Internal electron transfer within mitochondrial succinate-cytochrome c reductase.

Internal electron transfer within succinate-cytochrome C reductase from pigeon breast muscle mitochondria was followed by the pulse radiolytic technique. The electron equivalent is transferred from an unknown donor to b type cytochrome(s) in a first order process with a rate constant of: 660±150 s^?1. This process might be the rate determining step of electron transfer in mitochondria, since it is similar in rate to the turn over number of the mitochondrial respiratory chain.     

Interactions of cytochrome c with mitochondrial membranes. Binding to succinate-cytochrome c reductase

Methyl-4-azidobenzoimidate was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c bound to cytochrome c-depleted mitochondria with the same Kd as native cytochrome c and restored oxygen uptake to the same extent. Irradiation of cytochrome c-depleted mitochondrial membranes with 3- to 4-fold excess of photoaffinity-labeled cytochrome c over cytochrome c oxidase resulted in covalent binding of the derivative to the membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on an agarose Bio-Gel-A-5m showed that the labeled cytochrome c was bound covalently to succinate-cytochrome c reductase. The covalently bound cytochrome c was active in mediating electron transfer between its reductase and oxidase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the succinate-cytochrome c reductase containing photoaffinity-labeled 125I-cytochrome c showed that the reductase contained a protein binding site for cytochrome c. It is suggested that cytochrome c1 is the most likely site for the cytochrome c binding in mitochondria in situ.     

Excess no predisposes mitochondrial succinate-cytochrome c reductase to produce hydroxyl radical

Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O(2)(?-) generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate-cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/(?)OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron-H(2)O(2)-dependent Fenton reaction or O(2)(?-)-dependent Haber-Weiss mechanism. Direct EPR measurement of SCR at 77K indicated the formation of a nonheme iron-NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce (?)OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells.     

The triphasic reduction of cytochrome b in the succinate-cytochrome c reductase

In the succinate-cytochrome c reductase, the reduction of cytochrome b has been found to be triphasic: an initial rapid partial reduction was followed first by a rapid oxidation and then finally by a slow reduction. The initial reduction of cytochrome b was faster than that of cytochrome c₁ and the final slow reduction of cytochrome b began when cytochrome c₁ reduction was approaching completion. In presence of the inhibitors antimycin A or HQNO the reduction of cytochrome b became monophasic. Hysteresis or a kinetic cooperative effect of a factor controlling cytochrome b oxidation has been suggested as a possible explanation for the triphasic reduction of cytochrome b.     

Multiphasic oxidation-reduction of cytochrome b in the succinate-cytochrome c reductase

The triphasic course previously reported for the reduction of cytochrome b in the succinate-cytochrome c reductase by either succinate or duroquinol has been shown to be dependent on the redox state of the enzyme preparation. Prior reduction with increasing concentrations of ascorbate leads to partial reduction of cytochrome c1, and a gradual decrease in the magnitude of the oxidation phase of cytochrome b. At an ascorbate concentration sufficient to reduce cytochrome c1 almost completely, the reduction of cytochrome b by either succinate or duroquinol becomes monophasic. Owing to the presence of a trace amount of cytochrome oxidase in the reductase preparation employed, the addition of cytochrome c makes electron flow from substrate to oxygen possible. Under such circumstances, the addition of a limited amount of either succinate or duroquinol leads to a multiphasic reduction and oxidation of cytochrome b. After the initial three phases as described previously, cytochrome b becomes oxidized before cytochrome c1 when the limited amount of added substrate is being used up. However, at the end of the reaction when cytochrome c1 is being rapidly oxidized, cytochrome b becomes again reduced. The above observations support a cyclic scheme of electron flow in which the reduction of cytochrome b proceeds by two different routes and its oxidation controlled by the redox state of a component of the respiratory chain.     

