Ortega J M, Dohse B, Oesterhelt D, Mathis P
Section de Bioénergétique, DBCM (CNRS, URA 2096), CEA-Saclay, Gif-sur-Yvette, France.
Biophys J. 1998 Mar;74(3):1135-48. doi: 10.1016/S0006-3495(98)77831-2.
Electron transfer from the tetraheme cytochrome c to the special pair of bacteriochlorophylls (P) has been studied by flash absorption spectroscopy in reaction centers isolated from seven strains of the photosynthetic purple bacterium Rhodopseudomonas viridis, where the residue L162, located between the proximal heme c-559 and P, is Y (wild type), F, W, G, M, T, or L. Measurements were performed between 294 K and 8 K, under redox conditions in which the two high-potential hemes of the cytochrome were chemically reduced. At room temperature, the kinetics of P+ reduction include two phases in all of the strains: a dominant very fast phase (VF), and a minor fast phase (F). The VF phase has the following t(1/2): 90 ns (M), 130 ns (W), 135 ns (F), 189 ns (Y; wild type), 200 ns (G), 390 ns (L), and 430 ns (T). These data show that electron transfer is fast whatever the nature of the amino acid at position L162. The amplitudes of both phases decrease suddenly around 200 K in Y, F, and W. The effect of temperature on the extent of fast phases is different in mutants G, M, L, and T, in which electron transfer from c-559 to P+ takes place at cryogenic temperatures in a substantial fraction of the reaction centers (T, 48%; G, 38%; L, 23%, at 40 K; and M, 28%, at 60 K), producing a stable charge separated state. In these nonaromatic mutants the rate of VF electron transfer from cytochrome to P+ is nearly temperature-independent between 294 K and 8 K, remaining very fast at very low temperatures (123 ns at 60 K for M; 251 ns at 40 K for L; 190 ns at 8 K for G, and 458 ns at 8 K for T). In all cases, a decrease in amplitudes of the fast phases is paralleled by an increase in very slow reduction of P+, presumably by back-reaction with Q(A)-. The significance of these results is discussed in relation to electron transfer theories and to freezing at low temperatures of cytochrome structural reorganization.
通过闪光吸收光谱法，在从光合紫色细菌绿假单胞菌的七个菌株中分离出的反应中心里，研究了从四血红素细胞色素c到特殊对细菌叶绿素（P）的电子转移，其中位于近端血红素c-559和P之间的残基L162为酪氨酸（野生型）、苯丙氨酸、色氨酸、甘氨酸、甲硫氨酸、苏氨酸或亮氨酸。测量在294K至8K之间进行，处于氧化还原条件下，其中细胞色素的两个高电位血红素被化学还原。在室温下，所有菌株中P+还原的动力学包括两个阶段：一个占主导的非常快速的阶段（VF）和一个次要的快速阶段（F）。VF阶段的半衰期如下：90纳秒（甲硫氨酸）、130纳秒（色氨酸）、135纳秒（苯丙氨酸）、189纳秒（酪氨酸；野生型）、200纳秒（甘氨酸）、390纳秒（亮氨酸）和430纳秒（苏氨酸）。这些数据表明，无论L162位氨基酸的性质如何，电子转移都很快。在酪氨酸、苯丙氨酸和色氨酸中，两个阶段的幅度在200K左右突然下降。温度对快速阶段程度的影响在突变体甘氨酸（G）、甲硫氨酸（M）、亮氨酸（L）和苏氨酸（T）中有所不同，在这些突变体中，从c-559到P+的电子转移在低温下在相当一部分反应中心发生（苏氨酸，48%；甘氨酸，38%；亮氨酸，23%，在40K时；甲硫氨酸，28%，在60K时），产生稳定的电荷分离态。在这些非芳香族突变体中，从细胞色素到P+的VF电子转移速率在294K至8K之间几乎与温度无关，在非常低的温度下仍然非常快（甲硫氨酸在60K时为123纳秒；亮氨酸在40K时为251纳秒；甘氨酸在8K时为190纳秒，苏氨酸在8K时为458纳秒）。在所有情况下，快速阶段幅度的降低与P+非常缓慢还原的增加同时出现，推测是由于与Q(A)-的反向反应。结合电子转移理论以及细胞色素结构重组在低温下的冻结情况，讨论了这些结果的意义。
