-shift in the α-peak of the reduced enzyme (a²⁺ a₃³⁺H₂S), similar to but smaller than that induced by azide.4. The reduced sulphide-inhibited system shows a much higher Soret peak at 445 nm than the corresponding cyanide and azide complexes, suggesting that partial electron transfer from sulphide to haem may occur in the complex. No evidence was obtained for the formation of any sulfhaem derivatives of cytochromea₃.5. The influence of energization on the spectrum of mitochondrial cytochrome oxidase, and the effects of calcium on the α-peak of isolated cytochromeaa₃ (Wikstro¨m, M. K. F. (1974) Ann. N. Y. Acad. Sci. 227, 146–158) are distinct from the action of the cytochromea₃ligands.6. A classification of peak shifts in the α-region in terms of isosteric and allosteric ligands is proposed.     

Inhibition of Respiration in Prototheca zopfii by Light

Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of the cells. The inhibition of respiration was correlated with a photodestruction of cytochrome c(551), cytochrome b(559), and cytochrome a₃. Cytochrome c(549), cytochrome b(555), and cytochrome b(564) were unaffected by the irradiation treatment. The α-band of reduced cytochrome a was shifted from 599 to 603 nm by irradiation, an effect similar to that observed when methanol was added to nonirradiated cells. The presence of oxygen was required during irradiation for both photoinhibition of respiration and photodestruction of the cytochromes. Cytochrome a₃ was protected against photodestruction by cyanide. Photodestruction of these same cytochromes also occurred when washed mitochondria of P. zopfii were irradiated.     

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.     

Studies on the orientations of the mitochondrial redox carriers I. Orientation of the hemes of cytochrome c oxidase with respect to the plane of a cytochrome oxidase-lipid model membrane

The liganded derivatives of mitochondrial cytochrome c oxidase have been prepared in hydrated oriented multilayers of membranous cytochrome c oxidase. The optical spectra of the liganded derivatives recorded at an angle of 45° between the incident light beam and the normal to the planes of the membranes in the multilayers show dichroic ratios of almost 2 in the visible region and 1.2–1.4 in the Soret region. The dichroic ratios were found to be similar for both cytochromes a and a₃. Electron paramagnetic resonance spectra of the azide, sulfide, and formate complexes of cytochrome c oxidase obtained as a function of the orientation of the applied magnetic field relative to the planes of the membranes in the multilayer confirm the optical data and demonstrate that both hemes of cytochrome c oxidase are oriented such that the angle between the heme normal and the membrane normal is approximately 90°.     

Some properties of the redox components of cytochrome c oxidase and their interactions.

The electron paramagnetic resonance (epr) properties of cytochrome c oxidase have been examined with special attention to the effect of added ligands and of interactions between the redox components. The fully oxidized preparations have a very small g6 signal which increases greatly as the redox potential is made more negative, a process exactly paralleling the disappearance of the g3 signal. The potential for half appearance or disappearance (E_m), respectively, is 380 mV at pH 7.0 and 300 mV at pH 8.5. This identifies the changes as accompanying reduction of cytochrome a₃ because the E_m of the “invisible copper” is 340 mV and pH independent. Nitric oxide (NO) binds reduced cytochrome a₃ to form a paramagnetic species. This resulting epr signal is strongly dependent on the redox state of cytochrome a, another expression of heme-heme interaction in cytochrome oxidase. The NO compound is also unique in that under the appropriate conditions three of the four redox components (cytochrome a₃, cytochrome a, and the “visible” copper) are epr active. In potentiometric titrations in the presence of azide the formation of the azide compound responsible for the g2.9 signal appears to require reduction of both cytochrome a₃ and the “invisible copper.” An internal sulfur compound is present which, at alkaline pH values, can bind the heme responsible for the g6 signal and change it to a low-spin sulfur compound with a signal at approximately g2.6. Evidence is also presented for the cytochrome c oxidase in situ being an equilibrium mixture of two different conformational states.     

The Cytochromes of Prototheca zopfii

The respiratory pigments of Prototheca zopfii include seven cytochromes: two c-type cytochromes, a soluble c(549) and a membrane bound c(551); three b-type cytochromes, b(555), b(559) and b(564); and cytochromes a and a₃. Cytochromes a and a₃ could be resolved spectrally in the α-band region by reducing the cells in the presence of methanol and cyanide. Methanol shifted the absorption maximum of cytochrome a from 598 to 603 nanometers and permitted dithionite (or substrate) to reduce the cyanide-cytochrome a₃ complex to give a well defined 595-nanometer absorption band. Methanol did not interfere with CO binding by cytochrome a₃, and CO did not alter the methanol effect on cytochrome a. Azide and cyanide, which partially inhibited exogenous respiration, stimulated endogenous respiration. Frozen steady states of the electron transport chain in the presence of cyanide and azide indicated that the stimulation by these inhibitors was due to an increased autooxidation of one of the b-type cytochromes, possibly b(564).     

