title: HoxE--a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria.
abstract: NAD(P)(+)-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 micromol H(2) evolved (min mg protein)(-1) and 15 micromol H(2) evolved (min mg protein)(-1), respectively. H(2)-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 degrees C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)(2).
authors.affiliation: Botanisches Institut der Universität Köln, Gyrhofstr. 15, D-50931 Cologne, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schmitz
authors.forename: Gudrun
authors.initials: G
authors.lastname: Boison
authors.forename: Heike
authors.initials: H
authors.lastname: Salzmann
authors.forename: Hermann
authors.initials: H
authors.lastname: Bothe
authors.forename: Kathrin
authors.initials: K
authors.lastname: Schütz
authors.forename: Shu-hua
authors.initials: SH
authors.lastname: Wang
authors.forename: Thomas
authors.initials: T
authors.lastname: Happe
mesh_terms: D000595:Amino Acid Sequence; D000458:Cyanobacteria / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits
chemical_list: D021122:Protein Subunits; D009245:NADH Dehydrogenase