A new species of bound ubisemiquinone anion in QH2: cytochrome c oxidoreductase

Using a combination of EPR and low temperature diffuse reflectance spectroscopy, a new species of semiquinone anion has been detected in QH2:cytochrome c oxidoreductase in submitochondrial particles under conditions of oxidant-induced extra reduction of cytochrome b. In contrast to the previously detected semiquinone anion, this new species is insensitive to antimycin but sensitive to treatment with 2,3-dimercaptopropanol and O2. The two species can easily be distinguished on the basis of their respective EPR properties since they differ in g-value, line width, and microwave power saturation behavior. It is concluded that the two species of semiquinone anion are bound to different domains on QH2:cytochrome c oxidoreductase. The existence of two different semiquinone anions in the enzyme strongly supports a mechanism of electron flow as proposed in the Q-cycle.     

Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria contains one cooperative ubiquinone-reduction centre

Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria was isolated as a protein-Triton complex and free of ubiquinol (Q). The enzyme was incorporated into phosphatidylcholine membranes together with Q. The effects of varying the molar ratio of Q to enzyme on the electron transfer from duroquinol (DHQ2) to the cytochromes c, c1 and b were studied. The rate of electron flow from DQH2 to cytochrome c was 15 times increased by Q and was maximal when one molecule of Q was bound to one enzyme dimer. The apparent Km value for DQH2 of the Q-free enzyme was 5 microM and of the Q-supplemented enzyme 25 microM. The pre-steady-state rate of electron transfer from DQH2 to cytochrome c1 was also 15 times increased by Q and was maximal with one Q molecule bound to one enzyme dimer. This effect of Q was inhibited by antimycin. The pre-steady-state rate of electron transfer from DQH2 to cytochrome b was 5 times decreased when Q was bound to the enzyme and this effect of Q was insensitive to myxothiazol. The H+/2e- stoichiometry with DQH2 as substrate of the Q-supplemented enzyme was 3.6. These results are interpreted in accordance with a Q-cycle mechanism operating in a dimeric cytochrome reductase. Each enzyme monomer catalyses a single electron transfer from the QH2-oxidation centre to the Q-reduction centre and the two monomers cooperate in the reduction of Q to QH2 at one Q-reduction centre. This centre contains two different binding sites for Q. DQH2 does not properly react at the QH2-oxidation centre. DQH2, however, binds to the loose Q-binding site of the Q-reduction centre and reduces the Q bound to the tight Q-binding site of the centre. The QH2 thus formed at the Q-reduction centre serves as electron donor for the QH2-oxidation centre.     

The effect of pH, ubiquinone depletion and myxothiazol on the reduction kinetics of the prosthetic groups of ubiquinol:cytochrome c oxidoreductase

(1) The kinetics of the reduction by duroquinol of the prosthetic groups of QH2:cytochrome c oxidoreductase and of the formation of ubisemiquinone have been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) The formation of the antimycin-sensitive ubisemiquinone anion parallels the reduction of both high-potential and low-potential cytochrome b-562. (3) The rates of reduction of both the [2Fe-2S] clusters and cytochromes (c + c1) are pH dependent. There is, however, a pH-dependent discrepancy between their rate of reduction, which can be correlated with the difference in pH dependencies of their midpoint potentials. (4) Lowering the pH or the Q content results in a slower reduction of part of the [2Fe-2S] clusters. It is suggested that one cluster is reduced by a quinol/semiquinone couple and the other by a semiquinone/quinone couple. (5) Myxothiazol inhibits the reduction of the [2Fe-2S] clusters, cytochrome c1 and high-potential cytochrome b-562. (6) The results are consistent with a Q-cycle model describing the pathway of electrons through a dimeric QH2:cytochrome c oxidoreductase.     

The multiplicity and stoichiometry of the prosthetic groups in QH2: cytochrome c oxidoreductase as studied by EPR.

