Shinkarev V P, Ugulava N B, Takahashi E, Crofts A R, Wraight C A
Department of Plant Biology, University of Illinois at Urbana-Champaign, 265 Morrill Hall, 505 South Goodwin Avenue, Urbana, Illinois 61801, USA.
Biochemistry. 2000 Nov 21;39(46):14232-7. doi: 10.1021/bi001179t.
N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit steady-state proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. In chromatophores of the purple bacterium Rhodobacter sphaeroides, this has been associated with the specific labeling of a surface-exposed aspartate-187 of the cytochrome b subunit of the bc(1) complex [Wang et al. (1998) Arch. Biochem. Biophys. 352, 193-198]. To explore the possible role of this amino acid residue in the protonogenic reactions of cytochrome 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 asparagine (mutant B187 DN). The kinetics and amplitude of phase III of the electrochromic shift of carotenoids, reflecting electrogenic reactions in the bc(1) complex, and of the redox changes of cytochromes and reaction center, were similar (+/- 15%) in both WT and B187DN chromatophores. DCCD effectively inhibited phase III of the carotenoid bandshift in both B187DN and WT chromatophores. The dependence of the kinetics and amplitude of phase III of the 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 cytochrome bc(1) complex. Furthermore, no evidence for differential inhibition of electrogenesis and electron transport was found in either strain. We conclude 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 significant effects on either the electrogenic reactions of bc(1) complex, as revealed by phase III of the electrochromic shift of carotenoids, or sensitivity of turnover to DCCD.
据报道，N,N'-二环己基碳二亚胺（DCCD）可抑制细胞色素bc(1)和b(6)f复合物的稳态质子转运，而不会显著改变电子传递速率，这一过程被称为解偶联。在紫色细菌球形红杆菌的色素细胞中，这与bc(1)复合物细胞色素b亚基表面暴露的天冬氨酸-187的特异性标记有关[Wang等人（1998年），《生物化学与生物物理学报》352卷，第193 - 198页]。为了探究该氨基酸残基在细胞色素bc(1)复合物质子生成反应中的可能作用，我们研究了DCCD修饰对野生型（WT）和突变体细胞（其中细胞色素b的天冬氨酸-187（Asp(B187)）已被替换为天冬酰胺（突变体B187 DN））中球形红杆菌色素细胞的闪光诱导电子传递和类胡萝卜素电致变色带移的影响。反映bc(1)复合物中电化学反应的类胡萝卜素电致变色位移第三阶段的动力学和幅度，以及细胞色素和反应中心的氧化还原变化，在WT和B187DN色素细胞中相似（±15%）。DCCD有效抑制了B187DN和WT色素细胞中类胡萝卜素带移的第三阶段。电致变色位移第三阶段的动力学和幅度对DCCD浓度的依赖性在WT和B187DN色素细胞中相同，表明Asp(B187)的共价修饰并非DCCD诱导细胞色素bc(1)复合物效应的特异性原因。此外，在任一菌株中均未发现电生成和电子传递存在差异抑制的证据。我们得出结论，Asp(B187)在bc(1)复合物的质子生成反应中不发挥关键作用，因为其被天冬酰胺取代对bc(1)复合物的电化学反应（如类胡萝卜素电致变色位移第三阶段所揭示）或周转对DCCD的敏感性均未产生任何显著影响。
