Soriano G M, Ponamarev M V, Piskorowski R A, Cramer W A
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Biochemistry. 1998 Oct 27;37(43):15120-8. doi: 10.1021/bi9807714.
The prominent basic patch seen in the atomic structure of the lumen-side domain of turnip cytochrome f, consisting of Arg209 and Lys187, 58, 65, and 66, was proposed to be an electrostatically complementary docking site for its physiological electron acceptor, plastocyanin [Martinez, S. E., Huang, D., Szczepaniak, A., Cramer, W. A., and Smith, J. L. (1994) Structure 2, 95-105]. This proposal agrees with solution studies on the cytochrome f/plastocyanin electron-transfer reaction that showed a major contribution of electrostatic interactions to the docking, but not with studies on the oxidation rate of cyt f in vivo using mutants in which the basic patch of cyt f was neutralized. The apparent contradiction might be explained by an unknown electron acceptor protein for cyt f. However, (i) flash-induced oxidation of cyt f is absent in a PC-deficient mutant. (ii) Lys58, 65, and 66 in the large domain and Lys188 and 189 in the small domain are major contributors to the ionic strength dependence of the electron-transfer reaction in solution. Replacement of Lys58 and 65 by neutral residues and of Lys66 by the acidic residue Glu66 resulted in a >10-fold decrease in the rate of electron transfer in solution and complete loss of its ionic strength dependence. Replacement of Lys188 and Lys189 in the small domain of cyt f resulted in a 3-4-fold decrease in the second-order rate constant and a smaller dependence of the overall rate of electron transfer on ionic strength, corresponding to a loss of two positive charges. (iii) Acidification of the thylakoid lumen cannot explain the absence of electrostatic interactions. (iv) Changing the five lysines to acidic residues did not result in any significant retardation of the rate of cyt f oxidation in vivo. If the docking of cyt f and plastocyanin in vivo is mediated by basic residues of cyt f, they are different from those that mediate electron transfer in vitro or that are implicated by simulations of electrostatic interactions of the docking. Alternatively, docking of cyt f/PC in vivo is limited by spatial constraints or release of PC from P700 that precludes a rate-limiting mediation of the cyt f/PC reaction by specific electrostatic interactions. The cyt f/PC system in Chlamydomonas reinhardtii is the first electron-transfer couple for which the role of electrostatics in mediating the docking reaction has been studied both in vitro and in vivo.
在芜菁细胞色素f腔侧结构域的原子结构中发现的由Arg209和Lys187、58、65及66组成的显著碱性斑块，被认为是其生理电子受体质体蓝素的静电互补对接位点[Martinez, S. E., Huang, D., Szczepaniak, A., Cramer, W. A., and Smith, J. L. (1994) Structure 2, 95 - 105]。这一观点与细胞色素f/质体蓝素电子转移反应的溶液研究结果相符，该研究表明静电相互作用对对接起主要作用，但与利用细胞色素f碱性斑块被中和的突变体进行的细胞色素f体内氧化速率研究结果不符。这种明显的矛盾可能是由细胞色素f未知的电子受体蛋白所解释。然而，(i)在缺乏质体蓝素的突变体中不存在闪光诱导的细胞色素f氧化。(ii)大结构域中的Lys58、65和66以及小结构域中的Lys188和189是溶液中电子转移反应离子强度依赖性的主要贡献者。用中性残基取代Lys58和65，并用酸性残基Glu66取代Lys66，导致溶液中电子转移速率下降超过10倍，并完全丧失其对离子强度的依赖性。在细胞色素f的小结构域中取代Lys188和Lys189导致二级速率常数下降3 - 4倍，电子转移总速率对离子强度的依赖性减小，相当于失去两个正电荷。(iii)类囊体腔的酸化不能解释静电相互作用的缺失。(iv)将五个赖氨酸变为酸性残基并未导致细胞色素f体内氧化速率有任何显著减慢。如果细胞色素f和质体蓝素在体内的对接是由细胞色素f的碱性残基介导的，那么它们与在体外介导电子转移的残基或对接静电相互作用模拟所涉及的残基不同。或者，细胞色素f/质体蓝素在体内的对接受到空间限制或质体蓝素从P700的释放的限制，这排除了特定静电相互作用对细胞色素f/质体蓝素反应的限速介导作用。莱茵衣藻中的细胞色素f/质体蓝素系统是第一个在体外和体内都研究了静电在介导对接反应中作用的电子转移偶联。
