Chitnis V P, Jungs Y S, Albee L, Golbeck J H, Chitnis P R
Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901,USA.
J Biol Chem. 1996 May 17;271(20):11772-80. doi: 10.1074/jbc.271.20.11772.
The ADC4 mutant of the cyanobacterium Synechocystis sp. PCC 6803 was studied to determine the structural and functional consequences of the absence of PsaD in photosystem I. Isolated ADC4 membranes were shown to be deficient in ferredoxin-mediated NADP(+) reduction, even though charge separation between P700 and FA/FB occurred with high efficiency. Unlike the wild type, FB became preferentially photoreduced when ADC4 membranes were illuminated at 15 K, and the EPR line shapes were relatively broad. Membrane fragments oriented in two dimensions on thin mylar films showed that the g tensor axes of FA- and FB- were identical in the ADC4 and wild type strains, implying that PsaC is oriented similarly on the reaction center. PsaC and the FA/FB iron-sulfur clusters are lost more readily from the ADC4 membranes after treatment with Triton X-100 or chaotropic agents, implying a stabilizing role for PsaD. The specific role of Lys106 of PsaD, which can be crosslinked to Glu93 of ferredoxin (Lelong et al. (1994) J. Biol. Chem. 269, 10034-10039), was probed by site-directed mutagenesis. Chemical cross-linking and protease treatment experiments did not reveal any drastic alterations in the conformation of the mutant PsaD proteins. The EPR spectra of FA and FB in membranes of the Lys106 mutants were similar to those of the wild type. Membranes of all Lys106 mutants showed wild type rates of flavodoxin reduction and flavodoxin-mediated NADP+ reduction, but had 10-54% decrease in the ferredoxin-mediated NADP+ reduction rates. This implies that Lys106 is a dispensable component of the docking site on the reducing side of photosystem I and an ionic interaction between Lys106 of PsaD and Glu93 of ferredoxin is not essential for electron transfer to ferredoxin. These results demonstrate that PsaD serves distinct roles in modulating the EPR spectral characteristics of FA and FB, in stabilizing PsaC on the reaction center, and in facilitating ferredoxin-mediated NADP+ photoreduction on the reducing side of photosystem I.
对蓝藻集胞藻6803的ADC4突变体进行了研究，以确定光系统I中缺失PsaD的结构和功能后果。结果表明，分离出的ADC4膜在铁氧化还原蛋白介导的NADP(+)还原方面存在缺陷，尽管P700与FA/FB之间的电荷分离高效发生。与野生型不同，当在15K下照射ADC4膜时，FB优先发生光还原，且电子顺磁共振（EPR）谱线形状相对较宽。在聚酯薄膜上二维取向的膜片段显示，FA-和FB-的g张量轴在ADC4和野生型菌株中是相同的，这意味着PsaC在反应中心的取向相似。在用Triton X-100或离液剂处理后，PsaC和FA/FB铁硫簇更容易从ADC4膜上丢失，这意味着PsaD具有稳定作用。通过定点诱变探究了PsaD的Lys106的特定作用，该位点可与铁氧化还原蛋白的Glu93交联（勒隆等人，(1994)《生物化学杂志》269, 10034 - 10039）。化学交联和蛋白酶处理实验未揭示突变型PsaD蛋白构象有任何剧烈变化。Lys106突变体膜中FA和FB的EPR谱与野生型相似。所有Lys106突变体的膜显示出野生型的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，但铁氧化还原蛋白介导的NADP+还原速率降低了10 - 54%。这意味着Lys106是光系统I还原侧对接位点的一个可有可无的组分，且PsaD的Lys106与铁氧化还原蛋白的Glu93之间的离子相互作用对于向铁氧化还原蛋白的电子转移并非必不可少。这些结果表明，PsaD在调节FA和FB的EPR光谱特征、在稳定反应中心的PsaC以及在促进光系统I还原侧铁氧化还原蛋白介导的NADP+光还原方面发挥着不同的作用。
