cytochromes-c1 and 5-n-undecyl-6-hydroxy-4-7-dioxobenzothiazole

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.

Topics: Animals; Cattle; Cytochromes c1; Electron Transport; Electron Transport Complex III; Heme; Hydrogen-Ion Concentration; Iron; Iron-Sulfur Proteins; Oxidation-Reduction; Polyenes; Stilbenes; Sulfur; Thiazoles; Time Factors

Based on the high electron transfer rate between the [2Fe-2S] cluster and heme c(1) and the elevation of the redox midpoint potential of iron sulfur protein (ISP) upon binding of certain Qo inhibitors, the binding rate constants of stigmatellin and UHDBT to the cytochrome bc(1) complex were determined using a stopped-flow rapid scanning technique. Assuming that the intramolecular electron transfer from ISP to cytochrome c(1) is much faster than the binding of inhibitors, the rate of the inhibitor binding can be determined by the rate of cytochrome c(1) oxidation. The binding rate constants were calculated to be 1.0x10(5) and 2.3x10(5) M(-1) s(-1) at pH 7.5 for stigmatellin and UHDBT, respectively. The binding rate constant of UHDBT is pH dependent and that of stigmatellin is not.

Topics: Animals; Cattle; Cytochromes c1; Electron Transport; Electron Transport Complex III; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Oxidation-Reduction; Polyenes; Protein Binding; Spectrophotometry; Thiazoles; Time Factors

Crystallographic structures for the mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from different sources, and with different inhibitors in cocrystals, have revealed that the extrinsic domain of the iron sulfur subunit is not fixed [Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y.-I., Kim, K. K., Hung, L.-W., Crofts, A. R., Berry, E. A., and Kim, S.-H. (1998) Nature (London), 392, 677-684], but moves between reaction domains on cytochrome c(1) and cytochrome b subunits. We have suggested that the movement is necessary for quinol oxidation at the Q(o) site of the complex. In this paper, we show that the electron-transfer reactions of the high-potential chain of the complex, including oxidation of the iron sulfur protein by cytochrome c(1) and the reactions by which oxidizing equivalents become available at the Q(o) site, are rapid compared to the rate-determining step. Activation energies of partial reactions that contribute to movement of the iron sulfur protein have been measured and shown to be lower than the high activation barrier associated with quinol oxidation. We conclude that the movement is not the source of the activation barrier. We estimate the occupancies of different positions for the iron sulfur protein from the crystallographic electron densities and discuss the parameters determining the binding of the iron sulfur protein in different configurations. The low activation barrier is consistent with a movement between these locations through a constrained diffusion. Apart from ligation in enzyme-substrate or inhibitor complexes, the binding forces in the native structure are likely to be < = RT, suggesting that the mobile head can explore the reaction interfaces through stochastic processes within the time scale indicated by kinetic measurements.

Topics: Animals; Binding Sites; Crystallography; Cytochrome b Group; Cytochromes c1; Electron Transport Complex III; Iron-Sulfur Proteins; Kinetics; Oxidation-Reduction; Protein Conformation; Temperature; Thermodynamics; Thiazoles; Ubiquinone

The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

Topics: Animals; Cyanides; Cytochrome b Group; Cytochromes c1; Glycerolphosphate Dehydrogenase; Mitochondria; Naphthoquinones; Oxygen Consumption; Salicylamides; Thiazoles; Trypanosoma brucei brucei