Properties of the two terminal oxidases of Escherichia coli.

Proton translocation coupled to oxidation of ubiquinol by O2 was studied in spheroplasts of two mutant strains of Escherichia coli, one of which expresses cytochrome d, but not cytochrome bo, and the other expressing only the latter. O2 pulse experiments revealed that cytochrome d catalyzes separation of the protons and electrons of ubiquinol oxidation but is not a proton pump. In contrast, cytochrome bo functions as a proton pump in addition to separating the charges of quinol oxidation. E. coli membranes and isolated cytochrome bo lack the CuA center typical of cytochrome c oxidase, and the isolated enzyme contains only 1Cu/2Fe. Optical spectra indicate that high-spin heme o contributes less than 10% to the reduced minus oxidized 560-nm band of the enzyme. Pyridine hemochrome spectra suggest that the hemes of cytochrome bo are not protohemes. Proteoliposomes with cytochrome bo exhibited good respiratory control, but H+/e- during quinol oxidation was only 0.3-0.7. This was attributed to an "inside out" orientation of a significant fraction of the enzyme. Possible metabolic benefits of expressing both cytochromes bo and d in E. coli are discussed.     

Proton translocation by the mitochondrial cytochrome b-c1 complex is inhibited by NN''-dicyclohexylcarbodi-imide.

NN'-Dicyclohexylcarbodi-imide at low concentrations decreases the H+/2e ratio for rat liver mitochondria over the span succinate to oxygen from 5.9 +/- 0.3 (mean +/- S.E.M.) to 4.0 +/- 0.1 and for the cytochrome b-c1 complex from 3.8 +/- 0.2 to 1.9 +/- 0.1, but has little effect on the H+/2e ratio of cytochrome oxidase. The decrease in stoicheiometry is due, not to uncoupling or inhibition of electron transport, but to inhibition of proton translocation. NN'-Dicyclohexylcarbodi-imide thus 'decouples' proton translocation in the cytochrome b-c1 complex.     

Cytochrome o (bo) is a proton pump in Paracoccus denitrificans and Escherichia coli

Spheroplasts from aerobically grown wild-type Paracoccus denitrificans cells respire with succinate despite specific inhibition of the cytochrome bc1 complex by myxothiazol. Coupled to this activity, which involves only b-type cytochromes, there is translocation of 1.5-1.9 h+/e- across the cytoplasmic membrane. Similar H+ translocation ratios are observed during oxidation of ubiquinol in spheroplasts from aerobically grown mutants of Paracoccus lacking cytochrome c oxidase, or deficient in cytochrome c, as well as in a strain of E. coli from which cytochrome d was deleted. These observations show that the cytochrome o complex is a proton pump much like cytochrome aa3 to which it is structurally related.     

What is the essential proton-translocating molecular machinery in cytochrome oxidase?

Pulses of O2 added to anaerobic mitochondria in the presence of antimycin, but in the absence of exogenous reductants, led to H+ translocation until the amount of oxidizing equivalents exceeded the number of endogenous reducing equivalents capable of rapid reduction of cytochrome oxidase. This demonstrates that either the heme of cytochrome alpha or that CuA is the redox center, the function of which is coupled to proton translocation in cytochrome oxidase. Chemical labeling of subunit III of cytochrome oxidase by dicyclocarbodiimide (DCCD), or removal of this subunit by treatment of the enzyme at high pH, results in loss of proton translocation by the isolated and membrane-reconstituted enzyme. Possible roles of subunit III in proton translocation are discussed.     

