Venturoli G, Drepper F, Williams J C, Allen J P, Lin X, Mathis P
Dipartimento di Biologia, Università di Bologna, Italy.
Biophys J. 1998 Jun;74(6):3226-40. doi: 10.1016/s0006-3495(98)78029-4.
The kinetics of electron transfer from cytochrome c2 to the primary donor (P) of the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides have been investigated by time-resolved absorption spectroscopy. Rereduction of P+ induced by a laser pulse has been measured at temperatures from 300 K to 220 K in a series of specifically mutated reaction centers characterized by altered midpoint redox potentials of P+/P varying from 410 mV to 765 mV (as compared to 505 mV for wild type). Rate constants for first-order electron donation within preformed reaction center-cytochrome c2 complexes and for the bimolecular oxidation of free cytochrome c2 have been obtained by multiexponential deconvolution of the kinetics. At all temperatures the rate of the fastest intracomplex electron transfer increases by more than two orders of magnitude as the driving force -deltaGo is varied over a range of 350 meV. The temperature and deltaGo dependences of the rate constant fit the Marcus equation well. Global analysis yields a reorganization energy lambda = 0.96 +/- 0.07 eV and a set of electronic matrix elements, specific for each mutant, ranging from 1.2 10(-4) eV to 2.5 10(-4) eV. Analysis in terms of the Jortner equation indicates that the best fit is obtained in the classical limit and restricts the range of coupled vibrational modes to frequencies lower than approximately 200 cm(-1). An additional slower kinetic component of P+ reduction, attributed to electron transfer from cyt c2 docked in a nonoptimal configuration of the complex, displays a Marcus type dependence of the rate constant upon deltaGo, characterized by a similar value of lambda (0.8 +/- 0.1 eV) and by an average electronic matrix element smaller by more than one order of magnitude. In all of the mutants, as the temperature is decreased below 260 K, both intracomplex reactions are abruptly inhibited, their rate being negligible at 220 K. The free energy dependence of the second-order rate constant for oxidation of cyt c2 in solution suggests that the collisional reaction is partially diffusion controlled, reaching the diffusion limit at exothermicities between 150 and 250 meV over the temperature range investigated.
通过时间分辨吸收光谱研究了光合紫色细菌球形红杆菌反应中心从细胞色素c2到初级供体（P）的电子转移动力学。在一系列经过特定突变的反应中心中，测量了激光脉冲诱导的P+再还原，这些反应中心的特征是P+/P的中点氧化还原电位从410 mV到765 mV不等（野生型为505 mV）。通过动力学的多指数去卷积获得了预形成的反应中心 - 细胞色素c2复合物内一级电子供体的速率常数以及游离细胞色素c2的双分子氧化速率常数。在所有温度下，随着驱动力 -ΔGo在350 meV范围内变化，最快的复合物内电子转移速率增加了两个以上数量级。速率常数的温度和ΔGo依赖性与Marcus方程拟合良好。全局分析得出重组能λ = 0.96 ± 0.07 eV以及一组特定于每个突变体的电子矩阵元，范围从1.2×10⁻⁴ eV到2.5×10⁻⁴ eV。根据Jortner方程分析表明，在经典极限下获得了最佳拟合，并将耦合振动模式的范围限制在低于约200 cm⁻¹的频率。P+还原的另一个较慢的动力学成分，归因于从以复合物非最佳构型对接的细胞色素c2进行的电子转移，显示出速率常数对ΔGo的Marcus型依赖性，其特征是λ值相似（0.8 ± 0.1 eV）且平均电子矩阵元小一个以上数量级。在所有突变体中，当温度降至260 K以下时，两个复合物内反应均突然受到抑制，在220 K时其速率可忽略不计。溶液中细胞色素c2氧化的二级速率常数的自由能依赖性表明，碰撞反应部分受扩散控制，在所研究的温度范围内，在放热150至250 meV之间达到扩散极限。
