title: Metal binding activity of the Escherichia coli hydrogenase maturation factor HypB.
abstract: The formation of the [NiFe] metallocenter of Escherichia coli hydrogenase 3 requires the participation of proteins encoded by the hydrogenase pleiotropy operon hypABCDEF. The insertion of Ni(II) into the precursor enzyme follows the incorporation of the iron center and is the function of HypA, a Zn(II)-binding protein, and HypB, a GTPase. The Ni(II) donor and the mechanism of transfer of Ni(II) into the hydrogenase precursor protein are not known. In this study, we demonstrate that HypB is a nickel-binding protein capable of binding 1 equiv of Ni(II) with a K(d) in the sub-picomolar range. In addition, HypB has a weaker metal-binding site that is not specific for Ni(II) over Zn(II). Examination of the isolated C-terminal GTPase domain revealed that the high-affinity metal binding capability was severely abrogated but the low-affinity site was intact. By mutating conserved cysteine and histidine residues in E. coli HypB, we have localized the high-affinity Ni(II)-binding site to an N-terminal CXXCGC motif and the low-affinity metal-binding site to the GTPase domain. A model for the function of HypB during the Ni(II) loading of hydrogenase is proposed.
authors.affiliation: Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Leach
authors.forename: Shaifali
authors.initials: S
authors.lastname: Sandal
authors.forename: Haowei
authors.initials: H
authors.lastname: Sun
authors.forename: Deborah B
authors.initials: DB
authors.lastname: Zamble
mesh_terms: D001051:Apoenzymes / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009532:Nickel / Q000378:metabolism*; D013057:Spectrum Analysis; D017186:Titrimetry; D015032:Zinc / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C490324:HypB protein, E coli; D009532:Nickel; D006864:Hydrogenase; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D015032:Zinc; D003545:Cysteine