Proton to electron stoichiometry in electron transport of spinach thylakoids

According to the concept of the Q-cycle, the H+/e- ratio of the electron transport chain of thylakoids can be raised from 2 to 3 by means of the rereduction of plastoquinone across the cytochrome b6f complex. In order to investigate the H+/e- ratio we compared stationary rates of electron transport and proton translocation in spinach thylakoids both in the presence of the artificial electron acceptor ferricyanide and in the presence of the natural acceptor system ferredoxin+NADP. The results may be summarised as follows: (1) a variability of the H+/e- ratio occurs with either acceptor. H+/e- ratios of 3 (or even higher in the case of the natural acceptor system, see below) are decreased towards 2 if strong light intensity and low membrane permeability are employed. Mechanistically this could be explained by proton channels connecting the plastoquinol binding site alternatively to the lumenal or stromal side of the cytochrome b6f complex, giving rise to a proton slip reaction at high transmembrane DeltapH. In this slip reaction protons are deposited on the stromal instead of the lumenal side. In addition to the pH effect there seems to be a contribution of the redox state of the plastoquinone pool to the control of proton translocation; switching over to stromal proton deposition is favoured when the reduced state of plastoquinone becomes dominant. (2) In the presence of NADP a competition of both NADP and oxygen for the electrons supplied by photosystem I takes place, inducing a general increase of the H+/e- ratios above the values obtained with ferricyanide. The implications with respect to the adjustment of a proper ATP/NADPH ratio for CO2 reduction are discussed.     

Inhibition by dicyclohexylcarbodiimide of proton ejection but not electron transfer in rat liver mitochondria

The primary effect of dicyclohexylcarbodiimide (DCCD) at the cytochrome b-c1 region of the respiratory chain of rat liver mitochondria is an inhibition of proton translocation. No significant decrease was observed in the rate of electron flow from succinate to cytochrome c when measured as cytochrome c reductase, K3Fe(CN)6 reductase, or the rate of H+ release in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone after treatment with sufficient DCCD to abolish completely electrogenic proton ejection. The inhibitory effects of DCCD were time and concentration dependent and affected by the pH of the medium. Lowering the pH from 7.3 to 6.7 resulted in a progressively faster rate and extent of inhibition of proton ejection by DCCD. At pH 6.9, the H+/2e- decreased by 50% within 30 s after DCCD addition; however, at pH 7.3, a 50% decrease was not observed until 2 min after DCCD addition. DCCD did not act as an uncoupler as both the rate of proton ejection and back decay were decreased after incubation with DCCD. Treatment of rat liver mitochondria with DCCD under these same conditions also resulted in a broadening of the sharp spectral shift of cytochrome b observed after antimycin addition to mitochondria previously reduced with succinate suggesting that DCCD may modify cytochrome b in such a way that the binding of antimycin is altered.     

Characteristics of energy-linked proton translocation in liposome reconstituted bovine cytochrome bc1 complex. Influence of the protonmotive force on the H+/e- stoichiometry.

A study is presented on the H+/e- stoichiometry for proton translocation by the isolated cytochrome bc1 complex under level-flow and steady-state conditions. An experimental procedure was used which allows the determination of pure vectorial proton translocation in both conditions in a single experiment. The results obtained indicate an H+/e- ratio of 1 at level-flow and 0.3 at steady-state. The ratios appear to be independent of the rate of electron transfer through the complex. Making use of pyranine-entrapped bc1 vesicles, a respiration-dependent steady-state delta pH value of 0.4 was determined in the presence of valinomycin. This value could be either decreased by subsaturating concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or increased by introducing bovine serum albumin in the assay mixture. The steady-state H+/e- ratio appeared to be in linear inverse correlation with the delta pH. This indicates that delta pH exerts a control on the proton pump of the bc1 complex at the steady state. The effect of valinomycin-mediated potassium-diffusion potential on electron-transfer and proton-translocation activities is also shown. The experiments presented show that the H+/e- ratio is unaffected, both at level flow and steady state, by an imposed diffusion potential up to around 100 mV. At higher potential values the level-flow H+/e- ratio slightly decreased. Measurements as a function of imposed membrane potential of the rate of electron transfer at level flow and of the rate of the pre-steady-state reduction of b and c1 cytochromes in the complex indicate activation of electron transfer at potential values of 40-50 mV. This activation appears, however, to involve a rate-limiting step which remains normally coupled to proton translocation.     

