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 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.     

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 oxidation-reduction kinetics of cytochromes b, c1 and c in initially fully reduced mitochondrial membranes are in agreement with the Q-cycle hypothesis

Stopped-flow experiments were performed to distinguish between two hypotheses, the Q-cycle and the SQ-cycle, each describing the pathway of electron transfer in the QH2:cytochrome c oxidoreductases. It was observed that, when mitochondrial membranes from the yeast Saccharomyces cerevisiae were poised at a low redox potential with appropriate amounts of sodium dithionite to completely reduce cytochrome b, the kinetics of oxidation of cytochrome b showed a lag period of maximally 100 ms. Under the same experimental conditions, the oxidation-reduction kinetics of cytochromes c + c1 showed transient behaviour. These results do not support the presence of a mobile species of semiquinone in the QH2:cytochrome c oxidoreductases, as envisaged in the SQ-cycle, but are consistent with a Q-cycle mechanism in which the two quinone-binding domains do not exchange electrons directly on the timescale of turnover of the enzyme.     

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.     

Determination of the position of the Qi.- quinone binding site from the protein surface of the cytochrome bc1 complex in Rhodobacter capsulates chromatophores.

The technique of distance measurement, utilizing spin relaxation enhancement by an external probe, has been extended to the study of intrinsic semiquinone radicals through the use of holmium-EDTA complexes and continuous wave electron paramagnetic resonance spectroscopy. This technique has been used to determine the distance of the semiquinone anion, Qi (also designated as Qn.- or Qc.-), from the surface of the ubiquinone cytochrome c oxidoreductase, consisting of only three subunits, in membrane particles from Rhodobacter capsulates. The location of the semiquinone anion is 6-10 A from the N side protein, establishing that there are two separate quinone reaction sites, i.e., 'Qi' and 'Qo', within this complex on opposite sides of the membrane. The results are discussed in relation to reported ENDOR, EPR, and optical studies of the mitochondrial counterpart.     

The effect of ring substituents on the mechanism of interaction of exogenous quinones with the mitochondrial respiratory chain

In uncoupled pig-heart mitochondria the rate of the reduction of duroquinone by succinate in the presence of cyanide is inhibited by about 50% by antimycin. This inhibition approaches completion when myxothiazol is also added or British anti-Lewisite-treated (BAL-treated) mitochondria are used. If mitochondria are replaced by isolated succinate:cytochrome c oxidoreductase, the inhibition by antimycin alone is complete. The reduction of a plastoquinone homologue with an isoprenoid side chain (plastoquinone-2) is strongly inhibited by antimycin with either mitochondria or succinate:cytochrome c reductase. The reduction by succinate of plastoquinone analogues with an n-alkyl side chain in the presence of mitochondria is inhibited neither by antimycin nor by myxothiazol, but is sensitive to the combined use of these two inhibitors. On the other hand, the reduction of the ubiquinone homologues Q2, Q4, Q6 and Q10 and an analogue, 2,3-dimethoxyl-5-n-decyl-6-methyl-1,4-benzoquinone, is not sensitive to any inhibitor of QH2:cytochrome c reductase tested or their combined use, either in normal or BAL-treated mitochondria or in isolated succinate:cytochrome c reductase. It is concluded that quinones with a ubiquinone ring can be reduced directly by succinate:Q reductase, whereas those with a plastoquinone ring can not. Reduction of the latter compounds requires participation of either center i or center o (Mitchell, P. (1975) FEBS Lett. 56, 1-6) or both, of QH2:cytochrome c oxidoreductase. It is proposed that a saturated side chain promotes, while an isoprenoid side chain prevents reduction of these compounds at center o.     

Photosynthetic membrane development in Rhodopseudomonas sphaeroides. Spectral and kinetic characterization of redox components of light-driven electron flow in apparent photosynthetic membrane growth initiation sites

