HoxE—a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria

详细信息    查看全文

• 作者：Schmitz ; Oliver ; Boison ; Gudrun ; Salzmann ; Heike ; Bothe ; Hermann ; Schü ; tz ; Kathrin ; Wang ; Shu-hua ; et. al.
• 关键词：Cyanobacteria ; Bidirectional hydrogenase ; Hydrogen metabolism ; HoxE ; NADH dehydrogenase ; MV ; methyl viologen ; APPS ; advanced protein purification system
• 刊名：Biochimica et Biophysica Acta (BBA)/Bioenergetics
• 出版年：2002
• 出版时间：April 22, 2002
• 年：2002
• 卷：1554
• 期：1-2
• 页码：66-74
• 全文大小：279 K

文摘

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 _6;mol H₂ evolved (min mg protein)⁻¹ and 15 _6;mol H₂ evolved (min mg protein)⁻¹, respectively. H₂-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 °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.