Yu J, Vassiliev I R, Jung Y S, Golbeck J H, McIntosh L
DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312, USA.
J Biol Chem. 1997 Mar 21;272(12):8032-9. doi: 10.1074/jbc.272.12.8032.
Two [4Fe-4S] clusters, FA and FB, function as terminal electron carriers in Photosystem I (PS I), a thylakoid membrane-bound protein-pigment complex. To probe the function of these two clusters in photosynthetic electron transport, site-directed mutants were created in the transformable cyanobacterium Synechocystis sp. PCC 6803. Cysteine ligands in positions 14 or 51 to FB and FA, respectively, were replaced with aspartate, serine, or alanine, and the effect on the genetic, physiological, and biochemical characteristics of PS I complexes from the mutant strains were studied. All mutant strains were unable to grow photoautotrophically, and compared with wild type, mixotrophic growth was inhibited under normal light intensity. The mutant cells supported lower rates of whole-chain photosynthetic electron transport. Thylakoids isolated from the aspartate and serine mutants have lower levels of PS I subunits PsaC, PsaD, and PsaE and lower rates of PS I-mediated substrate photoreduction compared with the wild type. The alanine and double aspartate mutants have no detectable levels PsaC, PsaD, and PsaE. Electron transfer rates, measured by cytochrome c6-mediated NADP+ photoreduction, were lower in purified PS I complexes from the aspartate and serine mutants. By measuring the P700(+) kinetics after a single turnover flash, a large percentage of the backreaction in the aspartate and serine mutants was found to be derived from A1 and FX, indicating an inefficiency at the FX --> FA/FB electron transfer step. The alanine and double aspartate mutants failed to show any backreaction from [FA/FB]-. These results indicate that the various mutations of the cysteine 14 and 51 ligands to FB and FA affect biogenesis and electron transfer differently depending on the type of substitution, and that the effects of mutations on biogenesis and function can be biochemically separated and analyzed.
两个[4Fe-4S]簇，即FA和FB，作为光系统I（PS I）中的末端电子载体发挥作用，PS I是一种类囊体膜结合的蛋白质色素复合物。为了探究这两个簇在光合电子传递中的功能，在可转化的蓝藻集胞藻PCC 6803中创建了定点突变体。分别将FB和FA的第14位和第51位的半胱氨酸配体替换为天冬氨酸、丝氨酸或丙氨酸，并研究了这些替换对突变菌株PS I复合物的遗传、生理和生化特性的影响。所有突变菌株均无法进行光合自养生长，与野生型相比，在正常光照强度下混合营养生长受到抑制。突变细胞支持的全链光合电子传递速率较低。与野生型相比，从天冬氨酸和丝氨酸突变体中分离出的类囊体中PS I亚基PsaC、PsaD和PsaE的水平较低，PS I介导的底物光还原速率也较低。丙氨酸和双天冬氨酸突变体中未检测到PsaC、PsaD和PsaE的水平。通过细胞色素c6介导的NADP+光还原测量的电子传递速率，在天冬氨酸和丝氨酸突变体的纯化PS I复合物中较低。通过测量单次周转闪光后的P700(+)动力学，发现天冬氨酸和丝氨酸突变体中很大一部分反向反应源自A1和FX，表明在FX --> FA/FB电子传递步骤效率低下。丙氨酸和双天冬氨酸突变体未显示出[FA/FB]-的任何反向反应。这些结果表明，FB和FA的第14位和第51位半胱氨酸配体的各种突变根据取代类型对生物合成和电子传递的影响不同，并且突变对生物合成和功能的影响可以通过生化方法分离和分析。