1. The EPR signal in the g = 2 region of the reduced QH2: cytochrome c oxidoreductase as present in submitochondrial particles and the isolated enzyme is an overlap of two signals in a 1 : 1 weighted ratio. Both signals are due to [2Fe-2S]+1 centers. 2. From the signal intensity it is computed that the concentration of each Fe-S center is half that of cytochrome c1. 3. The line shape of one of the Fe-S centers, defined as center 1, is reversibly dependent on the redox state of the b-c1 complex. The change of the line shape cannot be correlated with changes of the redox state of any of the cytochromes in QH2: cytochrome c oxidoreductase. 4. Lie the optical spectrum, the EPR spectrum of the cytochromes is composed of the absorption of at least three different b cytochromes and cytochrome c1. 5. The molar ratio of the prosthetic groups was found to be c1 : b-562 : b-566 : b-558 : center 1 : center 2 = 2 : 2 : 1 : 1 : 1 : 1. The consequences of this stoichiometry are discussed in relation to the basic enzymic unit of QH2 : cytochrome c oxidoreductase.     

The kinetics of reoxidation of yeast complex III. An evaluation of the Q-cycle

The reoxidation of reduced yeast Complex III by oxidants believed to react with cytochrome c1 exhibited multiple phases for both cytochrome c1 and the cytochromes b; the reoxidation of cytochrome b, but not cytochrome c1, was markedly slowed by the presence of antimycin. The data are consistent with the Q-cycle or any other scheme which proposes a branched path for electron transport between the cytochrome b centers and the endogenous Q6, provided certain constraints are relaxed. The reoxidation of the endogenous quinone proceeded at a rate comparable to that of the rapidly reacting cytochrome b and appeared to be complete within 100 ms. Removal of the endogenous quinone did not change the rate or extent of reoxidation of any of the heme centers, demonstrating that quinone is not required for electron transport between cytochromes b and the iron-sulfur cluster. This result is inconsistent with the requirements of the Q-cycle. Funiculosin completely inhibited the reoxidation of cytochrome b whereas the reoxidation of cytochrome c1 exhibited simple first-order kinetics in the presence of this inhibitor, implying that the iron-sulfur cluster is on the direct path of electron transfer from cytochrome b to cytochrome c1. Potent inhibition of cytochrome b oxidation was also observed with myxothiazol and mucidin. The reaction of reduced Complex III with Q1 also exhibited multiple phases in the oxidation of the cytochrome b centers; these phases were unaffected by the presence of myxothiazol. Addition of antimycin, or removal of the endogenous quinone, eliminated the rapid phases; only one of the cytochrome b centers was oxidized under these conditions. Epr showed that it is the low-potential cytochrome b that is the species rapidly oxidized.     

Proton translocation by the cytochrome bc1 complexes of phototrophic bacteria: introducing the activated Q-cycle.

The cytochrome bc1 complexes are proton-translocating, dimeric membrane ubiquinol:cytochrome c oxidoreductases that serve as "hubs" in the vast majority of electron transfer chains. After each ubiquinol molecule is oxidized in the catalytic center P at the positively charged membrane side, the two liberated electrons head out, according to the Mitchell's Q-cycle mechanism, to different acceptors. One is taken by the [2Fe-2S] iron-sulfur Rieske protein to be passed further to cytochrome c1. The other electron goes across the membrane, via the low- and high-potential hemes of cytochrome b, to another ubiquinone-binding site N at the opposite membrane side. It has been assumed that two ubiquinol molecules have to be oxidized by center P to yield first a semiquinone in center N and then to reduce this semiquinone to ubiquinol. This review is focused on the operation of cytochrome bc1 complexes in phototrophic purple bacteria. Their membranes provide a unique system where the generation of membrane voltage by light-driven, energy-converting enzymes can be traced via spectral shifts of native carotenoids and correlated with the electron and proton transfer reactions. An "activated Q-cycle" is proposed as a novel mechanism that is consistent with the available experimental data on the electron/proton coupling. Under physiological conditions, the dimeric cytochrome bc1 complex is suggested to be continually primed by prompt oxidation of membrane ubiquinol via center N yielding a bound semiquinone in this center and a reduced, high-potential heme b in the other monomer of the enzyme. Then the oxidation of each ubiquinol molecule in center P is followed by ubiquinol formation in center N, proton translocation and generation of membrane voltage.     

