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.     

Rapid reduction of cytochrome c1 in the presence of antimycin and its implication for the mechanism of electron transfer in the cytochrome b-c1 segment of the mitochondrial respiratory chain

Antimycin, a specific and highly potent inhibitor of electron transfer in the cytochrome b-c1 segment of the mitochondrial respiratory chain, does not inhibit reduction of cytochrome c1 by succinate in isolated succinate-cytochrome c reductase complex under conditions where the respiratory chain complex undergoes one oxidation-reduction turnover. If a slight molar excess of cytochrome c is added to the isolated reductase complex in the presence of antimycin, there is rapid reduction of one equivalent of c type cytochrome by succinate, after which reduction of the remaining c type cytochrome is inhibited. Antimycin fully inhibits succinate-cytochrome c reductase activity of isolated succinate-cytochrome c reductase complex in which the b-c1 complex undergoes multiple turnovers in a catalytic fashion. In addition, when antimycin is added to isolated reductase complex in the presence of cytochrome c plus cytochrome c oxidase, the inhibitor causes a "crossover" in the steady state level of reduction of the cytochromes b and c1 comparable to this classical effect in mitochondria. On the basis of these results, it is suggested that linear schemes of electron transfer are not adequate to account for the site of antimycin inhibition and the mechanism of electron transfer in the cytochrome b-c1 segment of the respiratory chain. The effects of antimycin are consistent with cyclic electron transfer mechanisms such as the protonmotive Q cycle.     

Myxothiazol, a new inhibitor of the cytochrome b-c1 segment of th respiratory chain

Myxothiazol inhibited oxygen consumption of beef heart mitochondria in the presence and absence of 2,4-dinitrophenol, as well as NADH oxidation by submitochondrial particles. The doses required for 50% inhibition were 0.58 mol myxothiazol/mol cytochrome b for oxygen consumption of beef heart mitochondria, and 0.45 mol/mol cytochrome b for NADH oxidation by submitochondrial particles. Difference spectra with beef heart mitochondria and with cell suspensions of Saccharomyces cerevisiae revealed that myxothiazol blocked the electron transport within the cytochrome b-c1 segment of the respiratory chain. Myxothiazol induced a spectral change in cytochrome b which was different from and independent of the shift induced by antimycin. Myxothiazol did not give the extra reduction of cytochrome b typical for antimycin. Studies on the effect of mixtures of myxothiazol and antimycin on the inhibition of NADH oxidation indicated that the binding sites of the two inhibitors are not identical.     

The mode of action of stigmatellin,a new inhibitor of the cytochrome b-c1 segment of the respiratory chain

The new antibiotic stigmatellin, obtained from the myxobacterium Stigmatella aurantiaca, was found to block the electron flow in the respiratory chain of bovine heart submitochondrial particles at the site of the cytochrome b-c₁ segment. Its inhibitory potency was identical with that of antimycin and myxothiazol, and like these antibiotics, stigmatellin caused a shift in the spectrum of reduced cytochrome b. Difference spectroscopic studies with the three inhibitors in various combinations indicated that the binding site of stigmatellin was different from that of antimycin, but more or less identical with that of myxothiazol. Experiments with 14 synthesized derivatives of stigmatellin showed that good inhibitory activity can be expected only if the side chain was kept relatively lipophilic, and the keto and the hydroxy groups of the chromone system were left intact.     

Effect of electron transfer inhibitors on superoxide generation in the cytochrome bc1 site of the mitochondrial respiratory chain

Antimycin, 2-nonyl-4-hydroxyquinoline N-oxide and funiculosin induce O.2(-) generation by submitochondrial particles oxidizing succinate, whereas KCN, mucidin, myxothiazol or 2,3-dimercaptopropanol inhibit O.2(-) generation. Thenoyltrifluoroacetone does not induce superoxide production by itself but slightly stimulates the reaction initiated by antimycin. The results indicate that auto-oxidation of unstable ubisemiquinone formed in centre o of the Q-cycle generates most of the O.2(-) radicals in the cytochrome bc1-site of the mitochondrial respiratory chain.     

