Hanley J, Sétif P, Bottin H, Lagoutte B
Département de Biologie Cellulaire et Moléculaire, CEA-Service de Bioénergétique/CNRS-URA 1290, Gif sur Yvette, France.
Biochemistry. 1996 Jul 2;35(26):8563-71. doi: 10.1021/bi960399x.
The psaD gene isolated from the cyanobacterium Synechocystis sp. PCC 6803 has been mutated in the region encoding a cross-linking site for ferredoxin. A glucose tolerant strain of Synechocystis 6803 was first deleted for psaD, and the resulting PS-I was characterised by EPR and flash absorption spectroscopy. The major modification related to the absence of the PsaD subunit is the disappearance of the first order reduction of ferredoxin which is replaced by a second order reaction. Reconstitution of the deleted PS-I with the purified PsaD polypeptide restored 80% of the fast photoreduction of ferredoxin. The deletion of PsaD has no apparent effect on the main biochemical features of the resulting depleted PS-I complex, with the exception of minor modifications to the FA/FB centers. The deleted strain was transformed by a series of psaD genes mutated at three conserved residues, all located close to the ferredoxin cross-linking site. The resulting photosystem I complexes were extensively studied by flash absorption spectroscopy. Unexpectedly, the change of Lys 106 involved in the cross-linking of ferredoxin for an uncharged amino acid has almost no effect (mutation K106A). However, the functional consequences of more drastic substitutions of either Lys 106 or Arg 111 indicate a role for these two basic amino acids in the binding and submicrosecond reduction of ferredoxin. Various mutations of the unique His at position 97 show that this amino acid is involved in the increased affinity of PS-I for ferredoxin when the pH is lowered. This histidine could be central in regulating in vivo the rate of ferredoxin reduction as a precise sensor of the local proton concentration.
从蓝藻集胞藻PCC 6803中分离出的psaD基因，在编码铁氧化还原蛋白交联位点的区域发生了突变。首先对集胞藻6803的一个葡萄糖耐受菌株进行psaD缺失，然后通过电子顺磁共振（EPR）和闪光吸收光谱对产生的光系统I进行表征。与PsaD亚基缺失相关的主要变化是铁氧化还原蛋白一级还原的消失，取而代之的是二级反应。用纯化的PsaD多肽对缺失的光系统I进行重组，恢复了80%的铁氧化还原蛋白快速光还原。除了对FA/FB中心有轻微修饰外，PsaD的缺失对产生的耗尽型光系统I复合物的主要生化特性没有明显影响。缺失菌株被一系列在三个保守残基处发生突变的psaD基因转化，所有这些残基都位于靠近铁氧化还原蛋白交联位点的位置。通过闪光吸收光谱对产生的光系统I复合物进行了广泛研究。出乎意料的是，参与铁氧化还原蛋白交联的赖氨酸106突变为不带电荷的氨基酸几乎没有影响（K106A突变）。然而，赖氨酸106或精氨酸111更剧烈取代的功能后果表明，这两个碱性氨基酸在铁氧化还原蛋白的结合和亚微秒级还原中起作用。位于97位的独特组氨酸的各种突变表明，当pH降低时，该氨基酸参与了光系统I对铁氧化还原蛋白亲和力的增加。这个组氨酸可能作为局部质子浓度的精确传感器，在体内调节铁氧化还原蛋白还原速率方面起核心作用。
