Site-directed replacement of the coaxial heme ligands of bacterioferritin generates heme-free variants.
The bacterioferritin (BFR) of Escherichia coli is a heme-containing iron storage molecule. It is composed of 24 identical subunits, which form a roughly spherical protein shell surrounding a central iron storage cavity. Each of the 12 heme moieties of BFR possesses bis-methionine axial ligation, a heme coordination scheme so far only found in bacterioferritins. Members of the BFR family contain three partially conserved methionine residues (excluding the initiating methionine) and in this study each was substituted by leucine and/or histidine. The Met52 variants were devoid of heme, whereas the Met31 and Met86 variants possessed full heme complements and were spectroscopically indistinguishable from wild-type BFR. The heme-free Met52 variants appeared to be correctly assembled and were capable of accumulating iron both in vivo and in vitro. No major differences were observed in the overall rate of iron accumulation for BFR-M52H, BFR-M52L, and the wild-type protein. The iron contents of the Met52 variants, as isolated, were at least 4 times greater than for wild-type BFR. This study is consistent with the reported location of the BFR heme site at the 2-fold axis and shows that heme is unnecessary for BFR assembly and iron uptake.
Andrews
S C
SC
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, United Kingdom.
Le Brun
N E
NE
Barynin
V
V
Thomson
A J
AJ
Moore
G R
GR
Guest
J R
JR
Harrison
P M
PM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005293:Ferritins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D014644:Genetic Variation; D006418:Heme / Q000737:chemistry / Q000235:genetics*; D007501:Iron / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D017931:DNA Primers; D008024:