The H+/e? stoicheiometry of respiration-linked proton translocation in the cytochrome system of mitochondria

1. The -->H(+)/e(-) quotients for proton release from mitochondria associated with electron flow from succinate and duroquinol to O(2), ferricyanide or ferricytochrome c, and from NNN'N'-tetramethyl-p-phenylenediamine+ascorbate to O(2), were determined from rate measurements of electron flow and proton translocation. 2. Care was taken to avoid, or to take into account, unrelated electron flow and proton translocation, which might take place in addition to the oxido-reductions that were the subject of our analysis. Spectrophotometric techniques were chosen to provide accurate measurement of the rate of consumption of oxidants and reductants. The rate of proton translocation was measured with fast pH meters with a precision of 10(-3) pH unit. 3. The -->H(+)/O quotient for succinate or duroquinol oxidation was, at neutral pH, 4, when computed on the basis of spectrophotometric determinations of the rate of O(2) consumption or duroquinol oxidation. Higher -->H(+)/O quotients for succinate oxidation, obtained from polarographic measurements of O(2) consumption, resulted from underestimation of the respiratory rate. 4. The -->H(+)/2e(-) quotient for electron flow from succinate and duroquinol to ferricyanide or ferricytochrome c ranged from 3.9 to 3.6. 5. Respiration elicited by NNN'N'-tetramethyl-p-phenylenediamine+ascorbate by antimycin-inhibited mitochondria resulted in extra proton release in addition to that produced for oxidation of ascorbate to dehydroascorbate. Accurate spectrophotometric measurement of respiration showed that the -->H(+)/e(-) ratio was only 0.25 and not 0.7-1.0 as obtained with the inadequate polarographic assay of respiration. Proton release was practically suppressed when mitochondria were preincubated aerobically in the absence of antimycin. Furthermore, the rate of scalar proton consumption for water production was lower than that expected from the stoicheiometry. Thus the extra proton release observed during respiration elicited by NNN'N'-tetramethyl-p-phenylenediamine+ascorbate is caused by oxidation of endogenous hydrogenated reductants. 6. It is concluded that (i) the -->H(+)/O quotient for the cytochrome system is, at neutral pH, 4 and not 6 or 8 as reported by others; (ii) all the four protons are released during electron flow from quinol to cytochrome c; (iii) the oxidase transfers electrons from cytochrome c to protons from the matrix aqueous phase and does not pump protons from the matrix to the outer aqueous phase.     

Chemical modification of the mitochondrial bc1 complex by N,N'-dicyclohexylcarbodiimide inhibits proton translocation

We report here that N,N'-dicyclohexylcarbodiimide (DCCD) decreases the H/2e stoichiometry of the cytochrome bc1 complex from 3.8 +/- 0.2 (10) to 2.1 +/- 0.1 (8) but has only a minimal effect on the H/2e ratio of cytochrome oxidase under the relatively mild conditions used. The effect on the bc1 complex cannot be explained by uncoupling, by inhibition of electron transport or by selective mitochondrial damage. We conclude that DCCD is an inhibitor of proton translocation within the bc1 complex. There are three possible explanations of this effect: (a) DCCD could alter the pathway of electron flow, (b) DCCD could prevent one of the proton translocation reactions but not electron transport, (c) DCCD could prevent the conduction of the translocated proton to the external phase.     

Redox-linked proton translocation in the b-c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. General characteristics and control of electron flow by delta micro H+

A study is presented of the characteristics of redox-linked proton translocation in the b-c1 complex isolated from beef-heart mitochondria and reconstituted into phospholipid vesicles. Measurements of the H+/e- stoichiometry, with three different methods, show that four protons are released from the vesicles per 2e- flowing from quinols to cytochrome c, two of these protons formally deriving from scalar oxidation of quinols by cytochrome c. This H+/e- stoicheiometry is independent of the initial redox state of the b-c1 complex (fully reduced or oxidized) and the rate of electron flow through the complex. It does not change in the pH range 6.0 - 7.2, but declines to 1.5 going with pH from 7.2 - 8.3. This decrease is accompanied by enhancement of the rate of electron flow in the coupled state. Collapse of delta psi effected by valinomycin addition to turning-over b-c1 vesicles resulted in substantial oxidation of cytochrome b-566 and comparable reduction of cytochrome c1, with little oxidation of cytochrome b-562. Nigericin alone had no effect on the steady-state redox levels of b and c cytochromes. Its addition in the presence of valinomycin caused oxidation of b cytochromes but no change in the redox state of cytochrome c1. Valinomycin alone caused a marked enhancement of the rate of electron flow through the complex. Nigericin alone was ineffective, but caused further stimulation of electron flow when added in the presence of valinomycin. The data presented are discussed in terms of two mechanisms: the Q cycle and a model based on combination of protonmotive catalysis by special bound quinone and proton conduction along pathways in the apoproteins.     

