Enzymatic activities of the purified tetrameric and hexameric soluble [NiFe]-hydrogenase (SH)

Range Table - link
Organism Bacteria Ralstonia eutropha H16
Reference Burgdorf T et al., The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH. J Bacteriol. 2005 May187(9):3122-32. DOI: 10.1128/JB.187.9.3122-3132.2005 p.3127 table 4PubMed ID15838039
Method Abstract: "[Investigators] have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors."
Comments P.3126 right column bottom paragraph: "Enzymatic and infrared-spectroscopic characterization of the hexameric SH. The tetrameric SH consists of two cooperating enzymatic modules (refs 58, 41). Aside from the overall reaction, the H2-dependent reduction of NAD+, several module-specific enzymatic activities can be measured, e.g., the H2-dependent reduction of BV [benzyl viologen] or K3Fe(CN)6 and the oxidation of NAD(P)H with K3Fe(CN)6 as electron acceptor. Table 4 gives an overview of these activities for both the tetrameric and the hexameric SH. All assays were carried out in 50 mM Tris-HCl buffer (pH 8) since SH activities are maximal under these conditions (ref 58). The SH-HoxI complex dissociated after prolonged incubation at pH 8 but remained stable during the activity measurements. To compensate for the difference in molecular mass, turnover numbers were calculated as well. Since the specific activities of routine preparations can vary over a relatively wide range, the differences in turnover number observed for the two forms of the SH are not really significant. Remarkably, the hexameric SH showed a low but clearly detectable NADPH-K3Fe(CN)6 activity (0.6 U/mg), whereas the tetrameric form did not. Hydrogen oxidation with NADP+ as electron acceptor was absent in both preparations. Moreover, no H2 production from NADPH could be detected whereas both forms coupled the oxidation of NADH to proton reduction (data not shown)."
Entered by Uri M
ID 112820