Structural And Biological Analysis of the Metal Sites of Escherichia Coli Hydrogenase Accessory Protein

The [NiFe]-hydrogenase protein produced by many types of bacteria contains a dinuclear metal center that is required for enzymatic activity. Assembly of this metal cluster involves the coordinated activity of a number of helper proteins including the accessory protein, HypB, which is necessary for N...

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Bibliographic Details
Published in:Biochemistry (Easton) 2009-05, Vol.47 (46)
Main Authors: Dias, A.V., Mulvihill, C.M., Leach, M.R., Pickering, I.J., George, G.N., Zamble, D.B.
Format: Article
Language:English
Subjects:
ABSORPTION SPECTROSCOPY
BACTERIA
BASIC BIOLOGICAL SCIENCES
ENZYMES
ESCHERICHIA COLI
GEOMETRY
HYDROGENASES
MUTANTS
Other,OTHER
PRODUCTION
PROTEINS
RESIDUES
STRAINS
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Summary:The [NiFe]-hydrogenase protein produced by many types of bacteria contains a dinuclear metal center that is required for enzymatic activity. Assembly of this metal cluster involves the coordinated activity of a number of helper proteins including the accessory protein, HypB, which is necessary for Ni(II) incorporation into the hydrogenase proteins. The HypB protein from Escherichia coli has two metal-binding sites, a high-affinity Ni(II) site that includes ligands from the N-terminal domain and a low-affinity metal site located within the C-terminal GTPase domain. In order to determine the physiological relevance of the two separate sites, hydrogenase production was assessed in strains of E. coli expressing wild-type HypB, the isolated GTPase domain, or site-directed mutants of metal-binding residues. These experiments demonstrate that both metal sites of HypB are critical for the maturation of the hydrogenase enzymes in E. coli. X-ray absorption spectroscopy of purified proteins was used to examine the detailed coordination spheres of each nickel-loaded site. In addition, because the low-affinity metal site has a stronger preference for Zn(II) than Ni(II), the ligands and geometry for this metal were also resolved. The results from these experiments are discussed in the context of a mechanism for Ni(II) insertion into the hydrogenase protein.
ISSN:0006-2960
1520-4995