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 differential protein expression in the methane-oxidizing bacterium, Methylococcus 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 O(2). 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 O(2) may bind (Ile25, Phe59, Trp113, Leu114, Ile118). We were also able to model a three-dimensional structure of McHr maintaining the correct positioning of these residues. Furthermore, UV/vis spectrophotometric analysis demonstrated the presence of conjugated diiron atoms in McHr. A comprehensive genomic database search revealed 21 different prokaryotes containing the haemerythrin signature (PROSITE 00550), indicating that these putative haemerythrins may be a conserved prokaryotic subfamily.
Karlsen
Odd A
OA
Department of Molecular Biology, University of Bergen, Norway. Odd.Karlsen@mbi.uib.no
Ramsevik
Linda
L
Bruseth
Live J
LJ
Larsen
Øivind
Ø
Brenner
Annette
A
Berven
Frode S
FS
Jensen
Harald B
HB
Lillehaug
Johan R
JR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D006422:Hemerythrin / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000737:chemistry; D020619:Methylococcus capsulatus / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D001426:Bacterial Proteins; D006422:Hemerythrin; D008670:Metals; D011993:Recombinant Fusion Proteins