pH-induced Intramolecular Electron Transfer between the Iron-Sulfur Protein and Cytochrome c 1 in Bovine Cytochrome bc 1 Complex

Abstract

Structural analysis of the bc 1 complex suggests that the extramembrane 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 stoppedflow 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 Å from heme c 1 , making electron transfer very slow.The cytochrome bc 1 complex is a segment of the mitochondrial respiratory chain which catalyzes antimycin-sensitive electron transfer from ubiquinol to cytochrome c (1, 2). The reaction is coupled to the translocation of protons across the mitochondrial inner membrane to generate a proton gradient and membrane potential for ATP synthesis (2-4). The purified cytochrome bc 1 complex contains 11 protein subunits, 2165 amino acid residues, and 4 prosthetic groups with a total molecular mass of 248 kDa, without counting the bound phospholipids. The amino acid sequences of all subunits are available from either peptide or nucleotide sequencing (5, 6). The essential redox components of the complex are: two b-type cytochromes (heme b L , E m 1 ϭ Ϫ30 mV, heme b H, , E m ϭ 90 mV), one c-type cytochrome (heme c 1 , E m ϭ 227 mV), one high potential [2Fe-2S] cluster (FeS, E m ϭ 280 mV), and a ubiquinone. The three-dimensional structure of cytochrome bc 1 complex at 2.9 Å resolution was first reported in 1997 (5, 7). More complete structures, from a different crystalline form, have been reported recently (8 -10). The crystal structure of the complex exists as a closely interacting functional dimer. There are 13 transmembrane helices in each monomer, 8 belong to cytochrome b, and one each to cytochrome c 1 , ISP, and subunits 7, 10, and 11. The 21 Å between heme b L and heme b H and 27 Å between heme b L and the [2Fe-2S] cluster accommodate well the observed fast electron transfer between these redox centers (11, 12). However, the 31 Å between the [2Fe-2S] cluster and heme c 1 makes it difficult to explain the high electron transfer rates between them.During analysis of x-ray diffraction data of the crystalline complex (7), it was noticed that the extr...

Structure of the Yeast Cytochrome bc 1 Complex with a Hydroxyquinone Anion Qo Site Inhibitor Bound

“…4) are both competitive inhibitors. Kinetic studies show that stigmatellin is more tightly bound than UHDBT (44,47). Furthermore, stigmatellin binding raises the midpoint potential (E m ) of the Rieske protein by 250 mV (48), whereas for UHDBT an increase of only 70 mV was determined for the bovine enzyme (37) and for the yeast enzyme.…”

The Coenzyme Q10 Analog Decylubiquinone Inhibits the Redox-activated Mitochondrial Permeability Transition

“…The Respiratory Site of ROS Production Is Cytochrome bc 1 -To determine the cellular site regulating ROS-production after GSH depletion, cells were co-incubated with DEM, DEM ϩ rotenone, DEM ϩ stigmatellin, or DEM ϩ antimycin A. Rotenone selectively blocks respiratory complex I (38), stigmatellin blocks the Q o site of cytochrome bc 1 (39), and antimycin A blocks the Q i site of cytochrome bc 1 (40). Whereas rotenone and antimycin did not prevent ROS increase (Fig.…”

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome bL to bH

“…Cytochrome bc 1 complex was first reduced by a limited amount of ascorbate to give 50% cytochrome c 1 reduction, at pH 8. The pH of the partially reduced complex was adjusted to 7.0, under which condition ISP will become mostly reduced and cytochrome c 1 mostly oxidized [33]. The Pf inhibitor efficacy was then determined.…”