Rate enhancement of the internal electron transfer in cytochrome c oxidase by the formation of a peroxide complex; its implication on the reaction mechanism of cytochrome c oxidase

The oxidation of reduced cytochrome c oxidase by hydrogen peroxide was investigated with stopped-flow methods. It was reported by us previously (A.C.F. Gorren, H. Dekker and R. Wever (1986) Biochim. Biophys. Acta 852, 81-92) that at low H2O2 concentrations cytochrome a is oxidised simultaneously with cytochrome a3, but that at higher H2O2 concentrations the oxidation of cytochrome a is slower than that of cytochrome a3. We now report that for high peroxide concentrations (10-45 mM) the oxidation rate of cytochrome a increased linearly with the concentration of H2O2 (k = 700 M-1.S-1). Upon extrapolation to zero H2O2 concentration an intercept with a value of 16 s-1 (at 20 degrees C and pH 7.4) was found. A reaction sequence is described to explain these results; according to this model the rate constant (16 S-1) at zero H2O2 concentration represents the true value of the rate of electron transfer from cytochrome a to cytochrome a3 when the a3-CuB site is oxidised and unligated. However, when a complex of hydrogen peroxide with oxidised cytochrome a3 is formed, this rate is strongly enhanced. The slope (700 M-1.S-1) would then represent the rate of cytochrome a3(3+)-H2O2 complex formation. From experiments in which the pH was varied, we conclude that the reaction of H2O2 with cytochrome a3(2+) is independent of pH, whereas the electron-transfer rate from cytochrome a to cytochrome a3 gradually decreases with increasing pH. From the temperature dependence we could calculate values of 23 kJ.mol-1 and 45 kJ.mol-1 for the activation energies of the oxidations by H2O2 of cytochrome a3(2+) and cytochrome a2+, respectively. The similarity of the values that were obtained for cytochrome a oxidation both with H2O2 and with O2 as the electron acceptor suggests that the reactions share the same mechanism. In 2H2O the reactions studied decreased in rate. For the reaction of 2H2O2 with reduced cytochrome a3 in 2H2O, a small effect was found (15% decrease in rate constant). However, the internal electron-transfer rate from cytochrome a to cytochrome a3 decreased by 50%, Our results suggest that the internal electron transfer is associated with proton translocation.     

Bacterial respiration-linked proton translocation and its relationship to respiratory-chain composition.

1. The relationship between chain composition and the efficiency of respiration-linked proton translocation was studied in nine bacterial species of widely differing taxonomic and ecological status. 2. All the bacteria investigated contained respiratory chain dehydrogenases, ubiquinone and/or menaquinone, cytochrome b and cytochrome oxidase aa3 and/or o. In addition, some of these organisms also contained pyridine nucleotide transhydrogenase and/or cytochrome c. 3. leads to H+/O ratios of whole cell suspensions oxidising endogenous substrates were in the approximate range 4-8 mol H+ translocated per g-atom oxygen consumed. It was concluded from the observed leads to H+/O ratios of cells loaded with specific substrates that proton-translocating loops 1 and 2 were present in all of the organisms investigated, but that loops 0 and 3 were dependent upon the presence of pyridine nucleotide transhydrogenase and cytochrome c respectively. 4. The wide range in energy conservation efficiency which was observed in these organisms is discussed in relation to their respiratory chain composition and natural habitat.     

pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase. The role of H2O produced at the oxygen-reduction site

