Mühlenhoff U, Sétif P
Institut für Biologie II, Universität Freiburg, FRG.
Biochemistry. 1996 Feb 6;35(5):1367-74. doi: 10.1021/bi952381c.
The photoreduction of flavodoxin by trimeric photosystem I, both from the cyanobacterium Synechococcus sp. PCC 7002, was investigated by flash absorption spectroscopy. After addition of flavodoxin in darkness, single flash experiments show that the transient signals change between individual flashes. This behavior is assigned to a progressive accumulation of flavodoxin semiquinone, which is relatively stable under most experimental conditions. Different conditions were devised in order to study the reduction of the oxidized and semiquinone forms of flavodoxin separately. Both processes were identified by their differential spectra measured between 460 and 630 nm. Detailed kinetic characteristics of flavodoxin reduction were obtained at pH 8.0 in the presence of salts. The kinetics of reduction of oxidized flavodoxin displays a single-exponential component. The rate of this component increases with the flavodoxin concentration up to an asymptotic value of about 600 s-1. The semiquinone form of flavodoxin being protonated, this rate corresponds to a rate-limiting reaction which could be either an electron transfer reaction or a protonation reaction. In contrast, the reduction of flavodoxin semiquinone is biphasic. A fast first-order phase with t 1/2 approximately 10 microseconds is interpreted as an electron transfer process within a preformed complex. A dissociation constant of 2.64 microM is calculated for this complex by assuming a simple binding equilibrium between photosystem I and flavodoxin semiquinone. The slower phase observed for semiquinone reduction is concentration dependent, and a second-order rate constant of 1.7 x 10(8) M-1 s-1 is calculated. For both one-electron reduction steps, different optimal salt concentrations are observed indicating slightly different interactions between photosystem I and flavodoxin in its oxidized and semiquinone states.
利用闪光吸收光谱法研究了来自蓝藻聚球藻属PCC 7002的三聚体光系统I对黄素氧还蛋白的光还原作用。在黑暗中加入黄素氧还蛋白后，单次闪光实验表明，各次闪光之间的瞬态信号会发生变化。这种行为归因于黄素氧还蛋白半醌的逐步积累，在大多数实验条件下，它相对稳定。为了分别研究黄素氧还蛋白氧化形式和半醌形式的还原作用，设计了不同的条件。这两个过程都通过在460至630纳米之间测量的差示光谱来识别。在pH 8.0且有盐存在的情况下，获得了黄素氧还蛋白还原的详细动力学特征。氧化型黄素氧还蛋白的还原动力学呈现单指数成分。该成分的速率随黄素氧还蛋白浓度增加，直至达到约600 s-1的渐近值。由于黄素氧还蛋白的半醌形式被质子化，该速率对应于一个限速反应，它可能是电子转移反应或质子化反应。相比之下，黄素氧还蛋白半醌的还原是双相的。半衰期约为10微秒的快速一级相被解释为在预先形成的复合物内的电子转移过程。通过假设光系统I与黄素氧还蛋白半醌之间存在简单的结合平衡，计算出该复合物的解离常数为2.64 microM。观察到的半醌还原较慢的相依赖于浓度，计算出二级速率常数为1.7×108 M-1 s-1。对于两个单电子还原步骤，观察到不同的最佳盐浓度，这表明光系统I与处于氧化态和半醌态的黄素氧还蛋白之间的相互作用略有不同。
