title: Concerted action of two novel auxiliary proteins in assembly of the active site in a membrane-bound [NiFe] hydrogenase.
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.
authors.affiliation: Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Ludwig
authors.forename: Torsten
authors.initials: T
authors.lastname: Schubert
authors.forename: Ingo
authors.initials: I
authors.lastname: Zebger
authors.forename: Nattawadee
authors.initials: N
authors.lastname: Wisitruangsakul
authors.forename: Miguel
authors.initials: M
authors.lastname: Saggu
authors.forename: Angelika
authors.initials: A
authors.lastname: Strack
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology*; D017124:Conserved Sequence; D006864:Hydrogenase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011485:Protein Binding; D043367:Ralstonia / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D002352:Carrier Proteins; C043296:nickel-iron hydrogenase; D006864:Hydrogenase