A study is presented on the pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of purified cytochrome c oxidase (COX) from beef heart reconstituted in phospholipid vesicles (COV). Protons were shown to be released from COV both in the oxidative and reductive phases. In the oxidation by O2 of the fully reduced oxidase, the H+/COX ratio for proton release from COV (R --> O transition) decreased from approximately 2.4 at pH 6.5 to approximately 1.8 at pH 8.5. In the direct reduction of the fully oxidized enzyme (O --> R transition), the H+/COX ratio for proton release from COV increased from approximately 0.3 at pH 6.5 to approximately 1.6 at pH 8.5. Anaerobic oxidation by ferricyanide of the fully reduced oxidase, reconstituted in COV or in the soluble case, resulted in H+ release which exhibited, in both cases, an H+/COX ratio of 1.7-1.9 in the pH range 6.5-8.5. This H+ release associated with ferricyanide oxidation of the oxidase, in the absence of oxygen, originates evidently from deprotonation of acidic groups in the enzyme cooperatively linked to the redox state of the metal centers (redox Bohr protons). The additional H+ release (O2 versus ferricyanide oxidation) approaching 1 H+/COX at pH < or = 6.5 is associated with the reduction of O2 by the reduced metal centers. At pH > or = 8.5, this additional proton release takes place in the reductive phase of the catalytic cycle of the oxidase. The H+/COX ratio for proton release from COV in the overall catalytic cycle, oxidation by O2 of the fully reduced oxidase directly followed by re-reduction (R --> O --> R transition), exhibited a bell-shaped pH dependence approaching 4 at pH 7.2. A mechanism for the involvement in the proton pump of the oxidase of H+/e- cooperative coupling at the metal centers (redox Bohr effects) and protonmotive steps of reduction of O2 to H2O is presented.     

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Analysis of proton translocation associated with oxidation of endogenous ubiquinol.

A study is presented of the kinetics and stoichiometry of fast proton translocation associated to aerobic oxidation of components of the mitochondrial respiratory chain. 1. Aerobic oxidation of ubiquinol and b cytochromes is accompanied in EDTA particles, obtained by sonication of beef-heart mitochondria, by synchronous proton uptake. 2. The rapid proton uptake associated to oxidation and b cytochromes is greatly stimulated by valinomycin plus K+, but is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. 3. 4 gion H+ are taken up per mol ubiquinol oxidized by oxygen. This H+/2e- ratio, measured in the rapid anaerobic-aerobic transition of the particles is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. 4. Intact mitochondria aerobic oxidation of oxygen-terminal electron carriers is accompanied by antimycin-insensitive synchronous proton release, oxidation of ubiquinol and reduction of b cytochromes. The amount of protons released is in excess with respect to the amount of ubiquinol oxidized. 5. It is concluded that electron flow along complex III, from ubiquinol to cytochrome c, is directly coupled to vectorial proton translocation. The present data suggest that there exist(s) between ubiquinol and cytochrome c one (or two) respiratory carrier(s), whose oxido-reduction is directly linked to effective transmembrane proton translocation.     

-->H+/2e- stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles.

Tightly coupled bovine heart submitochondrial particles treated to activate complex I and to block ubiquinol oxidation were capable of rapid uncoupler-sensitive inside-directed proton translocation when a limited amount of NADH was oxidized by the exogenous ubiquinone homologue Q1. External alkalization, internal acidification and NADH oxidation were followed by the rapidly responding (t1/2 < or = 1 s) spectrophotometric technique. Quantitation of the initial rates of NADH oxidation and external H+ decrease resulted in a stoichiometric ratio of 4 H+ vectorially translocated per 1 NADH oxidized at pH 8.0. ADP-ribose, a competitive inhibitor of the NADH binding site decreased the rates of proton translocation and NADH oxidation without affecting -->H+/2e- stoichiometry. Rotenone, piericidin and thermal deactivation of complex I completely prevented NADH-induced proton translocation in the NADH-endogenous ubiquinone reductase reaction. NADH-exogenous Q1 reductase activity was only partially prevented by rotenone. The residual rotenone- (or piericidin-) insensitive NADH-exogenous Q1 reductase activity was found to be coupled with vectorial uncoupler-sensitive proton translocation showing the same -->H+/2e- stoichiometry of 4. It is concluded that the transfer of two electrons from NADH to the Q1-reactive intermediate located before the rotenone-sensitive step is coupled with translocation of 4 H+.     

