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 QH2:cytochrome c oxidoreductase studied by pre-steady-state kinetics

(1) The kinetic behaviour of the prosthetic groups and the semiquinones 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 Q^?_in, 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-2 S] 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 Q^?_in 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, Q^?_out (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 pathway of electron transfer in the dimeric QH2: Cytochromec oxidoreductase

The experimental data currently available suggest that QH₂: cytochromec oxidoreductase functions according to a Q-cycle type of mechanism. The molecular weight of the enzyme in a natural or artificial phospholipid bilayer or in solution corresponds to that of a dimer. The pre-steady state kinetics of reduction of the prosthetic groups indicate that the enzyme is functionally dimeric. A double Q cycle is proposed, describing the pathway of electron transfer in the dimeric QH₂: cytochromec oxidoreductase. According to this scheme, the two monomeric halves of the enzyme act in a cooperative fashion to complete the catalytic cycle. It is proposed that high-potential cytochromeb-562 and low-potential cytochromeb-562 act cooperatively, viz. as a functional pair, in the antimycin-sensitive reduction of ubiquinone to ubiquinol.     

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 site of inhibition by 5,5′-dithiobis(2-nitrobenzoate) in ubiquinol:Cytochrome c oxidoreductase

In 5,5′-dithiobis(2-nitrobenzoate) (DTNB)-treated succinate:cytochrome c reductase, the electron transfer from duroquinol to cytochrome c is inhibited due to the fact that the Rieske Fe-S cluster and, consequently, cytochrome c, are no longer reducible by substrate. The finding that, after this treatment, cytochrome b is still reducible by substrate in the absence of antimycin, but not in its presence, is consistent with a Q-cycle mechanism for the electron transfer through QH₂:cytochrome c oxidoreductase. The inhibitory effect of DTNB and its effect on the EPR spectrum of the [2Fe-2S] cluster suggest that it prevents either the binding of ubiquinone in the vicinity of this cluster or the interaction between the Fe-S protein and a ubiquinone-binding protein.     

Characterization of ubisemiquinone radical in the cytochrome b-c1 segment of the mitochondrial respiratory chain

The ubiquinone protein, QP-C, in reduced ubiquinone-cytochrome c reductase (the b?c₁-III complex) shows a stable ubisemiquinone radical when the enzyme is reduced by succinate in the presence of catalytic amounts of succinate dehydrogenase and QP-S. At room temperature using EPR technique the redox titration of the b?c₁-III complex in the presence of redox dyes or succinate/fumarate couple reveals that the ubisemiquinone radical has a midpoint potential of approximately +67 mV at pH 8.0. Further analysis yields E₁ of +83 mV and E₂ of +51 mV corresponding to ($\text{QH}{}_2\text{QH·}$) and ($\text{QH·}\text{Q}$) or other electronated forms, respectively. The equilibrium radical concentration has been found to be affected both by pH and succinate/fumarate couple. At pH 9.0 the radical shows the maximal amplitude and stability. Below pH 7.0, little radical was detected. The electron spin relaxation behavior of ubisemiquinone radical, as examined by microwave power saturation, indicates that the ubisemiquinone radical of QP-C is somewhat isolated from other paramagnetic centers. The effects of phospholipids, QP-S, and other agents on ubisemiquinone radical formation as well as the enzymatic activity of QP-C have been studied in detail.     

On the role of ubiquinone in the respiratory chain

(1) The V₁ (substrate-Q oxidoreductase activity) and V₂ (QH₂ oxidase activity) for the oxidation of substrates by submitochondrial particles have been measured by using heptylhydroxyquinoline N-oxide (HQNO) as inhibitor of V₂. (2) Partial destruction of the Rieske Fe-S cluster by treatment with BAL (2,3-dimercaptopropanol)+O₂ has the same effect on the QH₂ oxidase activity as partial saturation of the antimycin-binding site with HQNO. (3) The extent of the rapid reduction of cytochrome b in the presence of excess antimycin is proportional to the percentage of intact Rieske Fe-S cluster. (4) The measured rate of oxidation of endogenous ubiquinol (V₂) by submitochondrial particles is dependent on the substrate used to reduce ubiquinone, especially at low levels of ubiquinone. (5) Pool-function kinetics in the oxidation of substrate, found both in the presence and absence of free ubiquinone, are due both to the pool of free ubiquinone and to direct collision between Q-loaded Q-reducing and -oxidizing enzymes. At infinite Q content only the former mechanism is operative; at low Q content only the latter. (6) Duroquinone can be reduced directly by NADH dehydrogenase without mediation of ubiquinone, but duroquinol cannot be oxidized in the absence of ubiquinone. On the other hand, the reduction of cytochrome b by duroquinol does not require the presence of ubiquinone. (7) It is suggested that the need for ubiquinone for the oxidation of duroquinol is due to the requirement of ubisemiquinone for the oxidation of cytochrome b, duroquinol not being able to form a stabilized semiquinone.     

