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• W2032776586 endingPage "2651" @default.
• W2032776586 startingPage "2641" @default.
• W2032776586 abstract "ABSTRACT The development of cellular systems in which the enzyme hydrogenase is efficiently coupled to the oxygenic photosynthesis apparatus represents an attractive avenue to produce H 2 sustainably from light and water. Here we describe the molecular design of the individual components required for the direct coupling of the O 2 -tolerant membrane-bound hydrogenase (MBH) from Ralstonia eutropha H16 to the acceptor site of photosystem I (PS I) from Synechocystis sp. PCC 6803. By genetic engineering, the peripheral subunit PsaE of PS I was fused to the MBH, and the resulting hybrid protein was purified from R. eutropha to apparent homogeneity via two independent affinity chromatographical steps. The catalytically active MBH-PsaE (MBH PsaE ) hybrid protein could be isolated only from the cytoplasmic fraction. This was surprising, since the MBH is a substrate of the twin-arginine translocation system and was expected to reside in the periplasm. We conclude that the attachment of the additional PsaE domain to the small, electron-transferring subunit of the MBH completely abolished the export competence of the protein. Activity measurements revealed that the H 2 production capacity of the purified MBH PsaE fusion protein was very similar to that of wild-type MBH. In order to analyze the specific interaction of MBH PsaE with PS I, His-tagged PS I lacking the PsaE subunit was purified via Ni-nitrilotriacetic acid affinity and subsequent hydrophobic interaction chromatography. Formation of PS I-hydrogenase supercomplexes was demonstrated by blue native gel electrophoresis. The results indicate a vital prerequisite for the quantitative analysis of the MBH PsaE -PS I complex formation and its light-driven H 2 production capacity by means of spectroelectrochemistry." @default.
• W2032776586 created "2016-06-24" @default.
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• W2032776586 date "2010-04-15" @default.
• W2032776586 modified "2023-09-23" @default.
• W2032776586 title "Requirements for Construction of a Functional Hybrid Complex of Photosystem I and [NiFe]-Hydrogenase" @default.
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• W2032776586 doi "https://doi.org/10.1128/aem.02700-09" @default.
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