Protonmotive Q cycle pathway of electron transfer and energy transduction in the three-subunit ubiquinol-cytochrome c oxidoreductase complex of Paracoccus denitrificans

Ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex from Paracoccus denitrificans consists of only three polypeptide subunits (Yang, X., and Trumpower, B. L. (1986) J. Biol. Chem. 261, 12282-12289), whereas the analogous complexes of eukaryotic mitochondria consist of nine or more polypeptides (Schagger, H., Link, T. A., Engel, W. D., and von Jagow, G. (1986) Methods Enzymol. 126, 224-237). Using the purified three-subunit Paracoccus complex we have tested whether this simple cytochrome bc1 complex has the same electron transfer pathway and proton translocation activity as the bc1 complexes of mitochondria. Under presteady state conditions, the effects of inhibitors on reduction of cytochromes b and c1 by quinol and oxidant-induced reduction of cytochrome b indicate a cyclic electron transfer pathway and two routes of cytochrome b reduction in the three-subunit Paracoccus cytochrome bc1 complex. A novel method was developed to incorporate the cytochrome bc1 complex into liposomes with the detergent dodecyl maltoside. The enzyme reconstituted into liposomes translocated protons with an H+/2e value of 3.9. Carbonyl cyanide m-chlorophenylhydrazone eliminated proton translocation, while permitting the scalar release of protons from quinol, and thus reduced the H+/2e ratio to 2. These values agree with the predicted stoichiometries for proton translocation by a protonmotive Q cycle pathway. No inhibition of proton translocation by N',N'-dicyclohexylcarbodiimide was detected when the Paracoccus cytochrome bc1 complex was incubated with N',N'-dicyclohexylcarbodiimide before or after reconstitution into liposomes. Electron transfer in the three-subunit complex thus appears to occur by a protonmotive Q cycle pathway identical to that in mitochondrial cytochrome bc1 complexes. Only three polypeptides, cytochromes b, c1, and the Rieske iron-sulfur protein, are required for respiration and energy transduction in the cytochrome bc1 complex. The function of the supernumerary polypeptides in mitochondrial bc1 complexes is thus unclear.     

Structure and vectorial properties of proteoliposomes containing cytochrome oxidase in the submitochondrial orientation

Cytochrome oxidase proteoliposomes were prepared from bovine heart oxidase. Size distributions determined by quasi-elastic light scattering (QELS) showed that there was a small population of large vesicles (120-200-nm diameter) and a large population of small vesicles (50-100-nm diameter). Trapping cytochrome c inside the proteoliposomes did not significantly alter this size distribution. Separation of the vesicles by gel filtration, however, revealed that the cytochrome c/cytochrome a ratio is higher in the larger vesicles. Internally trapped cytochrome c can be reduced by the membrane-permeable reductants 2,3,5,6-tetramethyl-p-phenylenediamine (DAD) or N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). Respiration on internal cytochrome c generated a membrane potential of 53 mV (positive inside) and a pH gradient of 0.2 (acid inside) as monitored by the optical probes oxonol V and pyranine, respectively. But the true magnitude of these gradients in individual proteoliposomes is complicated by vesicle heterogeneity. The membrane potential increased biphasically with increasing concentration of reductant. Ionophore sensitivity was higher for the "low Km" phase, and respiration became increasingly uncoupled as the reductant concentration was increased. These findings are consistent with a kinetic heterogeneity such that vesicles respiring at lower reductant concentrations generate a higher proton motive force than those with a larger Km. The steady-state internal acidification induced by turnover of the internally facing enzyme is probably maintained by both cytochrome oxidase proton translocation and a TMPD+/H+ antiport present in these vesicles [Cooper, C. E., & Nicholls, P. (1987) FEBS. Lett. 223, 155-160].     

