Requirements for construction of a functional hybrid complex of photosystem I and [NiFe]-hydrogenase.
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.
Schwarze
Alexander
A
Institut für Biologie, Humboldt-Universität zu Berlin, Chausseestr. 117, D-10115 Berlin, Germany.
Kopczak
Marta J
MJ
Rögner
Matthias
M
Lenz
Oliver
O
D002846:Chromatography, Affinity; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008027:Light; D045331:Photosystem I Protein Complex / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics; D014867:Water / Q000378:metabolism
D045331:Photosystem I Protein Complex; D011993:Recombinant Fusion Proteins; D014867:Water; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase