Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi component of the aerobic delta microH+ (the sum of the proton chemical and electrical activities) exerts a pH-dependent constraint on forward electron flow from cytochrome b566 to cytochrome b562. This effect is explained as a consequence of anisotropic location of cytochromes b566 and b562 in the membrane and the pH-dependence of the redox function of these cytochromes. Transmembrane delta pH, on the other hand, exerts control on electron flow from cytochrome b562 to c cytochromes.     

Stepwise reduction of cytochromes b-562, b-566 and b-558 in rat liver mitochondria.

1. Addition of KCN to aerobic, rotenone-inhibited rat liver mitochondria with out addition of substrate caused reduction of cytochromes b-562 (having an alpha-band at 562 nm at room temperature), c + c1, and a + a3. The effect of KCN on cytochrome b-562 was reversed by pentachlorophenol, though the effect of KCN on cytochromes c+c1 and a+a3 was not reversed by this uncoupler.2. Addition of ATP to aerobic, rat liver mitochondria inhibited with 500 muM KCN under conditions were cytochromes b-562, c+c1 and a+a3 were reduced, caused reduction of cytochrome b-566. The absorbance spectrum of cytochrome b-566 had an alpha-band at 565.5 nm, a beta-band at 538 nm and a gamma-band at 431 nm, but no shoulder around 558 nm at room temperature. 3. Addition of succinate to rotenone-KCN-inhibited and ATP-treated rat liver mitochondria under conditions where cytochromes b-566, b-562, c+c1 and a+a3 were already fully reduced, caused reduction of cytochrome b-558 (having an alpha-band at 558 nm, a beta-band at 527 nm and a gamma-band at 426 nm at room temperature) after exhaustion of molecular oxygen in the reaction medium, without any contribution from a long-wavelength species (cytochrome b-566). 4. It was concluded that the 558-nm band is not a short-wavelength shoulder of cytochrome b-566, but is due to a different species from cytochrome b-566.     

The mechanism of proton translocation by the cytochrome system of mitochondria. Characterization of proton-transfer reactions associated with oxidoreductions of terminal respiratory carriers.

A direct kinetic analysis is presented of rapid proton-releasing reactions at the outer or C-side of the membrane, in ox heart and rat liver mitochondria, associated with aerobic oxidation of reduced terminal respiratory carriers in the presence of antimycin. Valinomycin plus K+ enhances the rate of cytochrome c oxidation and the rate and extent of H+ release. In the presence of valinomycin the leads to H+/e- ratio, computed on the basis of total electron flow from respiratory carriers to oxygen, varies with pH, remaining always lower than 1, and is unaffected by N-ethylmaleimide. 2-Heptyl-4-hydroxyquinoline N-oxide and 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, at concentrations which inhibit in the presence of antimycin the oxygen-induced reduction of b cytochromes, cause also a marked depression of the H+ release associated with aerobic oxidation of terminal respiratory carriers. Aerobic oxidation of the cytochrome system in mitochondria and of isolated b-c1 complex and cytochrome c oxidase results in scalar proton release from ionizable groups (redox Bohr effects). In mitochondria and submitochondrial particles, about 70% of the oxidoreductions of the components of the cytochrome system are linked to scalar proton transfer by ionizable groups. In isolated b-c1 complex scalar proton transfer, resulting from redox Bohr effect, amounts to 0.9H+ per Fe-S protein (190 muT). In isolated cytochrome c oxidase, Bohr protons amount to 0.8 per haem a + a3. The results presented indicate that the H+ release from mitochondria during oxidation of terminal respiratory carriers derives from residual antimycin-insensitive electron flow in the quinone-cytochrome c span and from redox Bohr effects in the b-c1 complex and cytochrome c oxidase. There is no sign of proton pumping by cytochrome oxidase during its transition from the reduced to the active 'pulsed' state and the first one or two turnovers.     

Sulphide as an inhibitor and electron donor for the cytochrome c oxidase system

Anomalies both kinetic and equilibrium in nature are described for the inhibition of cytochrome c oxidase activity by sulphide in the isolated enzyme and in submitochondrial particles. These anomalies are related to the involvement of more than 1 mol of sulphide in the blockage of one cytochrome aa3 centre. Sulphide reduces resting cytochrome a3, a reaction that results in oxygen uptake and the loss of a sulphide molecule. Sulphide can also reduce cytochromes c and a; in the former case, a part of the one-equivalent oxidation product, presumed to be the SH radical, reacts with oxygen. Such oxygen uptake is also seen under aerobic conditions when ferricyanide reacts with sulphide. Three phases are identified in the inhibitory interaction of sulphide with the cytochrome c oxidase enzyme itself: an initial rapid reaction involving sulphide oxidation, oxygen uptake, and conversion of cytochrome aa3 into the low-spin "oxyferri" form; a subsequent step in which sulphide reduces cytochrome a; and the final inhibitory step in which a third molecule of sulphide binds the a3 iron centre in the cytochrome a2+ a3 3+ (oxy) species to give cytochrome a2+ a3 3+ H2S. the initial events parallel some of the events in the interaction of the cytochrome c-cytochrome aa3 system with monothiols; the final inhibitory event resembles that with cyanide.     

Cytochrome c oxidation by the electron transport fraction of Azotobacter vinelandii

The spectrophotometric oxidation of horse heart ferrocytochrome c was examined by use of the particulate electron transport fraction (R(3)) of Azotobacter vinelandii strain O. Unlike cytochrome c, purified preparations of native Azotobacter cytochromes c(4) + c(5) were oxidized only slowly by the electron transport fraction. The oxidation of mammalian cytochrome c proceeded at an appreciable rate and displayed "apparent" first-order kinetics at a pH optimum of 9.0 with tris(hydroxymethyl)aminomethane-chloride buffer. The calculated V(max) value was 0.22 mumole of cytochrome c oxidized per min per mg of protein (25 C) and a K(m) value for cytochrome c of 2.3 x 10(-5)m was obtained. Ferricytochrome c was a "strict" competitive inhibitor for this oxidation. Cytochrome c oxidation by the Azotobacter electron transport system was markedly sensitive to cyanide, azide, and hydroxylamine, although carbon monoxide inhibition could not be demonstrated. It was sensitive also to high concentrations of phosphate, ethylenediaminetetraacetate, and some metal cations. "Aging" or prolonged storage of the Azotobacter R(3) fraction, at 4 C for 10 days, resulted in a threefold increase in specific activity. The cytochrome c peroxidase type of reaction did not occur with the R(3) electron transport fraction.     

Heterogeneous pools of cytochrome c2 in photo-denitrifying cells of Rhodobacter sphaeroides forma sp. denitrificans

When cells of the denitrifying phototrophic bacterium Rhodobacter sphaeroides forma sp. denitrificans were grown anaerobically under illumination in the presence of nitrate, the content of photosynthetic reaction centers per cellular protein was less than that in cells grown photosynthetically without nitrate under the same light intensity. The contents of cytochromes c1 and c2, which work in both photosynthetic and denitrifying electron transport systems, were almost constant, being independent of the presence of nitrate during growth. Consequently, the ratio of cytochromes c1 and c2 to the reaction center was more than three in the photo-denitrifying cells, whereas it was close to one in the photosynthetic cells under light-limiting conditions. In spite of the excess of cytochromes c1 + c2 over the reaction center in the photo-denitrifying cells, all cytochromes c1 + c2 were oxidized by illumination within hundreds of milliseconds in the presence of antimycin. When glycerol was added to increase the viscosity in the periplasm, biphasic oxidation of cytochromes c1 + c2 was apparent in the photo-denitrifying cells with repetitive flashes. The fast phase oxidation, which took place instantaneously (less than 1 ms) after the first and second flashes, showed a similar pattern to the oxidation in the light-limiting photosynthetic cells. The rate of the slow phase oxidation was sensitive to viscosity and was thought to reflect a diffusion-controlled second-order reaction between cytochrome c2 and the reaction center. The biphasic oxidation of cytochromes c1 + c2 suggests that these cytochromes exist in the photo-denitrifying cells as two different pools in relation to the reaction center.     

The Respiratory Chain of Plant Mitochondria: IV. Oxidation Rates of the Respiratory Carriers of Mung Bean Mitochondria in the Presence of Cyanide

The half-time for oxidation of cytochrome b(557) in mitochondria from etiolated mung bean (Phaseolus aureus) hypocotyls is 5.8 milliseconds at 24 Celsius in the absence or presence of 0.3 mm KCN, when the oxidation is carried out by injecting a small amount of oxygenated medium into a suspension of mitochondria made anaerobic in the presence of succinate plus malonate. Since oxygen is consumed by the alternate, cyanide-insensitive respiratory pathway of these mitochondria, cycles of oxidation and reduction can be obtained with the oxygen pulses when cyanide is present. Reduced cytochromes (a + a(3)) also become oxidized at nearly the uninhibited rate under these conditions, a(3) completely and a partially. The half-time for oxidation of c(547) is also unaffected by 0.3 mm KCN, but c(549) has a half-time equal to that of c(547) in the presence of KCN, compared to the shorter one observed in the absence of inhibitor. The maximum extent of oxidation of the cytochromes c is about 70% in the presence of 0.3 mm KCN; this oxidation is rapidly followed by an extensive reduction which is synchronous with the reduction of cytochrome a observed under the same conditions. In the presence of cyanide, it appears likely that the cytochromes c and b(557) are oxidized by cytochrome oxidase in oxygen pulse experiments, rather than by the alternate oxidase. The oxidation of cytochrome b(553) is partially inhibited by KCN, but complete oxidation is attained in the aerobic steady state with excess oxygen. If the oxygen pulse experiment is carried out in the presence of sufficient malonate so that entry of reducing equivalents into the respiratory chain occurs at a rate negligible compared to inter-carrier electron transport, the half-time for flavoprotein oxidation is unaffected by 0.3 mm KCN while that for ubiquinone oxidation is but 2-fold larger. The observed net oxidation rate of these two carriers in mung bean mitochondria is more sensitive to the entry rate of reducing equivalents, as set by succinate concentration and malonate to succinate ratio, then it is in skunk cabbage (Symplocarpus foetidus) mitochondria. These observations are interpreted in terms of a respiratory carrier Y, placed between flavoprotein plus ubiquinone and the cytochromes, which is the fork in the split respiratory pathway to the two terminal oxidases and which has lower electron transport capacity in mung bean mitochondria than in skunk cabbage mitochondria.     

Oxidation of cytochromes c and c2 by bacterial photosynthetic reaction centers in phospholipid vesicles. 1. Studies with neutral membranes

The oxidation of cytochrome c2 by photosynthetic reaction center isolated from Rhodopseudomonas sphaeroides and incorporated into unilamellar phosphatidylcholine vesicles was found to be kinetically similar to that observed earlier for reaction centers in low detergent solution [Overfield, R.E., Wraight, C.A., & DeVault, D. (1979) FEBS Lett. 105, 137-142]. At low ionic strength the kinetics were biphasic. The fast phase indicated the formation of a cytochrome-reaction center complex with an apparent binding constant, KB, of about 10(5) M-1. However, KB decreased dramatically with increasing salt concentration, and no fast oxidation was detectable in 0.1 M NaCl. The slow cytochrome oxidation was first order in both cytochrome and reaction centers and, thus, second order overall. Deviations from theoretical second-order behavior were observed when the rate of the first-order back reaction of the primary photoproducts was significant compared to the cytochrome oxidation. This can cause serious overestimation of the second-order rate constant. The slow oxidation of cytochrome c2 by reaction centers in phosphatidylcholine vesicles exhibited a 40% lower encounter frequency than with the solubilized reaction center. This was attributed to the much lower diffusion coefficient of the reaction center in the vesicle membrane than in solution. No effects of diminished dimensionality were detected with neutral vesicles. An activation energy of 8.0 +/- 0.4 kcal x mol-1 was determined for the slow phase of cytochrome c2 oxidation by reaction centers in solution and in vesicles of several different phosphatidylcholines, including dimyristoylphosphatidylcholine above and below its phase transition temperature. Thus, the physical state of the lipid did not appear to affect any rate-limiting steps leading to cytochrome oxidation. The ionic strength dependence of the slow kinetics of oxidation of cytochromes c and c2 confirmed the electrostatic nature of the cytochrome-reaction center interaction, and the pH dependence indicated the titration of a group or groups, important to this interaction, at pH 9.5.     

Reduction of cytochromes by nitrite in electron-transport particles from Nitrobacter winogradskyi: proposal of a mechanism for H+ translocation.

1. A novel component in the respiratory chain of Nitrobacter winogradskyi was identified. This component absorbs maximally at 552.5 nm when in its reduced form, has an Eo' (pH7.0) value of-110mV and undergoes reduction by a mechanism involving the transfer of a single electron. 2. Degrees of reduction of cytochromes c and a1 in electron-transport (ET) particles were monitored during the course of NO2- oxidation, and the effects of ADP together with Pi, oligomycin and of carbonyl cyanide phenylhydrazone were determined. 3. The influences of ionophorous antibiotics, NH4Cl and cyclohexylamine hydrochloride on the reductions of cytochromes c and a1 by NO2- indicate that the flow of reducing equivalents from cytochrome a1 (+350mV) to cytochrome c (+270mV) is facilitated by deltapsi, the electrical component of the protonmotive force. 4. Cytochromes c and a1 in ET particles are reduced by the non-physiological reductant KBH4 in a manner similar to that observed with the physiological reductant NO2-. 5. To account both for the observed cytochrome reductions and for the translocation of H+ ions which accompanies NO2- oxidation, a mechanism is proposed which involves the transfer of a hydride equivalent (H+ plus 2e) inward across the membrane of the ET particle in response to deltapsi.     

The development of cytochromes during the cell cycle of a glucose-repressed fission yeast,Schizosaccharomyces pombe 972h?

1. Spectrophotometric analysis of intact cells of Schizosaccharomyces pombe, harvested from exponentially growing cultures during the phase of glucose repression, revealed the presence of cytochromes a+a(3), c and at least two species of cytochrome b. 2. An absorption maximum at 554nm at 77 degrees K, previously attributed to cytochrome c(1), has been identified as a b-type cytochrome. 3. CO-difference spectra reveal the presence of cytochromes P-420 and P-450 in addition to cytochrome a(3). 4. The cell cycle was analysed by separation of cells into classes representing successive stages in the cell cycle by isopycnic zonal centrifugation. 5. Cytochromes c(548), b(554) and b(560) each exhibited a single broad maximum of synthesis during the cell cycle. 6. Amounts of cytochromes a+a(3) and b(563) (tentatively identified as cytochrome b(T) by its reaction on pulsing anaerobic cell suspensions with O(2)) oscillated in phase, and showed two maxima during the cycle; the second maximum of cytochromes a+a(3) was coincident with a maximum of activity of enzymically active cytochrome c oxidase. 7. The amount of cytochrome P-420 decreased during the first three-quarters of the cell-cycle, whereas that of cytochrome P-450 increased during this period. 8. The discrepancy between spectrophotometric and enzymic assay of cytochrome c oxidase, the changing ratio of cytochrome a(3)/cytochrome a and the relationship between changes in cellular content of cytochromes and previous observations on respiratory oscillations during the cell cycle are discussed.     

Cyclosporin A-Sensitive Cytochrome c Release and Activation of External Pathway of NADH Oxidation in Liver Mitochondria Due to Pore Opening by Acidification of Phosphate-Containing Incubation Medium

Acidification of a high phosphate incubation medium from pH 7.4 to 6.5 promotes increase in rates of succinate oxidation and exogenous NADH oxidation via external (rotenone- and myxothiazol-resistant) pathway by factors 2 and 2.3 respectively. Cyclosporin A prevents these effects. To measure the cytochrome c release, mitochondrial cytochrome c concentration was calculated from absorption spectrum of -band of cytochromes c+c₁. The cytochrome c release is shown to be equal to 27±4%, 40±12%, 70±5% at pH 7.4, 7.0, 6.5, respectively, the last value being reduced by cyclosporin A to 10±3%. Immunoblot method gives the similar results. It is concluded that acidification of the high phosphate medium induces release of a large part of the cytochrome c pool from liver mitochondria due to opening the Ca²⁺-dependent cyclosporin A-sensitive permeability transition pore and subsequent high amplitude swelling.     

Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c.

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Desferrioxamine enhances the reactivity of vanadium (IV) and vanadium (V) toward ferri- and ferrocytochrome c.

Ligands, especially desferrioxamine, affect the rate at which vanadium reduces or oxidizes cytochrome c. Whether reduction or oxidation occurs, and how fast, depends on the nature of the ligand, the state of reduction of the vanadium, the pH (6.0, 7.0, or 7.4), and the availability of oxygen. In general, oxidation of ferrocytochrome c was favored by (1) low pH, (2) an oxidized state of the vanadium, (3) the presence of oxygen, and (4) more strongly binding ligands (desferrioxamine much greater than histidine = ATP greater than EDTA greater than albumin greater than aquo). Thus, at pH 6.0, desferrioxamine accelerated the V(V)-catalyzed ferrocytochrome c oxidation 160-fold aerobically, and 3500-fold anaerobically. In general, strongly binding ligands slowed oxidations, especially at higher pH. Desferrioxamine was unique among the five ligands in that it not only accelerated oxidation of ferrocytochrome c at pH 6.0, but at pH 7.4 the redox balance shifted to the point where it paradoxically reduced ferricytochrome c. V(V) is an improbable electron donor, but desferrioxamine will reduce cytochrome c, and V(V) accelerates this process. Oxidation of cytochrome c by V(V):desferrioxamine was faster anaerobically, and reduction by V(IV):desferrioxamine was faster aerobically. Although V(V) did not oxidize ferrocytochrome c at pH 7.4, V(IV) did, provided oxygen and desferrioxamine were both present. V(IV):desferrioxamine almost completely reduced ferricytochrome c, and this reduction was followed by a slow, progressive oxidation. This latter oxidation of cytochrome c is mediated by active species generated in the reaction between V(IV):desferrioxamine and oxygen, because none of these reagents alone can induce oxidation at a comparable rate. The mediating species were transient, and generated in reactions with oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional intermediates in the reaction of membrane-bound cytochrome oxidase with oxygen.

Flash photolysis of the membrane-bound cytochrome oxidase/carbon monoxide compound in the presence of oxygen at low temperatures and in the frozen state leads to the formation of three types of intermediates functional in electron transfer in cytochrome oxidase and reduction of oxygen by cytochrome oxidase. The first category (A) does not involve electron transfer to oxygen between -125 degrees and -105 degrees, and includes oxy compounds which are spectroscopically similar for the completely reduced oxidase (Cu1+alpha3(2+)-O2) or for the ferricyanide-pretreated oxidase (Cu2+alpha3(3+)-O2). Oxygen is readily dissociated from compounds of type A. The second category (B) involves oxidation of the heme and the copper moiety of the reduced oxidase to form a peroxy compound (Cu2+alpha 3(3+)-O2=or Cu2+alpha3(2+)-O2H2) in the temperature range from -105 degrees to -60 degrees. Above -60 degrees, compounds of type B serve as effective electron acceptors from cytochromes a, c, and c1. The third category (C) is formed above -100 degrees from mixed valency states of the oxidase obtained by ferricyanide pretreatment, and may involve higher valency states of the heme iron (Cu2+alpha3(4+)-O2=). These compounds act as electron acceptors for the respiratory chain and as functional intermediates in oxygen reduction. The remarkable features of cytochrome oxidase are its highly dissociable "oxy" compound and its extremely effective electron donor reaction which converts this rapidly to tightly bound reduced oxygen and oxidized oxidase.     

Oxidation of reduced nicotinamide adenine dinucleotide by particles from Mycobacterium lepraemurium.

Particles from Mycobacterium lepraemurium catalysed the oxidation of NADH with oxygen as the terminal electron acceptor. The preparations contained cytochromes of the a + a3'b and c types, as well as CO-binding pigments. The NADH oxidase activity was sensitive to inhibitors of the flavoprotein system as well as to HQNO and antimycin A. In addition, a cytochrome oxidase sensitive to cyanide was also present. The system was inhibited by the thiol-binding agent, PCMB, and thus indicated the involvement of sulphydryl group in the enzymatic oxidation of NADH. The sensitivity of the NADH oxidase system to all the inhibitors of the respiratory chain and the effect of these inhibitors on the absorption spectra suggested that cytochromes of the b, c, a + a3 types are involved in the transfer of electrons in NADH oxidation.     

The oxidation of ubiquinol by the isolated Rieske iron-sulfur protein in solution

The pre-steady-state redox reactions of the Rieske iron-sulfur protein isolated from beef heart mitochondria have been characterized. The rates of oxidation by c-type cytochromes is much faster than the rate of reduction by ubiquinols. This enables the monitoring of the oxidation of ubiquinols by the Rieske protein through the steady-state electron transfer to cytochrome c in solution. The pH and ionic strength dependence of this reaction indicate that the ubiquinol anion is the direct reductant of the oxidized cluster of the iron-sulfur protein. The second electron from ubiquinol is diverted to oxygen by the isolated Rieske protein, and forms oxygen radicals that contribute to the steady-state reduction of cytochrome c. Under anaerobic conditions, however, the reduction of cytochrome c catalyzed by the protein becomes mechanicistically identical to the chemical reduction by ubiquinols. The present kinetic work outlines that: (i) the electron transfer between the ubiquinol anion and the Rieske cluster has a comparable rate when the protein is isolated or inserted into the parent cytochrome c reductase enzyme; (ii) the Rieske protein may be a relevant generator of oxygen radicals during mitochondrial respiration.     