Functional flexibility of electron flow between quinol oxidation Qo site of cytochrome bc1 and cytochrome c revealed by combinatory effects of mutations in cytochrome b,iron-sulfur protein and cytochrome c1

Transfer of electron from quinol to cytochrome c is an integral part of catalytic cycle of cytochrome bc₁. It is a multi-step reaction involving: i) electron transfer from quinol bound at the catalytic Q_o site to the Rieske iron-sulfur ([2Fe-2S]) cluster, ii) large-scale movement of a domain containing [2Fe-2S] cluster (ISP-HD) towards cytochrome c₁, iii) reduction of cytochrome c₁ by reduced [2Fe-2S] cluster, iv) reduction of cytochrome c by cytochrome c₁.In this work, to examine this multi-step reaction we introduced various types of barriers for electron transfer within the chain of [2Fe-2S] cluster, cytochrome c₁ and cytochrome c. The barriers included: impediment in the motion of ISP-HD, uphill electron transfer from [2Fe-2S] cluster to heme c₁ of cytochrome c₁, and impediment in the catalytic quinol oxidation. The barriers were introduced separately or in various combinations and their effects on enzymatic activity of cytochrome bc₁ were compared. This analysis revealed significant degree of functional flexibility allowing the cofactor chains to accommodate certain structural and/or redox potential changes without losing overall electron and proton transfers capabilities. In some cases inhibitory effects compensated one another to improve/restore the function. The results support an equilibrium model in which a random oscillation of ISP-HD between the Q_o site and cytochrome c₁ helps maintaining redox equilibrium between all cofactors of the chain. We propose a new concept in which independence of the dynamics of the Q_o site substrate and the motion of ISP-HD is one of the elements supporting this equilibrium and also is a potential factor limiting the overall catalytic rate.     

The Qrc Membrane Complex,Related to the Alternative Complex III,Is a Menaquinone Reductase Involved in Sulfate Respiration

Biological sulfate reduction is a process with high environmental significance due to its major contribution to the carbon and sulfur cycles in anaerobic environments. However, the respiratory chain of sulfate-reducing bacteria is still poorly understood. Here we describe a new respiratory complex that was isolated as a major protein present in the membranes of Desulfovibrio vulgaris Hildenborough. The complex, which was named Qrc, is the first representative of a new family of redox complexes. It has three subunits related to the complex iron-sulfur molybdoenzyme family and a multiheme cytochrome c and binds six hemes c, one [3Fe-4S]^+1/0 cluster, and several interacting [4Fe-4S]^2+/1+ clusters but no molybdenum. Qrc is related to the alternative complex III, and we show that it has the reverse catalytic activity, acting as a Type I cytochrome c₃:menaquinone oxidoreductase. The qrc genes are found in the genomes of deltaproteobacterial sulfate reducers, which have periplasmic hydrogenases and formate dehydrogenases that lack a membrane subunit for reduction of the quinone pool. In these organisms, Qrc acts as a menaquinone reductase with electrons from periplasmic hydrogen or formate oxidation. Binding of a menaquinone analogue affects the EPR spectrum of the [3Fe-4S]^+1/0 cluster, indicating the presence of a quinone-binding site close to the periplasmic subunits. Qrc is the first respiratory complex from sulfate reducers to have its physiological function clearly elucidated.     

Kinetics of cytochrome b reduction in submitochondrial particles

(1) In agreement with Eisenbach and Gutman (Eisenbach, M. and Gutman, M. (1975) Eur. J. Biochem. 52, 107–116) the reduction of cytochrome b in beef-heart submitochondrial particles by succinate in the presence of antimycin was found to be biphasic, the relative amounts of fast and slow phases being dependent on the redox state of a component located on the oxygen side of the antimycin block. (2) HQNO in a concentration sufficiently large to saturate the specific antimycin- and HQNO-binding sites can substitute for antimycin in these experiments. (3) The rate of the slow phase of the reduction of cytochrome b is decreased under anaerobic conditions and after pretreatment with 2,3-dimercaptopropanol (BAL). (4) In the presence of antimycin and cyanide, cytochrome b-562 is, to some extent, preferentially reduced in the rapid phase and b-566 in the slow phase. (5) The previously proposed regulatory effects of redox-sensitive components X and Y on the redox level and reduction kinetics, respectively, of cytochrome b are ascribed to the role of the Fe-S protein, when it is oxidized, in producing the reductant of cytochrome b by oxidation of QH₂, and by the fact that when QH₂ is bound to it, the reduced Fe-S protein cannot be oxidized by its natural oxidant, cytochrome c₁.     

