A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit |
| |
Authors: | Winter Gordon Buhrke Thorsten Lenz Oliver Jones Anne Katherine Forgber Michael Friedrich Bärbel |
| |
Affiliation: | Institut für Biologie/Mikrobiologie, Humboldt-Universit?t zu Berlin, Chausseestr. 117, D-10115 Berlin, Germany. |
| |
Abstract: | The large subunit HoxC of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni-Fe site maturation proteins HypC and HypB. Despite the presence of the Ni-Fe active site in some of the proteins, both forms, which lack the Fe-S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni-Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly. |
| |
Keywords: | RH, H2-sensing regulatory hydrogenase SH, soluble hydrogenase MBH, membrane-bound hydrogenase AAS, atomic absorption spectroscopy TRXF, total reflection X-ray fluorescence PAGE, polyacrylamide gel electrophoresis TCEP, Tris(2-carboxyethyl)- phosphine LB, Luria broth FGN, fructose-glycerol minimal medium CV, column volume Ve, elution volume V0, void volume BSA, bovine serum albumine ADH, alcohol dehydrogenase |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|