Ubiquinol:cytochrome c oxidoreductase (complex III). Effect of inhibitors on cytochrome b reduction in submitochondrial particles and the role of ubiquinone in complex III

Two sets of studies have been reported on the electron transfer pathway of complex III in bovine heart submitochondrial particles (SMP). 1) In the presence of myxothiazol, MOA-stilbene, stigmatellin, or of antimycin added to SMP pretreated with ascorbate and KCN to reduce the high potential components (iron-sulfur protein (ISP) and cytochrome c(1)) of complex III, addition of succinate reduced heme b(H) followed by a slow and partial reduction of heme b(L). Similar results were obtained when SMP were treated only with KCN or NaN(3), reagents that inhibit cytochrome oxidase, not complex III. The average initial rate of b(H) reduction under these conditions was about 25-30% of the rate of b reduction by succinate in antimycin-treated SMP, where both b(H) and b(L) were concomitantly reduced. These results have been discussed in relation to the Q-cycle hypothesis and the effect of the redox state of ISP/c(1) on cytochrome b reduction by succinate. 2) Reverse electron transfer from ISP reduced with ascorbate plus phenazine methosulfate to cytochrome b was studied in SMP, ubiquinone (Q)-depleted SMP containing 相似文献   

The pathway of electrons through OH2:cytochrome c oxidoreductase studied by pre-steady -state kinetics

The kinetic behaviour of the prosthetic groups and the semiquinones in in QH2:cytochrome c oxidoreductase has been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) In the absence of antimycin, cytochrome b-562 is reduced in two phases separated by a lag time. The initial very rapid reduction phase, that coincides with the formation of the antimycin-sensitive Qin, is ascribed to high-potential cytochrome b-562 and the slow phase to low-potential cytochrome b-562. the two cytochromes are present in a 1:1 molar ratio. The lag time between the two reduction phases decreases with increasing pH. Both the [2 Fe-2S] clusters and cytochrome c1 are reduced monophasically under these conditions, but at a rate lower than that of the initial rapid reduction of cytochrome b-562. (3) In the presence of antimycin and absence of oxidant, cytochrome b-562 is still reduced biphasically, but there is no lag between the two phases. No Qin is formed and both the Fe-S clusters and cytochrome c1 are reduced biphasically, one-half being reduced at the same rate as in the absence of antimycin and the other half 10-times slower. (4) In the presence of antimycin and oxidant, the recently described antimycin-insensitive species of semiquinone anion, Qout (De Vries, S., Albracht, S.P.J., Berden, J.A. and Slater, E.C. (1982) J. Biol. Chem. 256, 11996-11998) is formed at the same rate as that of the reduction of all species of cytochrome b. In this case cytochrome b is reduced in a single phase. (5) The reversible change of the line shape of the EPR spectrum of the [2Fe-2S] cluster 1 is caused by ubiquinone bound in the vicinity of this cluster. (6) The experimental results are consistent with the basic principles of the Q cycle. Because of the multiplicity, stoicheiometry and heterogeneous kinetics of the prosthetic groups, a Q cycle model describing the pathway of electrons through a dimeric QH2:cytochrome c oxidoreductase is proposed.     

Pre-steady-state reduction kinetics of QH2:cytochrome c oxidoreductase and the Q-pool: evidence for a special quinone not in rapid equilibrium with the Q-pool