Resolution and reconstitution of succinate-cytochrome c reductase: preparations and properties of high purity succinate dehydrogenase and ubiquinol-cytochrome c reductase

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c1 III complex). An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinae dehydrogenase fraction and the cytochrome b-c1 complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c1 complex was separated into chtochrome b-c1 III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate. The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 anbd 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low Km ferricyanide reductase activity of 85 mumol succinate oxidized per min per mg protein at 38 degrees C. Chemical composition analysis of cytochrome b-c1 III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation. When these three components were mixed in a proper ratio, a thenoyltrifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.     

Protein-ubiquinone interaction in bovine heart mitochondrial succinate-cytochrome c reductase. Synthesis and biological properties of fluorine substituted ubiquinone derivatives.

To investigate the protein-ubiquinone interaction in the bovine heart mitochondrial succinate-cytochrome c reductase region of the respiratory chain, three fluorine substituted ubiquinone derivatives, 2,3-dimethoxy-6-(9'-fluorodecyl)-1,4-benzoquinone (9FQ), 2-methoxy-5-trifluoromethyl-6-decyl-1,4-benzoquinone (TFQ), and 2-methoxy-5-trifluoromethyl-6-(9'-fluorodecyl)-1,4-benzoquinone (9FTFQ), were synthesized. 9FQ was synthesized by radical coupling of Q0 and bis(10-fluoroundecanoyl)peroxide. The latter was prepared by fluorination of undecylenic acid followed by thionylchloride treatment and peroxidation. TFQ was synthesized from 2,2,2-trifluoro-p-cresol by methylation, nitration, reduction, acetylation, nitration, reduction, oxidation, and radical alkylation. 9FTFQ was prepared by the radical alkylation of 2-methoxy-5-trifluoromethyl-1,4-benzoquinone with bis(10-fluoroundecanoyl)peroxide. All three fluoro-Q derivatives are active (greater than 50% the activity of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) when used as electron acceptors for succinate-ubiquinone reductase. However, only 9FQ is active when used as an electron donor for ubiquinol-cytochrome c reductase or as an electron mediator for succinate-cytochrome c reductase. Both TFQ and 9FTFQ are competitive inhibitors for ubiquinol-cytochrome c reductase. A 19FNMR peak-broadening effect was observed for 9FQ when it was reconstituted with ubiquinone-depleted ubiquinol-cytochrome c reductase. A drastic up-field chemical shift was observed for TFQ when it was reconstituted with ubiquinone-depleted reductase. These results indicate that the binding environments of the benzoquinone ring and the alkyl side chain of the Q molecule are different. The strong up-field chemical shift for TFQ, and lack of significant chemical shift for 9FQ, suggest that the benzoquinone ring is bound near the paramagnetic cytochrome b heme.     

The presence of multiple cytochrome b proteins in succinate-cytochrome c reductase.

Two cytochrome $\text{b}$ proteins were isolated from succinate-cytochrome $\text{c}$ reductase and the cytochrome $\text{b-c}{}_1$ complex. Their molecular weights were determined to be 37,000 and 17,000 daltons by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Spectral properties and amino acid composition of these two proteins are reported in the paper.     

Studies on the stabilized ubisemiquinone species in the succinate-cytochrome c reductase segment of the intact mitochondrial membrane system.

1. Evidence is presented for the presence of a stable ubisemiquinone pair in the vicinity of iron-sulphur centre S-3, based on its thermodynamic and spin relaxation properties. 2. These semiquinones are coupled by dipolar interaction; quantitative analysis of the signals of the spin-coupled semiquinones (at pH 7.4) gives midpoint redox potentials E1 (oxidized to semiquinone state) and E2 (semiquinone to fully reduced state) of 140 and 80mV, respectively, for individual ubiquinones. 3. Values of pKS (pK of the semiquinone form) below 6.5 and pKR (pK of the fully reduced ubiquinone) of about 8.0 or above were estimated from the pH-dependence of the midpoint potentials of the spin coupled signals. Thus the ubisemiquinone associated with succinate dehydrogenase (designated as SQS) functions mostly in the anionic form of the physiological pH range. 4. Theonyltrifluoroacetone, a specific inhibitor of the succinate-ubiquinone reductase segment of the respiratory chain, destabilized the intermediate redox state; thus it quenches both the g = 2.00 signal and ubisemiquinone (SQS) and split signals from the spin coupled pair. This inhibitor has no significant effect on another bound ubisemiquinone species present in the cytochrome bc1 region (designated as SQC). 5. The possible function and location of these stabilized ubisemiquinone species were discussed in connection with Site-II energy transduction.     