Dissecting the pattern of proton release from partial process involved in ubihydroquinone oxidation in the Q-cycle

A key feature of the modified Q-cycle of the cytochrome bc₁ and related complexes is a bifurcation of QH₂ oxidation involving electron transfer to two different acceptor chains, each coupled to proton release. We have studied the kinetics of proton release in chromatophore vesicles from Rhodobacter sphaeroides, using the pH-sensitive dye neutral red to follow pH changes inside on activation of the photosynthetic chain, focusing on the bifurcated reaction, in which 4H⁺are released on complete turnover of the Q-cycle (2H⁺/ubiquinol (QH₂) oxidized). We identified different partial processes of the Q_o-site reaction, isolated through use of specific inhibitors, and correlated proton release with electron transfer processes by spectrophotometric measurement of cytochromes or electrochromic response. In the presence of myxothiazol or azoxystrobin, the proton release observed reflected oxidation of the Rieske iron?sulfur protein. In the absence of Q_o-site inhibitors, the pH change measured represented the convolution of this proton release with release of protons on turnover of the Q_o-site, involving formation of the ES-complex and oxidation of the semiquinone intermediate. Turnover also regenerated the reduced iron-sulfur protein, available for further oxidation on a second turnover. Proton release was well-matched with the rate limiting step on oxidation of QH₂ on both turnovers. However, a minor lag in proton release found at pH?7 but not at pH?8 might suggest that a process linked to rapid proton release on oxidation of the intermediate semiquinone involves a group with a pK in that range.     

Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase

The inhibition by superoxide dismutase of cytochrome c reduction by a range of semiquinone radicals has been studied. The semiquinones were produced from the parent quinones by reduction with xanthine and xanthine oxidase. Most of the quinones studied were favored over O₂ as the enzyme substrate, and in air as well as N₂, semiquinone radicals rather than superoxide were produced and they caused the cytochrome c reduction. With all but one of the quinones (benzoquinone), cytochrome c reduction in air was inhibited by superoxide dismutase, but the amount of enzyme required for inhibition was up to 100 times greater than that required to inhibit reduction by superoxide. It was highest for the quinones with the highest redox potential. These results demonstrate how superoxide dismutase can inhibit cytochrome c reduction by species other than superoxide. They can be explained by the dismutase displacing the equilibrium: semiquinone + O₂ ? quinone + O₂^? to the right, thereby allowing the forward reaction to out-compete other reactions of the semiquinone. The implication from these findings that superoxide dismutase-inhibitable reduction of cytochrome c may not be a specific test for superoxide production is discussed.     

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.     

The role of the quinone pool in the cyclic electron-transfer chain of Rhodopseudomonas sphaeroides A modified Q-cycle mechanism

(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c₂ oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c₁ and c₂, the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a low-potential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0–3 mol ubiquinol/mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c₁ and c₂, and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycin-sensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 ix oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 became completely reduced following one flash in the presence of antimycin. The results are interpreted as showing that at potentials higher than 180 mV, ubiquinol stoichiometric with cytochrome b-561 reaches the complex from the reaction center. The increased extent of the carotenoid change, when one extra ubiquinol is available in the pool, is interpreted as showing that the ubiquinol oxidase site turns over twice, and the ubiquinone reductase sites turns over once, for a complete turnover of the ubiquinol:cytochrome c₂ oxidoreductase complex, and the net oxidation of one ubiquinol/complex. (7) The antimycin-sensitive reduction of cytochrome c₁ and c₂ is shown to reflect the second turnover of the ubiquinol oxidase site. (8) We suggest that, in the presence of antimycin, the ubiquinol oxidase site reaches a quasi equilibrium with ubiquinol from the pool and the high- and low-potential chains, and that the equilibrium constant of the reaction catalysed constrains the site to the single turnover under most conditions. (9) The results are discussed in the context of a detailed mechanism. The modified Q-cycle proposed is described by physicochemical parameters which account well for the results reported.     

Evidence of ubisemiquinone radicals in electron transfer at the cytochromes b and c1 region of the cardiac respiratory chain

