Abstract
Famoxadone is a new cytochrome bc(1) Q(o) site inhibitor that immobilizes the iron-sulfur protein (ISP) in the b conformation. The effects of famoxadone on electron transfer between the iron-sulfur center (2Fe-2S) and cyt c(1) were studied using a ruthenium dimer to photoinitiate the reaction. The rate constant for electron transfer in the forward direction from 2Fe-2S to cyt c(1) was found to be 16,000 s(-1) in bovine cyt bc(1). Binding famoxadone decreased this rate constant to 1,480 s(-1), consistent with a decrease in mobility of the ISP. Reverse electron transfer from cyt c(1) to 2Fe-2S was found to be biphasic in bovine cyt bc(1) with rate constants of 90,000 and 7,300 s(-1). In the presence of famoxadone, reverse electron transfer was monophasic with a rate constant of 1,420 s(-1). It appears that the rate constants for the release of the oxidized and reduced ISP from the b conformation are the same in the presence of famoxadone. The effects of famoxadone binding on electron transfer were also studied in a series of Rhodobacter sphaeroides cyt bc(1) mutants involving residues at the interface between the Rieske protein and cyt c(1) and/or cyt b.
Highlights
EXPERIMENTAL PROCEDURESMaterials—Ru2D was prepared by a modification of the method of Downard et al [24]. Bovine cyt bc was purified as described by Yu et al [25]
The cytochrome1 bc1 complex is an integral membrane protein in the electron transport chains of mitochondria and many respiratory and photosynthetic prokaryotes [1]
It has been difficult to determine the kinetics of electron transfer from 2Fe-2S to cyt c1 [34, 35] as well as the dynamics of iron-sulfur protein (ISP) conformational changes
Summary
Materials—Ru2D was prepared by a modification of the method of Downard et al [24]. Bovine cyt bc was purified as described by Yu et al [25]. Wild-type and mutant R. sphaeroides cyt bc were prepared as described by Tian et al [11]. Succinate cytochrome c reductase (SCR) was purified as described previously [27]. Growth of E. coli cells and plasmid-bearing R. sphaeroides cells were carried out as described previously [12]. Cluster in Mutant Cyt bc1—The cyt bc activity was determined in an assay mixture containing 100 mM Naϩ/Kϩ phosphate buffer, pH 7.4, 300 M EDTA, 100 M cyt c, and 25 M 2,3-dimethoxy-5-methyl-6-(10bromodecyl)-1,4-benzoquinol (Q0C10BrH2) at 23 °C using the method described by Xiao et al [12]. The redox potentials of 2Fe-2S in cyt bc mutants were determined as described previously [12]. Paraquat or [Co(NH3)5Cl]2ϩ was used as sacrificial acceptors, and Q0C10BrH2 was used to reduce cyt bc. The experiments were carried out aerobically to rapidly reoxidize the highly absorbing reduced paraquat
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