Respiratory metabolism of normal and divisionless strains of Candida albicans

Respiration of a normal strain of Candida albicans was compared with that of a divisionless mutant which has a biochemical lesion such that metabolically generated hydrogen "spills over," during growth, for non-specific dye reduction. This waste is not at expense of growth, since both strains grow at essentially similar rates, nor at expense of respiration, since the mutant reduces oxygen more rapidly than the normal strain. Respiration in both strains is qualitatively similar, and seemingly unique among highly aerobic organisms in that it is not mediated by cytochrome oxidase. In resting cells of both strains, respiration is not only resistant to, but markedly stimulated by, high concentrations of cyanide, carbon monoxide, and azide. In contrast, growth of these yeasts is inhibited by low concentrations of cyanide and azide. Cytochrome oxidase could not be detected in cell-free preparations; reduced cytochrome c was not oxidized by such preparations. Cytochrome bands could not be observed in thick cell suspensions treated with reducing agents. However, incorporation of superoptimal levels of zinc and iron into the culture medium resulted in growth of cells possessing distinct cytochrome bands; respiration of these cells remained insensitive to cyanide, monoxide, and azide, and the bands were maintained in a reduced form on oxygenation. In the divisionless yeast, tetrazolium dyes compete with oxygen for reduction; this is not the case in the normal strain. The firmness with which hydrogen transfer is channeled in the latter for reduction of disulfide bonds (of importance in the division mechanism) and of oxygen, is contrasted with the lack of such control in the mutant.     

The participation of cytochromes in the process of nitrate respiration in Klebsiella (Aerobacter) aerogenes

1. The participation of cytochromes in the membrane-bound, nitrate and oxygen respiratory systems of Klebsiella (Aerobacter) aerogenes has been investigated. The membrane preparations contained the NADH, succinate, lactate and formate oxidase systems, and in addition a high respiratory nitrate reductase activity.2. Difference spectra indicated the presence of cytochromes b, a₁, d, and o. Cytochromes of the c-type could not be detected in these membranes. Both cytochrome b content and respiratory nitrate reductase activity were the highest in bacteria grown anaerobically in the presence of nitrate.3. Cytochrome b was the only cytochrome which, after being reduced by NADH, could be partially reoxidized anaerobically in the presence of nitrate. Furthermore, nitrate caused a lower aerobic steady state reduction only of cytochrome b.4. NADH oxidase and NADH-linked respiratory nitrate reductase activities were both inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and KCN. NADH oxidase activity was selectively inhibited by CO, while azide was found to inhibit only the respiratory nitrate reductase. In the presence of azide, nitrate did not affect the level of reduction of cytochrome b.5. The evidence presented suggests that cytochrome b is a carrier in the electron transport systems to both nitrate and oxygen; from cytochrome b branching occurs, with one branch linked to the respiratory nitrate reductase and one branch linked to oxidase systems, containing the cytochromes a₁, d and o.     

Ligand Trapping by Cytochrome c Oxidase: IMPLICATIONS FOR GATING AT THE CATALYTIC CENTER*

Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe_a3³⁺–Cu_B²⁺) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme. Here we show that cytochrome oxidase pretreated with a low concentration of azide exhibits a significant increase in the apparent rate of cyanide binding relative to that of free enzyme. However, this increase in rate does not reflect a conformational change enhancing the rapid formation of a Fe_a3³⁺–CN–Cu_B²⁺ complex. Instead the cyanide-induced transition of a preformed Fe_a3³⁺–N₃–Cu_B²⁺ to the ternary complex of Fe_a3³⁺–N₃ Cu_B²⁺–CN is the most likely reason for the observed acceleration. Significantly, the slow rate of azide release from the ternary complex indicates that cyanide ligated to Cu_B blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at Cu_B and that, during catalysis, ligands present at this copper center control access to the iron of heme a₃ from the bulk medium.     

