A thermostable hybrid cluster protein from Pyrococcus furiosus: effects of the loss of a three helix bundle subdomain.
Pyrococcus furiosus hybrid cluster protein (HCP) was expressed in Escherichia coli, purified, and characterized. This is the first archaeal and thermostable HCP to be isolated. Compared with the protein sequences of previously characterized HCPs from mesophiles, the protein sequence of P. furiosus HCP exhibits a deletion of approximately 13 kDa as a single amino acid stretch just after the N-terminal cysteine motif, characteristic for class-III HCPs from (hyper)thermophilic archaea and bacteria. The protein was expressed as a thermostable, soluble homodimeric protein. Hydroxylamine reductase activity of P. furiosus HCP showed a K(m) value of 0.40 mM and a k(cat) value of 3.8 s(-1) at 70 degrees C and pH 9.0. Electron paramagnetic resonance spectroscopy showed evidence for the presence of a spin-admixed, S = 3/2 [4Fe-4S](+) cubane cluster and of the hybrid cluster. The cubane cluster of P. furiosus HCP is presumably coordinated by a CXXC-X(7)-C-X(5)-C motif close to the N-terminus, which is similar to the CXXC-X(8)-C-X(5)-C motif of the Desulfovibrio desulfuricans and Desulfovibrio vulgaris HCPs. Amino acid sequence alignment and homology modeling of P. furiosus HCP reveal that the deletion results in a loss of one of the two three-helix bundles of domain 1. Clearly the loss of one of the three-helix bundles of domain 1 does not diminish the hydroxylamine reduction activity and the incorporation of the iron-sulfur clusters.
Overeijnder
Marieke L
ML
Department of Biotechnology, Delft University of Technology, BC, Delft, The Netherlands.
Hagen
Wilfred R
WR
Hagedoorn
Peter-Leon
PL
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019766:Genes, Archaeal; D019811:Hydroxylamine / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D019811:Hydroxylamine; D013455:Sulfur; D007501:Iron