The kinetics of light-driven electron flow and the nature of redox centers at apparent photosynthetic membrane growth initiation sites in Rhodopseudomans sphaeroides were compared to those of intracytoplasmic photosynthetic membranes. In sucrose gradients, these membrane growth sites sediment more slowly than intracytoplasmic membrane-derived chromatophores and form an upper pigmented band. Cytochromes c1, c2, b561, and b566 were demonstrated in the upper fraction by redox potentiometry; c-type cytochromes were also detected electrophoretically. Signals characteristic of light-induced reaction center bacteriochlorophyll triplet and photooxidized reaction center bacteriochlorophyll dimer states were observed by EPR spectroscopy but the Rieske iron-sulfur signal of the ubiquinol-cytochrome c2 oxidoreductase was present at a 3-fold reduced level on a reaction center basis in comparison to chromatophores. Flash-induced absorbance measurements of the upper pigmented fraction demonstrated reaction center primary and secondary semiquinone anion acceptor signals, but cytochrome b561 photoreduction and cytochrome c1/c2 reactions occurred at slow rates. This fraction was enriched approximately 2- and 4-fold in total b- and c-type cytochromes, respectively, per reaction center over chromatophores, but photoreducible b-type cytochrome was lower. Measurements of respiratory activity indicated a 1.6-fold higher level of succinate-cytochrome c oxidoreductase/reaction center than in chromatophores, but the apparent turnover rates in both preparations were low. Overall, the results suggest that complete cycles of rapid, light-driven electron flow do not occur merely by introduction of newly synthesized reaction centers into respiratory membrane, but that subsequent synthesis and assembly of appropriate components of the ubiquinol-cytochrome c2 oxidoreductase is required.     

The kinetics and thermodynamics of the reduction of cytochrome c by substituted p-benzoquinols in solution

1. The mechanisms by which p-benzoquinol and its derivatives reduce cytochrome c in solution have been investigated. 2. The two major reductants are the species QH- (anionic quinol) and Q.- (anionic semiquinone). A minor route of electron transfer from the fully protonated QH2 species can also occur. 3. The relative contributions of these routes to the overall reduction rate are governed by pH, ionic strength and relative reactant concentrations. 4. For a series of substituted p-benzoquinols, the forward rate constant, k1, of the anionic quinol-mediatd reaction is related to the midpoint potential of the QH-/QH. couple involved in the rate-limiting step, as predicted by the theory of Marcus for outer-sphere electron transfer reactions in a bimolecular collision process. 5. A mechanism for the biological quinol oxidation reactions in mitochondria and chloroplasts is proposed based upon the findings with these reactions in solution.     

The thiol:disulfide oxidoreductase DsbB mediates the oxidizing effects of the toxic metalloid tellurite (TeO32-) on the plasma membrane redox system of the facultative phototroph Rhodobacter capsulatus

The highly toxic oxyanion tellurite (TeO3(2-)) is a well known pro-oxidant in mammalian and bacterial cells. This work examines the effects of tellurite on the redox state of the electron transport chain of the facultative phototroph Rhodobacter capsulatus, in relation to the role of the thiol:disulfide oxidoreductase DsbB. Under steady-state respiration, the addition of tellurite (2.5 mM) to membrane fragments generated an extrareduction of the cytochrome pool (c- and b-type hemes); further, in plasma membranes exposed to tellurite (0.25 to 2.5 mM) and subjected to a series of flashes of light, the rate of the QH2:cytochrome c (Cyt c) oxidoreductase activity was enhanced. The effect of tellurite was blocked by the antibiotics antimycin A and/or myxothiazol, specific inhibitors of the QH2:Cyt c oxidoreductase, and, most interestingly, the membrane-associated thiol:disulfide oxidoreductase DsbB was required to mediate the redox unbalance produced by the oxyanion. Indeed, this phenomenon was absent from R. capsulatus MD22, a DsbB-deficient mutant, whereas the tellurite effect was present in membranes from MD22/pDsbB(WT), in which the mutant gene was complemented to regain the wild-type DsbB phenotype. These findings were taken as evidence that the membrane-bound thiol:disulfide oxidoreductase DsbB acts as an "electron conduit" between the hydrophilic metalloid and the lipid-embedded Q pool, so that in habitats contaminated with subinhibitory amounts of Te(IV), the metalloid is likely to function as a disposal for the excess reducing power at the Q-pool level of facultative phototrophic bacteria.     

Pathways of hydrogen peroxide generation in guinea pig cerebral cortex mitochondria

The production of H2O2 by brain mitochondria was monitored employing a new technique based on the horseradish peroxidase dependent oxidation of acetylated ferrocytochrome c. It was shown that brain mitochondria release H2O2 by an intermediate autooxidation at the QH2-cytochrome c oxidoreductase level (induced by antimycin A and inhibited by myxothiazol). With both succinate and pyruvate plus malate this H2O2 release is inhibited at high substrate concentrations. With pyruvate plus malate a second source of H2O2 could be detected, apparently from autoxidation at the NADH dehydrogenase level. With alpha-glycerophosphate some H2O2 derives from autooxidation at the alpha-glycerophosphate dehydrogenase. The NADH dehydrogenase dependent, but not the QH2-cytochrome c oxidoreductase dependent H2O2 was significantly stimulated upon depletion of the mitochondrial glutathione.     

