Abstract
[NiFe] hydrogenases catalyze the reversible conversion of H2 into protons and electrons. The reaction takes place at the active site, which is composed of a nickel and an iron atom and three diatomic ligands, two cyanides and one carbon monoxide, bound to the iron. The NiFe(CN-)2CO cofactor is synthesized by an intricate posttranslational maturation process, which is mediated by a set of six conserved Hyp proteins. Depending on the cellular location and the physiological function, additional auxiliary proteins are involved in hydrogenase biosynthesis. Here we present evidence that the auxiliary proteins HoxL and HoxV assist in assembly of the Fe(CN-)2CO moiety. This unit was identified as a cofactor intermediate of the oxygen-tolerant membrane-bound [NiFe] hydrogenase (MBH) in the beta-proteobacterium Ralstonia eutropha H16. Both HoxL and HoxV proved to be essential for H2-oxidizing activity and MBH-driven growth on H2. Copurification studies revealed that HoxL and HoxV directly interact with the hydrogenase apoprotein. HoxV forms complexes with HoxL and HypC, a HoxL paralogue that is essential for cofactor assembly. These observations suggest that HoxL acts as a specific chaperone assisting the transfer of the Fe(CN-)2CO cofactor intermediate from the Hyp machinery to the MBH. This shuttle also involves the scaffold protein HoxV. Indeed, infrared spectroscopy and metal analysis identified for the first time a non-redox-active Fe(CN-)2CO intermediate coordinated to HoxV.
Highlights
Sfb498 and the Cluster of Excellence “Unifying Concepts in Catalysis” and the Fonds der Chemischen Industrie
Synthesis of hydrogenases is one of the most intriguing processes of metalloprotein biochemistry, requiring in addition to a complex biosynthetic apparatus for Fe-S cluster assembly (reviewed by Johnson et al [6] and Fontecave et al [52]) sets of components designed for assembly of the dinuclear metal centers
Recent studies have documented that these served cysteine residues as candidates for coordination of a gene products are essential for acquiring catalytic competency Fe(CNϪ)2CO moiety (Fig. 5)
Summary
Strains and Plasmids—Bacterial strains, plasmids, and primers used in this study are listed in Tables 1 and 2, respectively. The StrepTag II sequence was fused to the 3Ј-end of hoxG by PCR with primers CtermStrep-hoxG_RsrII-rev and CtermStrep-hoxG_RsrII-fwd with pCH1235 as template, which is a religated 4.20-kbp BsiWI fragment of pCH1229 (carrying parts of hoxG and hoxZ), resulting in a 4.24-kbp fragment. Using primers CtermStrep-hoxL_NheI-fwd and CtermStrep-hoxL_NheI-rev, the StrepTag II sequence was fused to the 3Ј-end of hoxL by inverse PCR with pCH1136 as template, a pBluescriptII KSϩ derivative harboring a 2-kbp Ecl136II-MfeI fragment of pCH462 in Ecl136II-EcoRI sites. The resulting PCR product was cut with NheI and religated, resulting in pCH1137, from which a 1.4-kbp XhoI fragment was transferred to SalI-digested pLO2, yielding pCH1138. PCH1138 was transferred to Re HF632 by conjugation, and the mutation was introduced by allele exchange [37], resulting in strain HF656.
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