Role of hydrophobic interactions and functional groups in the inhibitor binding to various sites of mitochondrial succinate-cytochrome c reductase

It was shown that the efficiency of succinate-cytochrome c reductase inhibitors, i. e. neutral polar substances, negatively charged phenols and 2-hydroxy-3-alkyl-1.4-naphthoquinones, is increased with an increase in their hydrophobicity. Plotting-lg C50 versus lg P for all the three groups of inhibitors, the role of functional groups of the inhibitors in their binding to the corresponding sites of the respiratory chain was determined. The efficiency of inhibition by neutral polar substances does not depend on the chemical nature of the inhibitors and is described by the equation-lg C50 = 0.864 lg P + 0.222 (r = 0.99). The negatively charged group of dissociated phenols determines the specificity of the inhibitor binding to the terminal site of the succinate dehydrogenase complex and is involved in the inhibitor binding to the enzyme. The carbonyl group of 2-hydroxy-3-alkyl-1.4-naphthoquinones selectively increases the affinity and efficiency of binding of these inhibitors to the b-c1 site of the respiratory chain.     

Effect of some cardiac and respiratory drugs on succinate-cytochrome c reductase

Succinate-cytochrome c reductase was inhibited in vitro and in vivo by phenobarbitone, aminophylline and neostigmine using both 2,6-dichlorophenolindophenol (DCIP) and cytochrome c (cyt c) as substrates. The enzyme was also activated by gallamine towards both substrates. In vitro, phenobarbitone and aminophylline inhibited the enzyme with respect to the reduction of DCIP and cyt c in a non-competitive manner with Ki values of 1.5 x 10(-5) and 5.7 x 10(-5)M, respectively. Moreover, neostigmine competitively inhibited the enzyme towards both substrates with Ki values of 1.36 x 10(-5) and 1.50 x 10(-5)M, respectively.     

Effect of specific lysine modification on the reduction of cytochrome c by succinate-cytochrome c reductase.

The reduction of cytochrome c by succinate-cytochrome c reductase was studied at very low cytochrome c concentrations where the reaction between cytochrome c1 and cytochrome c was rate limiting. The rate constant for the reaction was found to be independent of ionic strength up to 0.1 M chloride, and to decrease rapidly at higher ionic strength, suggesting that the interaction between cytochrome c1 and cytochrome c was primarily electrostatic. The reaction rates of cytochrome c derivatives modified at single lysine residues to form trifluoroacetylated or trifluoromethylphenylcarbamylated cytochromes c were studied to determine the role of individual lysines in the reaction. None of the modifications affected the reaction at low ionic strength, but at higher ionic strength the reaction rate was substantially decreased by modification of those lysines surrounding the heme crevice, lysine-8, -13, -27, -72, and -79. Modification of lysine-22, -25, -55, -99, and -100 had no effect on the rate. These results indicate that the binding site on cytochrome c for cytochrome c1 overlaps considerably with that for cytochrome oxidase, suggesting that cytochrome c might undergo some type of rotational diffusion during the electron-transport process.     