Proton pumping in the bc1 complex: a new gating mechanism that prevents short circuits

The Q-cycle mechanism of the bc1 complex explains how the electron transfer from ubihydroquinone (quinol, QH2) to cytochrome (cyt) c (or c2 in bacteria) is coupled to the pumping of protons across the membrane. The efficiency of proton pumping depends on the effectiveness of the bifurcated reaction at the Q(o)-site of the complex. This directs the two electrons from QH2 down two different pathways, one to the high potential chain for delivery to an electron acceptor, and the other across the membrane through a chain containing heme bL and bH to the Qi-site, to provide the vectorial charge transfer contributing to the proton gradient. In this review, we discuss problems associated with the turnover of the bc1 complex that center around rates calculated for the normal forward and reverse reactions, and for bypass (or short-circuit) reactions. Based on rate constants given by distances between redox centers in known structures, these appeared to preclude conventional electron transfer mechanisms involving an intermediate semiquinone (SQ) in the Q(o)-site reaction. However, previous research has strongly suggested that SQ is the reductant for O2 in generation of superoxide at the Q(o)-site, introducing an apparent paradox. A simple gating mechanism, in which an intermediate SQ mobile in the volume of the Q(o)-site is a necessary component, can readily account for the observed data through a coulombic interaction that prevents SQ anion from close approach to heme bL when the latter is reduced. This allows rapid and reversible QH2 oxidation, but prevents rapid bypass reactions. The mechanism is quite natural, and is well supported by experiments in which the role of a key residue, Glu-295, which facilitates proton transfer from the site through a rotational displacement, has been tested by mutation.     

Fractionation of cytochromes of phototrophically grown Chloroflexus aurantiacus. Is there a cytochrome bc complex among them?

The cytochrome-containing membrane complexes of the phototrophically grown green non-sulfur bacterium Chloroflexus aurantiacus were fractionated by anion exchange chromatography. Three cytochrome b and four cytochrome c peaks were observed. None of the separated complexes met the features of the cytochrome bc complex. Two main cytochrome b-containing complexes were further purified: a dimer of identical subunits with unknown function and a succinate:quinone oxidoreductase containing three subunit species. Two novel multisubunit complexes, similar to each other, with two heme c-bearing subunits were also purified.     

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.     

Characterization of mutants that change the hydrogen bonding of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli

The cytochrome bo3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol in the cytoplasmic membrane of Escherichia coli, and reduces O2 to water. This enzyme has a high affinity quinone binding site (QH), and the quinone bound to this site acts as a cofactor, necessary for rapid electron transfer from substrate ubiquinol, which binds at a separate site (QL), to heme b. Previous pulsed EPR studies have shown that a semiquinone at the QH site formed during the catalytic cycle is a neutral species, with two strong hydrogen bonds to Asp-75 and either Arg-71 or Gln-101. In the current work, pulsed EPR studies have been extended to two mutants at the QH site. The D75E mutation has little influence on the catalytic activity, and the pattern of hydrogen bonding is similar to the wild type. In contrast, the D75H mutant is virtually inactive. Pulsed EPR revealed significant structural changes in this mutant. The hydrogen bond to Arg-71 or Gln-101 that is present in both the wild type and D75E mutant oxidases is missing in the D75H mutant. Instead, the D75H has a single, strong hydrogen bond to a histidine, likely His-75. The D75H mutant stabilizes an anionic form of the semiquinone as a result of the altered hydrogen bond network. Either the redistribution of charge density in the semiquinone species, or the altered hydrogen bonding network is responsible for the loss of catalytic function.     