The pre-steady-state kinetics of the reduction of the prosthetic groups of QH2:cytochrome c oxidoreductase in bovine heart submitochondrial particles were studied in relation to the kinetics of the Q-10 reduction, using duroquinol as substrate. The prosthetic groups, including semiquinone, were measured with EPR and low-temperature-diffuse reflectance spectroscopy, the samples being prepared with the rapid-freeze quench technique. For the determination of the redox state of ubiquinone in the pre-steady state the rapid chemical quench technique was used as an extension of the rapid-freeze quench technique, and Q-10 and QH2-10 were measured with reversed-phase HPLC after extraction with petroleum ether. Ubiquinone was reduced biphasically, 8% of total Q-10 (equal to 1 mol Q-10/mol cytochrome c1), being reduced within 5 ms, and the rest, the Q-pool, at a much lower rate. The initial rapid reduction of this special Q-10 was accompanied by rapid formation of Qi and rapid reduction of a large part of the cytochrome b-562. Both semiquinone formation and reduction of b-562 showed transient kinetics due to a contribution of the reaction pathway via centre o when the iron-sulphur cluster and cytochrome c1 were oxidised. The majority of the special quinol was located at centre i, probably bound, but also at centre o some bound quinol was formed. This was visible when antimycin was present, the antimycin-insensitive bound quinol being totally sensitive to myxothiazol. Myxothiazol alone accelerated the reduction of the Q-pool via centre i, but also the equilibration of cytochrome b-562 with the Q-pool. Antimycin drastically lowered the rate of reduction of the Q-pool and additionally seemed to block the rapid electron transfer from part of the Rieske iron-sulphur cluster to cytochrome c1. It is concluded that, during the pre-steady-state, cytochrome b-562 is not in equilibrium with the Q-pool and that the rate of equilibration is probably determined by the rate of dissociation of the special bound quinol from centre i.     

Spectral analysis of the bc(1) complex components in situ: beyond the traditional difference approach

The cytochrome (cyt) bc(1) complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electron-transport chains. It is rich in prosthetic groups, many of which have significant but overlapping absorption bands in the visible spectrum. The kinetics of the cytochrome components of the bc(1) complex are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. This difference-wavelength (DW) approach has been used extensively in the development and testing of the Q-cycle mechanism of the bc(1) complex in Rhodobacter sphaeroides chromatophores. However, the DW approach does not fully compensate for spectral interference from other components, which can significantly distort both amplitudes and kinetics. Mechanistic elaboration of cyt bc(1) turnover requires an approach that overcomes this limitation. Here, we compare the traditional DW approach to a least squares (LS) analysis of electron transport, based on newly determined difference spectra of all individual components of cyclic electron transport in chromatophores. Multiple sets of kinetic traces, measured at different wavelengths in the absence and presence of specific inhibitors, were analyzed by both LS and DW approaches. Comparison of the two methods showed that the DW approach did not adequately correct for the spectral overlap among the components, and was generally unreliable when amplitude changes for a component of interest were small. In particular, it was unable to correct for extraneous contributions to the amplitudes and kinetics of cyt b(L). From LS analysis of the chromophoric components (RC, c(tot), b(H) and b(L)), we show that while the Q-cycle model remains firmly grounded, quantitative reevaluation of rates, amplitudes, delays, etc., of individual components is necessary. We conclude that further exploration of mechanisms of the bc(1) complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH2: cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands. 2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c1, band 5 and Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c1 and identified with the so-called oxidation factor, and band 7 a polypeptide peptide associated with cytochrome b. 3. The validity of molecular weight estimate for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively. 4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide band.     

The kinetics of reduction of yeast complex III by a substrate analog

The kinetics of reduction of the cytochrome and quinone constituents of yeast complex III by the substrate homolog Q1H2 have been measured under a variety of conditions. The maximum rates of reduction of cytochromes b and c1 and of the endogenous Q6 by Q1H2 were sufficiently fast to support the Vmax for the reduction of cytochrome c by this substrate. The absorbance at 562 nm showed an initial increase which was subsequently followed by a decrease. This decrease was synchronous with the appearance of reduced cytochrome c1 and is interpreted as reflecting the absorbance contribution of c1 at 562 nm under conditions where the steady state level of the b cytochromes is constant. Prereduction of c1 and the Fe/S cluster did not affect the initial very rapid reduction of b, but the second phase was eliminated. Antimycin abolished the very rapid rate of reduction of cytochrome b in untreated complex III and completely inhibited the reduction of cytochrome b in complex III in which c1 and the Fe/S cluster had been prereduced. However, the reduction of the endogenous quinone was essentially unaffected by these treatments. Antimycin had no effect on the reduction of c1. Funiculosin also suppressed the very rapid reduction of b while both myxothiazol and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole did not modify this phase of the reaction; no secondary decrease in absorbance was observed in the presence of any of these inhibitors. Most of the observed kinetic changes could be reproduced by simulation of the Q-cycle; simple linear and branched schemes were unable to reproduce the data.     