Direct observation of release of cytochrome c from lipid-encapsulated protein by peroxide and superoxide: a possible mechanism for drug-induced apoptosis

Release of cytochrome c from inside lipid vesicles and from inside proteoliposomes formed by cytochrome c oxidase has been studied by spectrophotometric methods. The protein encapsulated inside vesicles did not form complex with sodium azide solution added externally. Both hydrogen peroxide and superoxide were found to cause release of cytochrome c from the lipid encapsulated protein, which was detected from the distinct spectral changes due to the formation of the azide complex of cytochrome c in the solution. Cytochrome c encapsulated inside proteoliposomes containing cytochrome c oxidase (CcO) did not release the cytochrome c during enzymatic turnover of CcO. The anticancer drug, doxorubicin, was found to inhibit the biochemical function of cytochrome c oxidase and release of cytochrome c was observed from the proteoliposome encapsulating the protein during the enzymatic turnover in the presence of doxorubicin. The results indicated that the inhibition of enzymatic activity by doxorubicin possibly leads to the formation of reactive oxygen species, which induce the release of cytochrome c from inside to outside of the membrane.     

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 oxidation mechanisms of thiosulphate and sulphide in Chlorobium thiosulphatophilum: Roles of cytochrome c-551 and cytochrome c-553

A thiosulphate-cytochrome c reductase was highly purified from Chlorobium thiosulphatophilum and its properties were studied. The enzyme catalyses reduction with Na₂S₂O₃ of c cytochromes, including cytochrome c-551 of the bacterium. Cytochrome c (555, C. thiosulphatophilum) does not react directly with the enzyme at an appreciable rate but stimulates greatly the reduction by the enzyme of cytochrome c-551 with Na₂S₂O₃. The reduction of c cytochromes catalysed by the enzyme is strongly inhibited by cyanide and sulphite.Cytochrome c (553, C. thiosulphatophilum), a c-type cytochrome with covalently bound flavin, was found to catalyse reduction with sulphide of c cytochromes, including cytochrome c-555. The reaction is strongly inhibited by cyanide. Cyanide seems to combine strongly with cytochrome c-553 probably at the flavin moiety. Thus, the absorption spectrum attributable to flavin of the haemoprotein is changed on addition of cyanide, and neither the original spectrum nor the activity reappears even after the cyanide-treated cytochrome has been subjected to gel filtration with a Sephadex G-25 column or to isoelectric focusing.     

Cyanide-insensitive Respiration in Plant Mitochondria

Pathways of electron transport have been studied in mitochondria isolated from hypocotyls of etiolated mung bean seedlings and skunk cabbage spadices that show cyanide-resistant respiratory activity. The residual flux through cytochrome c oxidase is shown to be small in comparison with the flux through an unidentified alternative oxidase that is known to have a high affinity for oxygen. This alternative oxidase is not a cytochrome. Skunk cabbage and mung bean mitochondria contain cytochromes a and a₃ that have absorption peaks differing slightly from those of animal preparations. A slow oxidation-reduction of cytochrome a₃-CN has been demonstrated. Cytochromes b undergo oxidation and reduction in the presence of cyanide but play no essential role in the cyanide-resistant pathway. Antimycin inhibits to an extent similar to that of cyanide; the respiratory chain bifurcates on the substrate side of the antimycin-sensitive site. Evidence is presented for the selective inhibition by thiocyanate, α, α′-dipyridyl, and 8-hydroxyquinoline of the alternative oxidase pathway, which may therefore contain a non-heme iron protein.     

Electron-transfer processes in carboxy-cytochrome c oxidase after photodissociation of cytochrome a2+3 · CO

Under continuous illumination the CO binding curve of reduced carboxy-cytochrome c oxidase maintains the shape of the binding curve in the dark. The apparent dissociation constant calculated from the binding curves at various light intensities is a linear function of the light intensity.Marked differences are observed between the light-induced difference spectra of the fully reduced carboxy-cytochrome c oxidase and the mixed-valence carboxy-cytochrome c oxidase. These differences are enhanced in the presence of ferricyanide as an electron acceptor and are explained by partial oxidation of cytochrome a₃ in the mixed-valence enzyme after photodissociation.Upon addition of CO to partially reduced formate cytochrome c oxidase (a²⁺a³⁺₃ · HCOOH) the cytochrome a²⁺₃ · CO compound is formed completely with a concomitant oxidation of cytochrome a and the Cu associated with cytochrome a. During photodissociation of the CO compound the formate rebinds to cytochrome a₃ and cytochrome a and its associated Cu are simultaneously reduced. These electron transfer processes are fully reversible since in the dark the a³⁺₃ · HCOOH compound is dissociated slowly with a concomitant formation of the a²⁺₃ · CO compound and oxidation of cytochrome a.When these experiments are carried out in the presence of cytochrome c, both cytochrome c and cytochrome a are reduced upon illumination of the mixed-valence carboxy-cytochrome c oxidase. In the dark both cytochrome c and cytochrome a are reoxidized when formate dissociates from cytochrome a₃ and the a²⁺₃ · CO compound is formed back. Thus, in this system we are able to reverse and to modulate the redox state of the different components of the final part of the respiratory chain by light.