Direct and indirect roles of cytochrome b in the mediation of superoxide generation and NO catabolism by mitochondrial succinate-cytochrome c reductase

Mitochondrial superoxide (O2*-) production is an important mediator of oxidative cellular injury. Succinate-cytochrome c reductase (SCR) of the electron transport chain has been implicated as an essential part of the mediation of O2*- generation and an alternative target of nitric oxide (NO) in the regulation of mitochondrial respiration. The Q cycle mechanism plays a central role in controlling both events. In the present work, O2*- generation by SCR was measured with the EPR spin-trapping technique using DEPMPO (5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide) as the spin trap. In the presence of succinate, O2*- generation from SCR was detected as the spin adduct DEPMPO/*OOH. Inhibitors of the Q(o*-) site only marginally reduced (20-30%) this O2*- production, suggesting a secondary role of Q(o*-) in the mediation of O2*- generation. Addition of cyanide significantly decreased (approximately 70%) O2*- production, indicating the involvement of the heme component. UV-visible spectral analysis revealed that oxidation of ferrocytochrome b was accompanied by cytochrome c(1) reduction, and the reaction was mediated by the formation of an O2*- intermediate, indicating a direct role for cytochrome b in O2*- generation. In the presence of NO, DEPMPO/*OOH production was progressively diminished, implying that NO interacted with SCR or trapped the O2*-. The consumption of NO by SCR was investigated by electrochemical detection using an NO electrode. In the presence of succinate, SCR-mediated NO consumption was observed and inhibited by the addition of superoxide dismutase, suggesting the involvement of O2*-. Under the conditions of argon saturation, the NO consumption rate was not enhanced by succinate, suggesting a direct role for O2*- in the mediation of NO consumption. In the presence of succinate, oxidation of the ferrocytochrome b moiety of SCR was accelerated by the addition of NO, and was inhibited by argon saturation, indicating an indirect role for cytochrome b in the mediation of NO consumption.     

An antimycin-insensitive succinate-cytochrome c reductase activity in pure reconstitutively active succinate dehydrogenase

Antimycin-insensitive succinate-cytochrome c reductase activity has been detected in pure, reconstitutively active succinate dehydrogenase. The enzyme catalyzes electron transfer from succinate to cytochrome c at a rate of 0.7 mumole succinate oxidized per min per mg protein, in the presence of 100 microM cytochrome c. This activity, which is about 2% of that of reconstitutive (the ability of succinate dehydrogenase to reconstitute with coenzyme ubiquinone-binding proteins (QPs) to form succinate-ubiquinone reductase) or succinate-phenazine methosulfate activity in the preparation, differs from antimycin-insensitive succinate-cytochrome c reductase activity detected in submitochondrial particles or isolated succinate-cytochrome c reductase. The Km for cytochrome c for the former is too high to be measured. The Km for the latter is about 4.4 microM, similar to that of antimycin-sensitive succinate-cytochrome c activity in isolated succinate-cytochrome c reductase, suggesting that antimycin-insensitive succinate-cytochrome c activity of succinate-cytochrome c reductase probably results from incomplete inhibition by antimycin. Like reconstitutive activity of succinate dehydrogenase, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase is sensitive to oxygen; the half-life is about 20 min at 0 degrees C at a protein concentration of 23 mg/ml. In the presence of QPs, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase disappears and at the same time a thenoyltrifluoroacetone-sensitive succinate-ubiquinone reductase activity appears. This suggests that antimycin-insensitive succinate-cytochrome c reductase activity of succinate dehydrogenase appears when succinate dehydrogenase is detached from the membrane or from QPs. Reconstitutively active succinate dehydrogenase oxidizes succinate using succinylated cytochrome c as electron acceptor, suggesting that a low potential intermediate (radical) may be involved. This suggestion is confirmed by the detection of an unknown radical by spin trapping techniques. When a spin trap, alpha-phenyl-N-tert-butylnitrone (PBN), is added to a succinate oxidizing system containing reconstitutively active succinate dehydrogenase, a PBN spin adduct is generated. Although this PBN spin adduct is identical to that generated by xanthine oxidase, indicating that a perhydroxy radical might be involved, the insensitivity of this antimycin-insensitive succinate-cytochrome c reductase activity to superoxide dismutase and oxygen questions the nature of this observed radical.