A highly purified reduced ubiquinone-cytochrome c reductase preparation (the b-c₁III complex) has been made. The b-c₁III complex is not reconstitutively active with succinate dehydrogenase. When the complex at about 10 mg/ml is reduced by succinate in the presence of catalytic (nanomolar) amounts of SDH and a ubiquinone protein (required in the succinate dehydrogenase region i.e, OP-S), a ubisemiquinone radical(s) has been detected using EPR measurements. The formation of the radical(s) is concurrent with the reduction of cytochrome b after the complete reduction of cytochrome c₁. All these rates are dependent on the amounts of succinate dehydrogenase and QP-S used. The maximal concentration of the radical formed is independent of the amounts of succinate dehydrogenase and QP-S added but dependent on the amount of succinate present. The formation of the radical and the reduction of b and c₁ by succinate requires the presence of phospholipids. Addition of thenoyltrifluoroacetone not only prevents the formation of the ubisemiquinone but also abolishes the prior formed radical and causes the reoxidation of b. Antimycin A also diminishes the radical intensity but causes only slight reoxidation of prior reduced cytochrome b. Treatment of the b-c₁III complex with α-chymotrypsin results in the diminishing of the radical formation. Consideration of all these results presented collectively indicates the existence of a ubiquinone binding protein in the b-c₁III complex preparation.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes. Evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 × g particulate fraction of the postnuclear supernatant obtained from guinea-pig phagocytosing polymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O^?₂ generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH.2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1 : 1.3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O^?₂ generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation.4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O^?₂ formed during the oxidation of NADPH.5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH.6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH.7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible.8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase.9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O₂; an enzymatic, apparently non-univalent, O₂ reduction and a non-enzymatic chain reaction.10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O₂ is optimal at pH 7.0 with 1 mM NADPH.     

Cytochrome b oxidation and reduction reactions in the ubiquinone-cytochrome b/c2 oxidoreductase from Rhodopseudomonas Sphaeroides

1. The kinetics of cytochrome b reduction and oxidation in the ubiquinone-cytochrome b/c₂ oxidoreductase of chromatophores from Rhodopseudomonas sphaeroides Ga have been measured both in the presence and absence of anti-mycin, after subtraction of contributions due to absorption changes from cytochrome c₂, the oxidized bacteriochlorophyll dimer of the reaction center, and a red shift of the antenna bacteriochlorophyll.2. A small red shift of the antenna bacteriochlorophyll band centered at 589 nm has been identified and found to be kinetically similar to the carotenoid bandshift.3. Antimycin inhibits the oxidation of ferrocytochrome b under all conditions; it also stimulates the amount of single flash activated cytochrome b reduction 3- to 4-fold under certain if not all conditions.4. A maximum of approximately 0.6 cytochrome b-560 (E_m(7) = 50 mV, n = 1, previously cytochrome b₅₀) hemes per reaction center are reduced following activating flashes. This ratio suggests that there is one cytochrome b-560 heme functional per ubiquinone-cytochrome b/c₂ oxidoreductase.5. Under the experimental conditions used here, only cytochrome b-560 is observed functional in cyclic electron transfer.6. We describe the existence of three distinct states of reduction of the ubiquinone-cytochrome b/c₂ oxidoreductase which can be established before activation, and result in markedly different reaction sequences involving cytochrome b after the flash activation. Poising such that the special ubiquinone (Q_z) is reduced and cytochrome b-560 is oxidized yields the conditions for optimal flash activated electron transfer rates through the ubiquinone-cytochrome b/c₂ oxidoreductase. However when the ambient redox state is lowered to reduce cytochrome b-560 or raised to oxidize Q_z, single turnover flash induced electron transfer through the ubiquinone-cytochrome b/c₂ oxidoreductase appears impeded; the points of the impediment are tentatively identified with the electron transfer step from the reduced secondary quinone (Q_II) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Q_z, respectively.     

The cytochrome b lysine 329 residue is critical for ubihydroquinone oxidation and proton release at the Qo site of bacterial cytochrome bc1

The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc₁) is an important enzyme for photosynthesis and respiration. In bacteria like Rhodobacter capsulatus, this membrane complex has three subunits, the iron?sulfur protein (ISP) with its Fe₂S₂ cluster, cyt c₁ and cyt b, forming two catalytic domains, the Q_o (hydroquinone (QH₂) oxidation) and Q_i (quinone (Q) reduction) sites. At the Q_o site, the electron transfer pathways originating from QH₂ oxidation are known, but their associated proton release routes are less well defined. Earlier, we demonstrated that the His291 of cyt b is important for this latter process. In this work, using the bacterial cyt bc₁ and site directed mutagenesis, we show that Lys329 of cyt b is also critical for electron and proton transfer at the Q_o site. Of the mutants examined, Lys329Arg was photosynthesis proficient and had quasi-wild type cyt bc₁ activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc₁. In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH₂ oxidation were drastically impaired in Lys329Asp mutant. Furthermore, in silico docking studies showed that in this mutant the location and the H-bonding network around the Fe₂S₂ cluster of ISP on cyt b surface was different than the wild type enzyme. Based on these experimental findings and theoretical considerations, we propose that the presence of a positive charge at position 329 of cyt b is critical for efficient electron transfer and proton release for QH₂ oxidation at the Q_o site of cyt bc₁.