Measurement of photosystem I activity with photoreduction of recombinant flavodoxin.
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.
Zhao
J
J
Department of Plant Molecular Biology, College of Life Sciences, Peking University, Beijing, China. jzhao@pku.edu.cn
Li
R
R
Bryant
D A
DA
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D005418:Flavodoxin / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D010084:Oxidation-Reduction / Q000187:drug effects; D010788:Photosynthesis / Q000187:drug effects; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism*; D045331:Photosystem I Protein Complex; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D005418:Flavodoxin; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D011994:Recombinant Proteins; C079741:photosystem I, psaE subunit; D007544:Isopropyl Thiogalactoside; D014508:Urea; D008274:Magnesium