tailieunhanh - Báo cáo khoa học: Characterization of a prokaryotic haemerythrin from the methanotrophic bacterium Methylococcus capsulatus (Bath)

For a long time, the haemerythrin family of proteins was considered to be restricted to only a few phyla of marine invertebrates. When analysing dif-ferential protein expression in the methane-oxidizing bacterium, Methylo-coccus capsulatus(Bath), grown at a high and low copper-to-biomass ratio, respectively, we identified a putative prokaryotic haemerythrin expressed in high-copper cultures. | iFEBS Journal Characterization of a prokaryotic haemerythrin from the methanotrophic bacterium Methylococcus capsulatus Bath Odd A. Karlsen Linda Ramsevik Live J. Bruseth 0ivind Larsen Annette Brenner Frode S. Berven Harald B. Jensen and Johan R. Lillehaug Department of Molecular Biology University of Bergen Norway Keywords copper regulated methanotroph Methylococcus capsulatus prokaryotic haemerythrin two-dimensionalgel electrophoresis Correspondence O. A. Karlsen Department of Molecular Biology University of Bergen HIB Thormohlensgt. 55 5020 Bergen Norway. Fax 47 555 89683 Tel 47 555 84372 E-mail Present address Department of Biology University of Bergen Norway Received 6 December 2004 revised 25 February 2005 accepted 15 March 2005 doi For a long time the haemerythrin family of proteins was considered to be restricted to only a few phyla of marine invertebrates. When analysing differential protein expression in the methane-oxidizing bacterium Methylo-coccus capsulatus Bath grown at a high and low copper-to-biomass ratio respectively we identified a putative prokaryotic haemerythrin expressed in high-copper cultures. Haemerythrins are recognized by a conserved sequence motif that provides five histidines and two carboxylate ligands which coordinate two iron atoms. The diiron site is located in a hydrophobic pocket and is capable of binding O2. We cloned the M. capsulatus haemerythrin gene and expressed it in Escherichia coli as a fusion protein with NusA. The haemerythrin protein was purified to homogeneity cleaved from its fusion partner. Recombinant M. capsulatus haemerythrin McHr was found to fold into a stable protein. Sequence similarity analysis identified all the candidate residues involved in the binding of diiron His22 His58 Glu62 His77 His81 His117 Asp122 and the amino acids forming the hydrophobic pocket in which O2 may bind Ile25 Phe59 Trp113 Leu114 Ile118 . We were also able to model a .