On the mechanism of the Mn3(+)-induced neurotoxicity of dopamine:prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase

Dopamine (DA) is rapidly oxidized by Mn3(+)-pyrophosphate to its cyclized o-quinone (cDAoQ), a reaction which can be prevented by NADH, reduced glutathione (GSH) or ascorbic acid. The oxidation of DA by Mn3+, which appears to be irreversible, results in a decrease in the level of DA, but not in a formation of reactive oxygen species, since oxygen is neither consumed nor required in this reaction. The formation of cDAoQ can initiate the generation of superoxide radicals (O2-.) by reduction-oxidation cycling, i.e. one-electron reduction of the quinone by various NADH- or NADPH-dependent flavoproteins to the semiquinone (QH.), which is readily reoxidized by O2 with the concomitant formation of O2-.. This mechanism is believed to underly the cytotoxicity of many quinones. Two-electron reduction of cDAoQ to the hydroquinone can be catalyzed by the flavoprotein DT diaphorase (NAD(P)H:quinone oxidoreductase). This enzyme efficiently maintains DA quinone in its fully reduced state, although some reoxidation of the hydroquinone (QH2) is observed (QH2 + O2----QH. + O2-. + H+; QH. + O2----Q + O2-.). In the presence of Mn3+, generated from Mn2+ by O2-. (Mn2+ + 2H+ + O2-.----Mn3+ + H2O2) formed during the autoxidation of DA hydroquinone, the rate of autoxidation is increased dramatically as is the formation of H2O2. Furthermore, cDAoQ is no longer fully reduced and the steady-state ratio between the hydroquinone and the quinone is dependent on the amount of DT diaphorase present. The generation of Mn3+ is inhibited by superoxide dismutase (SOD), which catalyzes the disproportionation of O2-. to H2O2 and O2. It is noteworthy that addition of SOD does not only result in a decrease in the amount of H2O2 formed during the regeneration of Mn3+, but, in fact, prevents H2O2 formation. Furthermore, in the presence of this enzyme the consumption of O2 is low, as is the oxidation of NADH, due to autoxidation of the hydroquinone, and the cyclized DA o-quinone is found to be fully reduced. These observations can be explained by the newly-discovered role of SOD as a superoxide:semiquinone (QH.) oxidoreductase catalyzing the following reaction: O2-. + QH. + 2H+----QH2 + O2. Thus, the combination of DT diaphorase and SOD is an efficient system for maintaining cDAoQ in its fully reduced state, a prerequisite for detoxication of the quinone by conjugation with sulfate or glucuronic acid. In addition, only minute amounts of reactive oxygen species will be formed, i.e. by the generation of O2-., which through disproportionation to H2O2 and further reduction by ferrous ions can be converted to the hydroxyl radical (OH.). Absence or low levels of these enzymes may create an oxidative stress on the cell and thereby initiate events leading to cell death.     

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.     

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase

The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr = 4 x 39000) containing FMN as expected and not heme. It has high L-lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same Km for L(+)-lactate as flavocytochrome b2, but it has no L-lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b2. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated.     

Functional characterization of the mitochondrial cytochrome b-c1 complex: steady-state kinetics of the monomeric and dimeric forms

The QH2:cytochrome c oxidoreductase activity of the isolated bovine heart cytochrome b-c1 complex resolved into monomeric and dimeric form was titrated with three different inhibitors of electron transfer, antimycin, myxothiazol, and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). In all cases one inhibitor molecule per cytochrome c1 was found necessary to block completely the activity of both molecular forms of the enzyme. The antimycin-sensitive cytochrome c reduction catalyzed by the b-c1 complex was also studied as a function of increasing concentrations of either cytochrome c or quinol. Double-reciprocal plots of the activity of the monomeric enzyme were found linear either when the concentration of cytochrome c or of quinol derivatives, 2,3-dimetoxy-5-methyl-6-decyl-1,4-benzoquinol (DBH), and 2-methyl-3-undecyl-1,4-naphthoquinol (UNH), was changed. Cytochrome c reductase activity of the dimeric b-c1 complex also showed a linear Lineweaver-Burk plot as a function of cytochrome c concentrations. In contrast to the monomeric enzyme, however, dimers of the b-c1 complex express a clear nonlinear kinetic behavior toward quinol derivatives, with two apparent Km values differing approximately by one order of magnitude (about 3-4 and about 20-30 microM). At saturating quinol concentrations the activity of the dimeric enzyme becomes two to three times higher than that of monomers. The nonlinear kinetic plots were found to be the same at different temperatures and different cytochrome c concentrations. The data suggest that although the monomer of the b-c1 complex appears to be the functional unit of the enzyme, the dimer is more active. A regulatory role of the dimerization process resulting in an increase of the electrons flux through the enzyme is postulated.     

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.