Inhibition of electron transfer in the cytochromeb-c 1 segment of the mitochondrial respiratory chain by a synthetic analogue of ubiquinone

A synthetic analogue of ubiquinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, inhibits oxidation of succinate and NADH-linked substrates by rat liver mitochondria. Inhibition occurs both in the presence (state 3) and absence (state 4) of ADP. With isolated succinate-cytochromec reductase complex from bovine heart mitochondria the quinone analogue inhibits succinate-cytochromec reductase and ubiquinol-cytochromec reductase activities but does not inhibit succinate-ubiquinone reductase activity. Inhibition of cytochromec reductase activities is markedly dependent on pH in the range pH 7–8. At pH 7.0 inhibition occurs with an apparentK _i1×10^–8 M, while at pH 8.0 the apparentK _i is more than an order of magnitude greater than this. Spectrophotometric titrations of 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole show a visibly detectable pK _a at pH 6.5 attributable to ionization of the 6-hydroxy group. These results indicate that this quinone derivative is a highly specific and potent inhibitor of electron transfer in theb-c ₁ segment of the respiratory chain. Because of the structural analogy, it is likely that the mechanism of inhibition involves disruption of normal ubiquinone function. In addition, this inhibition depends on protonation of the ionizable hydroxy group of the inhibitory analogue or on protonation of a functional group in theb-c ₁ segment.     

Assembly of the iron-sulfur protein into the cytochrome b-c1, complex of yeast mitochondria.

The assembly of the iron-sulfur protein into the cytochrome bc1 complex after import and processing of the precursor form into mitochondria in vitro was investigated by immunoprecipitation of the radiolabeled iron-sulfur protein from detergent-solubilized mitochondria with specific antisera. After import in vitro, the labeled mature form of the iron-sulfur protein was immunoprecipitated by antisera against both the iron-sulfur protein and the entire bc1 complex from mitochondria solubilized with either Triton X-100 or dodecyl maltoside. After sodium dodecyl sulfate solubilization of mitochondria, however, the antiserum against the iron-sulfur protein, but not that against the bc1 complex, immunoprecipitated the radiolabeled iron-sulfur protein. These results suggest that in mitochondria the mature form of the iron-sulfur protein is assembled with other subunits of the bc1 complex that are recognized by the antiserum against the bc1 complex. By contrast, the intermediate and precursor forms of the iron-sulfur protein that accumulated in the matrix when proteolytic processing was blocked with EDTA and o-phenanthroline were not efficiently assembled into the bc1 complex. The import and processing of the iron-sulfur protein also occurred in mitochondria lacking either cytochrome b (W-267) or the iron-sulfur protein (JPJ1). The mature form of the iron-sulfur protein was immunoprecipitated by antisera against the bc1 complex or core protein I after import in vitro into these mitochondria, suggesting that the mature form is associated with other subunits of the bc1 complex in these strains.     

Triphasic reduction of cytochrome b and the protonmotive Q cycle pathway of electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain

Reduction of cytochrome b in isolated succinate-cytochrome c reductase is a triphasic reaction. Initially, there is a relatively rapid, partial reduction of the cytochrome b, the rate of which matches the rate of reduction of cytochrome c1. This is followed by partial or complete reoxidation of the b, which is then followed by slow rereduction. At very low concentrations of succinate, the initial partial reduction of b is followed by reoxidation, but the third (rereduction) phase is absent, owing to insufficient substrate to rereduce the cytochromes. If antimycin is added at various times during the triphasic reaction, it inhibits the reoxidation and also inhibits the rereduction phase. Antimycin does not inhibit the initial phase of b reduction and, if added before or during this phase, it causes reduction of b to proceed to completion as a monophasic reaction. Myxothiazol inhibits the first phase of b reduction and the subsequent reoxidation, but does not inhibit the third, slow phase of b reduction. The resulting monophasic reduction of b which is observed in the presence of myxothiazol is slower than that in the presence of antimycin. The combination of both inhibitors, whether added together or successively during the triphasic reaction, completely inhibits b reduction. The triphasic reduction of cytochrome b is consistent with electron transfer by a protonmotive Q cycle in which there are two pathways for cytochrome b reduction. One pathway allows the initial phase of cytochrome b reduction by a myxothiazol-sensitive reaction in which reduction of b by ubisemiquinone is linked to reduction of iron-sulfur protein and cytochrome c1 by ubiquinol. In the second phase of the triphasic reaction, the b cytochromes are reoxidized by ubiquinone or ubisemiquinone through an antimycin-sensitive reaction. If oxidation of ubiquinol by iron-sulfur protein is blocked, either by myxothiazol or by reduction of iron-sulfur protein and cytochrome c1, the b cytochromes can be reduced by reversal of the antimycin-sensitive pathway, thus accounting for the third phase of b reduction.     

pH-induced intramolecular electron transfer between the iron-sulfur protein and cytochrome c(1) in bovine cytochrome bc(1) complex

Structural analysis of the bc(1) complex suggests that the extra membrane domain of iron-sulfur protein (ISP) undergoes substantial movement during the catalytic cycle. Binding of Qo site inhibitors to this complex affects the mobility of ISP. Taking advantage of the difference in the pH dependence of the redox midpoint potentials of cytochrome c(1) and ISP, we have measured electron transfer between the [2Fe-2S] cluster and heme c(1) in native and inhibitor-treated partially reduced cytochrome bc(1) complexes. The rate of the pH-induced cytochrome c(1) reduction can be estimated by conventional stopped-flow techniques (t1/2, 1-2 ms), whereas the rate of cytochrome c(1) oxidation is too high for stopped-flow measurement. These results suggest that oxidized ISP has a higher mobility than reduced ISP and that the movement of reduced ISP may require an energy input from another component. In the 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT)-inhibited complex, the rate of cytochrome c(1) reduction is greatly decreased to a t1/2 of approximately 2.8 s. An even lower rate is observed with the stigmatellin-treated complex. These results support the idea that UHDBT and stigmatellin arrest the [2Fe-2S] cluster at a fixed position, 31 A from heme c(1), making electron transfer very slow.     

Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 °K) and potentiometric titration.Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix “N”) resolves into two single electron titrations with E_{m 7.2} values of ?380±20 mV and ?240±20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g_// = 2.03, g_⊥ = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with g_z = 2.03, g_y = 1.94 and g_x = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g_// = 2.05 and g_⊥ = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high E_{m 7.2} value (?20±20 mV).Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with E_{m 7.2} = ? 240±20 mV is defined as Center N-3 (g_z = 2.10, (g_y = 1.93?), g_x = 1.87); the ?405±20 mV component as Center N-4 (g_z = 2.11, (g_y = 1.93?), g_x = 1.88). At temperatures close to 4.2 °K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with E_{m 7.2} = ?260±20 mV. The multiplicity of peaks suggests the presence of at least two different ironsulfur centers having similar E_{m 7.2} values (?260±20 mV); hence, tentatively assigned as N-5 and N-6.Consistent with the individual E_{m 7.2} values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the E_{m 7.2} of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. E_{m 7.2} values and EPR lineshapes for the other ironsulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show E_{m 7.2} values around -250 mV; Center N-2 exhibits a high E_{m 7.2} (?20±20 mV), while Center N-1a shows a very low E_{m 7.2} (?380±20 mV).     

Domain conformational switch of the iron-sulfur protein in cytochrome bc1 complex is induced by the electron transfer from cytochrome bL to bH

Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc(1) complex in the past have led to the formulation of the "protonmotive Q-cycle" mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the Q(P) site with both electrons transferred simultaneously to ISP and cyt b(L) when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc(1) demonstrates that the reduced ISP-ED moves to the c(1)-position to reduce cyt c(1) only after the reduced cyt b(L) is oxidized by cyt b(H). However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of Q(P) site inhibitors, Pm and Pf, under various redox states of the bc(1) complex, suggest that the electron transfer from heme b(L) to b(H) is the driving force for the releasing of the reduced ISP-ED from the b-position to c(1)-position to reduce cyt c(1).     

Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart.

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 degrees K) and potentiometric titration. Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix "N") resolves into two single electron titratins with EM7.2 values of minus 380 plus or minus 20 mV and minus 240 plus or minus 20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g parellel = 2.03, g = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with gz = 2.03, gy = 1.94 and gx = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g parallel = 2.05 and g = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high EM7.2 value (minus 20 plus or minus 20 mV). Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with EM7.2 = minus 240 plus or minus 20 mV is defined as Center N-3 (gz = 2.10, (gz = 2.10, (gy = 1.93?), GX = 1.87); the minus 405 plus or minus 20 mV component as Center N-4 (gz = 2.11, (gy = 1.93?), gx = 1.88). At temperatures close to 4.2 degrees K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with EM7.2 = minus 260 plus or minus 20 mV. The multiplicity of peaks suggests the presence of at least two different iron-sulfur centers having similar EM7.2 values (minus 260 plus or minus 20 mV); HENCE, tentatively assigned as N-5 and N-6. Consistent with the individual EM7.2 values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the EM7.2 of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. EM7.2 values and EPR lineshapes for the other iron-sulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show EM7.2 values around minus 250 mV; Center N-2 exhibits a high EM7.2 (minus 20 plus or minus 20 mV), while Center N-1a shows a very low EM7.2 (minus 380 plus or minus 20 mV).     

Ubisemiquinone radicals from the cytochrome b-c1 complex of the mitochondrial electron transport chain--demonstration of QP-S radical formation

Stable ubisemiquinone radical(s) in the cytochrome $\text{b}\text{?}\text{c}{}_1$-II complex of bovine heart was observed following reduction by succinate in the presence of catalytic amounts of succinate dehydrogenase. The radical was abolished by addition of antimycin A, but a residual radical remained in the presence of excess exogenous Q₂. The radical showed an EPR signal of g = 2.0046 ± .003 at X band (～9.4 GHz) with no resolved hyperfine structure and had a line width of 8.1 ± .5 Gauss at 23°C. The Q band (35 GHz) spectra showed wellresolved $\text{g}$-anisotropy and had a field separation between derivative extrema of 26 ± 1 Gauss. This radical is evidently from QP-C. These observations substantiate that the radical is immobilized and bound to a protein. The QP-S radical was demonstrated in the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex only in the presence of more than a catalytic amount of succinate dehydrogenase and cytochrome $\text{b}\text{-}\text{c}{}_1$. This signal was not antimycin a inhibitory. The signal amplitude paralleled the reconstitutive enzymic activity of succinate-cytochrome $\text{c}$ reductase from succinate dehydrogenase and the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex.     

Interaction of cytochrome c with cytochrome bc1 complex of the mitochondrial respiratory chain

The binding of cytochrome c to the cytochrome bc1 complex of bovine heart mitochondria was studied. Cytochrome c derivatives, arylazido-labeled at lysine 13 or lysine 22, were prepared and their properties as electron acceptors from the bc1 complex were measured. Mixtures of bc1 complex with cytochrome c derivatives were illuminated with ultraviolet light and afterwards subjected to polyacrylamide gel electrophoresis. The gels were analysed using dual-wavelength scanning at 280 minus 300 and 400 minus 430 nm. It was found that illumination with ultraviolet light in the presence of the lysine 12 derivative produced a diminution of the polypeptide of the bc1 coplex having molecular weight 30 000 (band IV) and formation of a new polypeptide composed of band IV and cytochrome c. Band IV was identified as cytochrome c1, and it was concluded that this hemoprotein interacts with cytochrome c and contains its binding site in complex III of the mitochondrial respiratory chain. Illumination of the bc1 complex in presence of the lysine 22 derivative did not produce changes of the polypeptide pattern.