Witthuhn V C, Gao J, Hong S, Halls S, Rott M A, Wraight C A, Crofts A R, Donohue T J
Bacteriology Department, University of Wisconsin-Madison 53706, USA.
Biochemistry. 1997 Jan 28;36(4):903-11. doi: 10.1021/bi961648k.
Rhodobacter sphaeroides strains lacking cytochrome c2 (cyt c2), the normal electron donor to P870+ in light-oxidized reaction center (RC) complexes, are unable to grow photosynthetically. However, spd mutations that suppress the photosynthetic deficiency of cyt c2 mutants elevate levels of the cyt c2 isoform, isocyt c2. We monitored photosynthetic electron transfer in whole cells, in chromatophores, and with purified components to ascertain if and how isocyt c2 reduced light-oxidized RC complexes. These studies revealed that several fundamental aspects of photosynthetic electron transfer were similar in strains that use isocyt c2 and wild-type cells. For example, P870+ reduction accompanied cytochrome c oxidation. In addition, photosynthetic electron transfer was blocked by the well-known cyt bc1 complex inhibitors antimycin and myxothiazol. However, even at the increased isocyt c2 levels present in these strains (approximately 40% that of cyt c2 in wild-type cells), there was little, if any, of the rapid (< 5 microns) electron transfer to P870+ that is characteristic of cytochromes bound to RC complexes at the time of the light flash. Thus, it appears that isocyt c2 function limits the in vivo rate of P870+ reduction. Indeed, at low ionic strength in vitro, the apparent affinity of isocyt c2 for RC complexes (KD approximately 40 microM) is significantly lower than that of cyt c2 (KD approximately 1.0 microM). This reduced affinity does not appear to result from an altered mode of RC binding by isocyt c2 since electrostatic interactions make similar overall contributions to the binding of both cyt c2 and isocyt c2 to this membrane-bound redox partner. Thus, sequence, structural, or local conformational differences between cyt c2 and isocyt c2 significantly alter their apparent affinities for this physiologically relevant redox partner.
缺乏细胞色素c2（cyt c2）的球形红杆菌菌株无法进行光合生长，细胞色素c2是光氧化反应中心（RC）复合物中P870+的正常电子供体。然而，抑制cyt c2突变体光合缺陷的spd突变会提高cyt c2同工型即异细胞色素c2的水平。我们监测了全细胞、载色体以及纯化组分中的光合电子传递，以确定异细胞色素c2是否以及如何还原光氧化的RC复合物。这些研究表明，在使用异细胞色素c2的菌株和野生型细胞中，光合电子传递的几个基本方面是相似的。例如，细胞色素c氧化伴随着P870+还原。此外，光合电子传递被著名的细胞色素bc1复合物抑制剂抗霉素和粘噻唑阻断。然而，即使在这些菌株中异细胞色素c2水平升高（约为野生型细胞中cyt c2水平的40%）的情况下，几乎没有（如果有的话）快速（<5微秒）的电子传递到P870+，而这种快速电子传递是光闪光时与RC复合物结合的细胞色素的特征。因此，似乎异细胞色素c2的功能限制了体内P870+还原的速率。实际上，在体外低离子强度下，异细胞色素c2对RC复合物的表观亲和力（KD约为40 microM）明显低于cyt c2（KD约为1.0 microM）。这种降低的亲和力似乎不是由于异细胞色素c2与RC结合模式的改变，因为静电相互作用对cyt c2和异细胞色素c2与这种膜结合氧化还原伙伴的结合总体贡献相似。因此，cyt c2和异细胞色素c2之间的序列、结构或局部构象差异显著改变了它们对这种生理相关氧化还原伙伴的表观亲和力。