Characterization and phenotypic control of the cytochrome content of Escherichia coli

1. Electron-transport particles derived from Escherichia coli grown aerobically contain three b-type cytochromes with mid-point oxidation-reduction potentials at pH7 of +260mV, +80mV and -50mV, with n=1 for each. The variation of these values with pH was determined. 2. E. coli develops a different set of b-type cytochromes when grown anaerobically on glycerol with fumarate or nitrate as terminal electron acceptor. Electron-transport particles of fumarate-grown cells contain b-type cytochromes with mid-point potentials at pH7 of +140mV and +250mV (n=1). These two cytochromes are also present in cells grown with nitrate as terminal acceptor, where an additional cytochrome b with a mid-point potential of +10mV (n=1) is developed. 3. The wavelengths of the alpha-absorption-band maxima of the b-type cytochromes at 77K were: (a) for aerobically grown cells, cytochrome b (E(m7) +260mV), 556nm and 563nm, cytochrome b (E(m7) +80mV), 556nm and cytochrome b (E(m7)-50mV), 558nm; (b) for anaerobically grown cells, cytochrome b (E(m7) +250mV), 558nm, cytochrome b (E(m7) +40mV), 555nm and cytochrome b (E(m7) +10mV), 556nm. 4. Cytochrome d was found to have a mid-point potential at pH7 of +280mV (n=1). 5. Cytochrome a(1) was resolved as two components of equal magnitude with mid-point potentials of +260mV and +160mV (n=1). 6. Redox titrations performed in the presence of CO showed that one of the b-type cytochromes in the aerobically grown cultures was reduced, even at the upper limits of our range of electrode potentials (above +400mV). Cytochrome d was also not oxidizable in the presence of CO. Neither of the cytochromes a(1) was affected by the presence of CO.     

Oxidation-reduction potentials of respiratory chain components in Thiobacillus A2

(1) Cells of Thiobacillus A2 grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a3-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 (Em,7.0) and spectral maxima of c-, b-, a- and a3-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the 'invisible', or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a3 complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.     

Pronounced and differential effects of ionic strength and pH on testosterone oxidation by membrane-bound and purified forms of rat liver microsomal cytochrome P-450

The aim of this study was to determine the effects of ionic strength and pH on the different pathways of testosterone oxidation catalyzed by rat liver microsomes. The catalytic activity of cytochromes P-450a (IIA1), P-450b (IIB1), P-450h (IIC11) and P-450p (IIIA1) was measured in liver microsomes from mature male rats and phenobarbital-treated rats as testosterone 7 alpha-, 16 beta-, 2 alpha- and 6 beta-hydroxylase activity, respectively. An increase in the concentration of potassium phosphate (from 25 to 250 mM) caused a marked decrease in the catalytic activity of cytochromes P-450a (to 8%), P-450b (to 22%) and P-450h (to 23%), but caused a pronounced increase in the catalytic activity of cytochrome P-450p (up to 4.2-fold). These effects were attributed to changes in ionic strength, because similar but less pronounced effects were observed with Tris-HCl (which has approximately 1/3 the ionic strength of phosphate buffer at pH 7.4). Testosterone oxidation by microsomal cytochromes P-450a, P-450b, P-450h and P-450p was also differentially affected by pH (over the range 6.8-8.0). The pH optima ranged from 7.1 (for P-450a and P-450h) to 8.0 (for P-450p), with an intermediate value of 7.4 for cytochrome P-450b. Increasing the pH from 6.8 to 8.0 unexpectedly altered the relative amounts of the 3 major metabolites produced by cytochrome P-450h. The decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h that accompanied an increase in ionic strength or pH could be duplicated in reconstitution systems containing purified P-450a, P-450b or P-450h, equimolar amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. This result indicated that the decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h was a direct effect of ionic strength and pH on these enzymes, rather than a secondary effect related to the increase in testosterone oxidation by cytochrome P-450p. Similar studies with purified cytochrome P-450p were complicated by the atypical conditions needed to reconstitute this enzyme. However, studies on the conversion of digitoxin to digitoxigenin bisdigitoxoside by liver microsomes, which is catalyzed specifically by cytochrome P-450p, provided indirect evidence that the increase in catalytic activity of cytochrome P-450p was also a direct effect of ionic strength and pH on this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)