Rapid redox equilibrium between the mitochondrial Q pool and cytochrome b during triphasic reduction of cytochrome b by succinate

The reliability of monitoring the redox reactions of cytochrome b using the different wavelengths employed by different authors has been reexamined. It was found that 562-575 nm is suitable in succinate: cytochrome c reductase but not in mitochondria, in which case 562-540 nm is a better pair. Direct optical measurements of the redox reaction kinetics of the mitochondrial Q pool using a commercial dual-wavelength spectrophotometer are possible when succinate is used as the electron donor. Using the correct wavelength pair, and with malonate to slow down the electron input, the reduction course of cytochrome b was still triphasic but a plateau or a turn replaced the oxidation phase previously reported by several authors. At the same time, the reduction course of the Q pool was also triphasic, and in perfect match with that of cytochrome b. Destruction of the Rieske iron-sulfur cluster by British anti-Lewisite (BAL) + O2 treatment or prereduction of the high-potential components made the reduction of both Q and b monophasic. The plot of log (Q/QH2) against log (b3+/b2+) gave a straight line with an n value of 1.7 for cytochrome b at pH 7.4. This n value rose to 2.0 at pH 6.5 and dropped to 1.4 at pH 8.5. On the other hand, the mid-point potential of cytochrome b relative to that of the Q pool remained essentially unchanged between pH 6.5 and 8.4. BAL treatment had a small effect on the midpoint potential of cytochrome b relative to that of the Q pool and had no effect on the n value. Addition of quinone homologues and analogues extended the plateau phase in the reduction of cytochrome b, but exogenous quinones did not equilibrate rapidly with cytochrome b. It was concluded that the appearance of the plateau between the two reduction phases of Q and b is caused by the rapid delivery of electrons to the high-potential components of the respiratory chain as envisaged in the Q cycle; the unexpected n value for cytochrome b suggests a concerted reduction by QH2 of two species of cytochromes b-562.     

Assembly of the mitochondrial membrane system. Analysis of structural mutants of the yeast coenzyme QH2-cytochrome c reductase complex

Coenzyme QH2-cytochrome c reductase is a multisubunit complex of the mitochondrial respiratory chain. Mutants of Saccharomyces cerevisiae with lesions in cytochromes b, c1, the non-heme iron protein, and the noncatalytic subunits have been used to study several aspects of the assembly of the complex. Strains with mutations in single subunits exhibit a variety of different phenotypes. Mutants in the 17-kDa (core 3) subunit grow normally on a nonfermentable substrate indicating that this component is not essential for either enzymatic activity or assembly of the enzyme. Mutations in all the other subunits express a respiratory-deficient phenotype and the absence of detectable enzyme activity. Among the respiratory-defective strains, some have mature cytochrome b (non-heme iron protein and cytochrome c1 mutants), while other mutants lack spectrally detectable cytochrome b and have reduced levels of the apoprotein (mutants in the 44-, 40-, 14-, and 11-kDa core subunits). Mutations in single subunits exert different effects on the concentrations of their partner proteins. These may be summarized as follows: 1) No substantial loss in the 44- or 40-kDa core subunits is seen in single mutants; 2) the concentration of cytochrome c1 is also relatively unaffected by mutations in the other subunits except for the cytochrome b mutant which has 60% of the wild type level of cytochrome c1; 3) all the single mutants have only 15-20% of the normal amount of non-heme iron protein; 4) mutations in the non-heme iron protein have no appreciable effect on the concentrations of the other subunits; 5) mutations in single subunits cause parallel decreases in the concentrations of cytochrome b, the 14-, and the 11-kDa subunits. These results indicate that the synthesis or stability of a subset of subunits depends on the presence of other subunit polypeptides of the complex. At present we favor the idea that the observed changes in the concentrations of some subunits are due to higher turnover rates of the proteins in a partially assembled complex. Based on the mutant phenotypes, a tentative model for the assembly of coenzyme QH2-cytochrome c reductase is proposed. According to this model it is envisioned that the subunits interact with one another in the lipid bilayer. Maturation of apocytochrome b occurs after it is assembled with the nonstructural subunits to form a core structure. This intermediate complex interacts with the non-heme iron protein to form the active holoenzyme.     

