Zhao J, Li R, Bryant D A
Department of Plant Molecular Biology, College of Life Sciences, Peking University, Beijing, China.
Anal Biochem. 1998 Nov 15;264(2):263-70. doi: 10.1006/abio.1998.2845.
Flavodoxin can function as an alternative electron acceptor for photosystem I (PSI) in place of ferredoxin under iron-limiting conditions. The isiB gene, encoding the flavodoxin in Synechococcus sp. PCC 7002, was overexpressed in Escherichia coli. Under the conditions employed, most recombinant flavodoxin (rFlvd) was in soluble form with cofactor correctly inserted. The absorption spectrum of rFlvd was identical to that of the native flavodoxin of the cyanobacteria. Photoreduction of rFlvd by PSI particles and thylakoid membranes was determined directly by monitoring the absorption change at 467 nm. The optimal conditions for rFlvd photoreduction were determined. Compared to other methods currently employed to measure PSI activity such as oxygen uptake in the presence of methyl viologen and NADP+ photoreduction in the presence of ferredoxin and ferredoxin:NADP+ oxidoreductase, measurement of PSI activity with flavodoxin as an electron acceptor has several advantages. It measures the full-chain electron transfer chain of PSI since flavodoxin accepts electrons from FA/FB and it is much simpler than the method with NADP+ photoreduction. With this method, we found that the affinity of wild-type PSI for rFlvd was 35% higher than that of the PsaE-less PSI, showing that this method is sensitive to structural changes of PSI. Our results demonstrate that rFlvd photoreduction is an effective and simple method for PSI activity measurement.
在铁限制条件下，黄素氧还蛋白可替代铁氧还蛋白作为光系统I（PSI）的替代电子受体。编码聚球藻属PCC 7002中黄素氧还蛋白的isiB基因在大肠杆菌中过表达。在所采用的条件下，大多数重组黄素氧还蛋白（rFlvd）呈可溶形式，辅因子正确插入。rFlvd的吸收光谱与蓝细菌天然黄素氧还蛋白的吸收光谱相同。通过监测467nm处的吸收变化直接测定PSI颗粒和类囊体膜对rFlvd的光还原作用。确定了rFlvd光还原的最佳条件。与目前用于测量PSI活性的其他方法（如在甲基紫精存在下的氧气摄取以及在铁氧还蛋白和铁氧还蛋白：NADP+氧化还原酶存在下的NADP+光还原）相比，以黄素氧还蛋白作为电子受体测量PSI活性具有几个优点。由于黄素氧还蛋白从FA/FB接受电子，因此它测量的是PSI的全链电子传递链，并且比NADP+光还原方法简单得多。通过这种方法，我们发现野生型PSI对rFlvd的亲和力比缺失PsaE的PSI高35%，表明该方法对PSI的结构变化敏感。我们的结果表明，rFlvd光还原是一种有效且简单的PSI活性测量方法。