Characteristics of the redox-linked proton ejection in beef-heart cytochrome c oxidase reconstituted in liposomes

In this paper a study is presented of the characteristics of redox-linked proton ejection exhibited by isolated beef-heart cytochrome c oxidase incorporated in asolectin vesicles. The enzyme was 90% oriented 'right-side out' as in the mitochondrial membrane. The effects on the H+/e- stoichiometry of the modalities of activation of electron flow, the pH of the medium and its ionic composition were investigated. The results obtained show that, whilst ferrocytochrome c pulses of the aerobic oxidase vesicles at neutral pH and in the presence of saturating concentrations of valinomycin and K+ to ensure charge compensation produced H+/e- ratios around 1 (as has been shown previously), oxygen pulses of reduced anaerobic vesicles supplemented with cytochrome c, gave H+/e- ratios around 0.3. The H+/e- ratios exhibited, with both reductant and oxidant pulses, a marked pH dependence. Maximum values were observed at pH 7.0-7.7, which decreased to negligible values at acidic pH with apparent pKa of 6.7-6.3. Mg2+ and Ca2+ caused a marked depression of the H+/e- ratio, which in the presence of these cations and after a few ferrocytochrome pulses, became negligible. Analysis of cytochrome c oxidation showed that the modalities of activation of electron flow and divalent cations exerted profound effects on the kinetics of cytochrome c oxidation by oxidase vesicles. The observations presented seem to provide interesting clues for the nature and mechanism of redox-linked proton ejection in reconstituted cytochrome c oxidase.     

Oxygen-limited continuous culture and respiratory energy conservation in Escherichia coli.

Escherichia coli B was cultured continuously in succinate-minimal medium under conditions of oxygen limitation in the phauxostat. With decreasing oxygenation and consequent decreasing growth rates, the complement of terminal cytochrome oxidases changed as follows: high growth rates, cytochrome o; intermediate growth rates, cytochromes o and d; lowest growth rates, cytochromes o, d, and a1. Respiratory kinetics exhibited by nongrowing cell suspensions obtained from continuous cultures indicated that terminal oxidase activity was exhibited by cytochrome o (Km for O2 = 0.2 micron; Vmax = 1.1 to 1.5 mumol of O2 per nmol of cytochrome o per min) and cytochrome d (Km for O2 = 0.024 micron; Vmax = 0.7 mumol of O2 per nmol of cytochrome d per min). During oxygen-limited growth, the molar growth yield referred to respiration, and corrected for maintenance respiration [Yo(max)], was 12.6 g (dry weight) per g-atom of oxygen, not significantly different from the succinate-limited value of 12.0 g (dry weight) per g-atom of oxygen. The rate of maintenance respiration of the oxygen-limited culture was only 3.4 mg-atoms of O per g (dry weight) per h, some threefold less than that of the succinate-limited culture. Respiration-driven proton extrusion did not vary with the growth rate or with the complement of terminal oxidases (H+/O = 3.7; standard deviation, 0.07). We conclude that the content of terminal oxidases is without effect on the efficiency of respiratory energy conservation.     

Deuterium kinetic isotope effects in the p-side pathway for quinol oxidation by the cytochrome b(6)f complex.

Plastoquinol oxidation and proton transfer by the cytochrome b(6) f complex on the lumen side of the chloroplast thylakoid membrane are mediated by high and low potential electron transport chains. The rate constant for reduction, k(bred), of cytochrome b(6) in the low potential chain at ambient pH 7.5-8 was twice that, k(fred), of cytochrome f in the high potential chain, as previously reported. k(bred) and k(fred) have a similar pH dependence in the presence of nigericin/nonactin, decreasing by factors of 2.5 and 4, respectively, from pH 8 to an ambient pH = 6, close to the lumen pH under conditions of steady-state photosynthesis. A substantial kinetic isotope effect, k(H2O)/k(D2O), was found over the pH range 6-8 for the reduction of cytochromes b(6) and f, and for the electrochromic band shift associated with charge transfer across the b(6)f complex, showing that isotope exchange affects the pK values linked to rate-limiting steps of proton transfer. The kinetic isotope effect, k(bred)(H2O)/k(bred) (D2O) approximately 3, for reduction of cytochrome b in the low potential chain was approximately constant from pH 6-8. However, the isotope effect for reduction of cytochrome f in the high potential chain undergoes a pH-dependent transition below pH 6.5 and increased 2-fold in the physiological region of the lumen pH, pH 5.7-6.3, where k(fred)(H2O)/k(fred)(D2O) approximately 4. It is proposed that a rate-limiting step for proton transfer in the high potential chain resides in the conserved, buried, and extended water chain of cytochrome f, which provides the exit port for transfer of the second proton derived from p-side quinol oxidation and a "dielectric well" for charge balance.