Xu Q, Jung Y S, Chitnis V P, Guikema J A, Golbeck J H, Chitnis P R
Division of Biology, Kansas State University, Manhattan 66506-4901.
J Biol Chem. 1994 Aug 26;269(34):21512-8.
The subunit requirements for NADP+ reduction by photosystem I were assessed in mutants of Synechocystis sp. PCC 6803 created by targeted inactivation of the psaD, psaE, psaF, and psaL genes. The PsaE-less, PsaF-PsaJ-less, and PsaL-less mutants showed normal photoautotrophic growth, while the growth of PsaD-less mutants was slower without glucose. In isolated wild-type membranes, the rate of flavodoxin reduction and flavodoxin-mediated NADP+ reduction were 800 and 480 mumol/mg of chlorophyll/h, respectively. The rate of ferredoxin-mediated NADP+ photoreduction was 460 mumol/mg of chlorophyll/h. There was no diminution in NADP+ photoreduction in membranes isolated from the PsaF-less and PsaL-less mutants. The rates of ferredoxin-mediated NADP+ photoreduction in membranes of the PsaE-less mutants were 25 mumol/mg of chlorophyll/h. However, the rate of flavodoxin reduction was 380 mumol/mg of chlorophyll/h, and that of flavodoxin-mediated NADP+ photoreduction was 170 mumol/mg of chlorophyll/h. PsaD-less membranes showed < 20% of the wild-type rates of flavodoxin-mediated NADP+ photoreduction, but were completely deficient in ferredoxin-mediated NADP+ photoreduction. Therefore, the roles of PsaE and PsaD are more crucial for "docking" of ferredoxin than of flavodoxin. Proteolysis studies showed that while PsaD was susceptible to rapid in vitro degradation by thermolysin, the number and sizes of protease-resistant fragments were not affected by the absence of PsaE. Protease accessibility studies further indicated that the C-terminal domain of PsaD is surface-exposed on the n-side. These results suggest that PsaE and the C-terminal domain of PsaD generate the docking site for the electron acceptors of photosystem I.
通过对集胞藻6803（Synechocystis sp. PCC 6803）中psaD、psaE、psaF和psaL基因进行靶向失活所构建的突变体，评估了光系统I还原NADP⁺所需的亚基。缺失PsaE、缺失PsaF - PsaJ和缺失PsaL的突变体表现出正常的光合自养生长，而缺失PsaD的突变体在没有葡萄糖的情况下生长较慢。在分离的野生型膜中，黄素氧还蛋白还原速率和黄素氧还蛋白介导的NADP⁺还原速率分别为800和480 μmol/（mg叶绿素·h）。铁氧还蛋白介导的NADP⁺光还原速率为460 μmol/（mg叶绿素·h）。从缺失PsaF和缺失PsaL的突变体中分离的膜中，NADP⁺光还原没有减少。缺失PsaE的突变体膜中铁氧还蛋白介导的NADP⁺光还原速率为25 μmol/（mg叶绿素·h）。然而，黄素氧还蛋白还原速率为380 μmol/（mg叶绿素·h），黄素氧还蛋白介导的NADP⁺光还原速率为170 μmol/（mg叶绿素·h）。缺失PsaD的膜中黄素氧还蛋白介导的NADP⁺光还原速率不到野生型的20%，但完全缺乏铁氧还蛋白介导的NADP⁺光还原。因此，PsaE和PsaD对铁氧还蛋白“对接”的作用比对黄素氧还蛋白更关键。蛋白酶解研究表明，虽然PsaD易受热溶素在体外快速降解，但抗蛋白酶片段的数量和大小不受PsaE缺失的影响。蛋白酶可及性研究进一步表明，PsaD的C末端结构域在n侧表面暴露。这些结果表明，PsaE和PsaD的C末端结构域为光系统I的电子受体产生了对接位点。