Spectral and kinetic resolution of the bc1 complex components in situ: a simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc(1) complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The "traditional" set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c(tot) (cyt c(1)+cyt c(2)), cyt b(L), cyt b(H), and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c(1) and c(2) is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c(1), c(2), b(L), and b(H)) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc(1) complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc(1) complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.     

Localization in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme QH2-cytochrome c reductase.

1. Three methods are described for the genetic analysis of yeast cytoplasmic mutants (mit- mutants) lacking cytochrome oxidase or coenzyme QH2-cytochrome c reductase. The procedures permit mutations in mitochondrial DNA to be mapped relative to each other and with respect to drug-resistant markers. The first method is based upon the finding that crosses of mit- mutants with some but not other isonuclear q- mutants lead to the restoration of respiratory functions. Thus a segment of mitochondrial DNA corresponding to a given mit- mutation or to a set of mutations can be delineated. The second method is based on the appearance of wild-type progeny in mit- X mit- crosses. The third one is based on the analysis of various recombinant classes issued from crosses between mit-, drug-sensitive and mit+, drug-resistant mutants. Representative genetic markers of the RIBI, OLII, OLI2 and PAR1 loci were used for this purpose. 2. The three methods when applied to the study of 48 mit- mutants gave coherent results. At least three distinct regions on mitochondrial DNA in which mutations cause loss of functional cytochrome oxidase have been established. A fourth region represented by closely clustered mutants lacking coenzyme QH2-cytochrome c reductase and spectrally detectable cytochrome b has also been studied. 3. The three genetic regions of cytochrome oxidase and the cytochrome b region were localized by the third method on the circular map, in spans of mitochondrial DNA defined by the drug-resistant markers. The results obtained by this method were confirmed by analysis of the crosses between selected mit- mutants and a large number of q- clones whose retained segments of mitochondrial DNA contained various combinations of drug-resistant markers. 4. All the genetic data indicate that the various regions studied are dispersed on the mitochondrial genome and in some instances regions or clusters of closely linked mutations involved in the same respiratory function (cytochrome oxidase) are separated by other regions which code for entirely different functions such as ribosomal RNA.     

Dynamic control on the rate of the reduction of the b type cytochromes in submitochondrial particles.

1. In the presence of antimycin and KCN the reduction of cytochrome b in phosphorylating submitochondrial particles followed a biphasic first-order kinetics. The transition from the first, rapid phase to the second, slow phase occurred while the reduction of chtochromes c + c1 and a through or around the antimycin block was still linear with time. Thus, the phase transition was due to a fall-off in the rate of cytochrome b reduction. 2. The biphasic reduction of cytochrome b was observed over a wide temperature range (0--30 degrees C), with succinate of NADH as electron donors and with phosphorylating particles or coupled rat-heart mitochondria. With rat-heart mitochondria the same biphasic reduction was observed in the presence of either carbonyl cyanide p-trifluoromethoxyphenylhydrazone or oligomycin. 3. In both the rapid and the slow phases, the rate of reduction of cytochrome b-561 was equal to that of b-565. Thus both cytochromes b-561 and b-565 were affected by the mechanism which determined the reduction-rate. Furthermore, each of these cytochromes could be reduced individually with rate constants typical of the slow phase. 4. The proportion of rapidly reduced to slowly reduced cytochrome b was independent of the degree of its reducibility and could be controlled by teh experimental conditions. When antimycin was used as the only inhibitor, 96% of the b-type cytochromes were reduced in the rapid phase. If the c and a-type cytochromes were first reduced by ascorbate and tetramethyl-p-phenylenediamine in the presence of KCN and antimycin, all the b-type cytochromes were fully reduced at the slow-rate. 5. With succinate, the rate of the rapid phase depended on the activation level of the succinic-dehydrogenase. The rate constant of the second phase was unaffected by the succinic dehydrogenase activity, if the preparation was more than 20% active. Furthermore, the rate constant of the slow reduction was the same with succinate, NADH, or even with durohydroquinone (which reacted directly with cytochromes b). 6. It is suggested that cytochrome b can exist in two forms: kinetically active or sluggish. The active form is rapidly reduced by the endogenous quinone (QH2) or durohydroquinone. The rate of the reduction of the active form by succinate or NADH is probably determined by the rate of the reduction of Q by the dehydrogenases. The second form of cytochrome b is characterized by its sluggish reduction by QH2 or durohydroquinone. 7. It is proposed that the transformation from the active to the sluggish form is induced by the reduction of a controlling group, named Y, located on the oxygen side of the antimycin inhibition site. When Y is oxidized, cytochrome b is in its active form, and when Y is reduced, cytochrome b is in its sluggish form. The nature of this kinetic control and a comparison with the mechanism controlling the reducibility of cytochrome b are discussed.     

