Aspartate-187 of cytochrome b is not needed for DCCD inhibition of ubiquinol: Cytochrome c oxidoreductase in Rhodobacter sphaeroides chromatophores

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit steady-st ate proton translocation by cytochrome bc(1) and b(6)f complexes without si gnificantly altering the rate of electron transport, a process referred to as decoupling. In chromatophores of the purple bacterium Rhodobacter sphaer oides, this has been associated with the specific labeling of a surface-exp osed aspartate-187 of the cytochrome b subunit of the bc(1) complex [Wang e t al. (1998) Arch. Biochem. Biophyss. 352, 193-198]. To explore the possibl e role of this amino acid residue in the protonogenic reactions of cytochro me bc(1) complex, we investigated the effect of DCCD modification on flash- induced electron transport and the electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores from wild type (WT) and mutant cells, in which aspartate-187 of cytochrome b (Asp(B187)) has been changed to asparag ine (mutant B187 DN). The kinetics and amplitude of phase III of the electr ochromic shift of carotenoids, reflecting electrogenic reactions in the bc( 1) complex, and of the redox changes of cytochromes and reaction center, we re similar (+/- 15%) in both WT and B187DN chromatophores. DCCD effectively inhibited phase III of the carotenoid bandshift in both B187DN and WT chro matophores, The dependence of the kinetics and amplitude of phase LII of th e electrochromic shift on DCCD concentration was identical in WT and B187DN chromatophores, indicating that covalent modification of Asp(B187) is not specifically responsible for the effect of DCCD-induced effects of cytochro me bc(1) complex. Furthermore, no evidence for differential inhibition of e lectrogenesis and electron transport was found in either strain. We conclud e that Asp(B187) plays no crucial role in the protonogenic reactions of bc( 1) complex, since its replacement by asparagine does not lead to any signif icant effects on either the electrogenic reactions of bc(1) complex, as rev ealed by phase III of the electrochromic shift of carotenoids, or sensitivi ty of turnover to DCCD.