An Engineered Cytochrome b6c1 Complex with a Split Cytochrome b Is Able To Support Photosynthetic Growth of Rhodobacter capsulatus

The ubihydroquinone-cytochrome c oxidoreductase (or the cytochrome bc1 complex) from Rhodobacter capsulatus is composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits encoded by petA(fbcF), petB(fbcB), and petC(fbcC) genes organized as an operon. In the work reported here, petB(fbcB) was split genetically into two cistrons, petB6 and petBIV, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome b, respectively. These polypeptides resembled the cytochrome b6 and su IV subunits of chloroplast cytochrome b6f complexes, and together with the unmodified subunits of the cytochrome bc1 complex, they formed a novel enzyme, named cytochrome b6c1 complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of R. capsulatus. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of petBIV, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome b6c1 complex were similar to those of the cytochrome bc1 complex. In particular, it was highly sensitive to inhibitors of the cytochrome bc1 complex, including antimycin A, and the redox properties of its b- and c-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome bL heme was modified in a way reminiscent of that of a cytochrome b6f complex. Based on the work described here and that with Rhodobacter sphaeroides (R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280-16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome bc1 complexes and the chloroplast cytochrome b6f complexes are direct consequences of splitting cytochrome b into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome bc oxidoreductases.     

Isolation and RNA sequence analysis of cytochrome b mutants resistant to funiculosin, a center i inhibitor of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae

Funiculosin is a well-known inhibitor of the mitochondrial respiratory chain, probably acting at the ubiquinone reducing site or center i of QH2-cytochrome c reductase. We report here the isolation, mapping and RNA sequence analysis of yeast apo-cytochrome b mutants resistant to this inhibitor. Funiculosin-resistance was found to be conferred, in 4 independent isolates, upon replacement of a leucine residue by phenylalanine in position 198 of the cytochrome b polypeptide chain.     

Reduction of cytochrome b-561 through the antimycin-sensitive site of the ubiquinol-cytochrome c2 oxidoreductase complex of Rhodopseudomonas sphaeroides

Cytochrome b-561 of the ubiquinol-cytochrome c2 oxidoreductase complex of Rhodopseudomonas sphaeroides is reduced after flash illumination in the presence of myxothiazol in an antimycin-sensitive reaction. Flash-induced reduction was observed over the redox range in which cytochrome b-561 and the Q-pool are both oxidized before the flash. The extent of reduction increased with increasing pH, and was maximal at pH greater than 10.0 where the extent approached that observed in the presence of antimycin following a group of flashes. Reduction of cytochrome b-561 in the presence of myxothiazol showed a lag of approximately 1 ms after the flash, followed by reduction with t 1/2 approximately 6 ms; by analogy with the similar kinetics of the quinol oxidase site, we suggest that the rate is determined by collision with the QH2 produced in the pool on flash excitation.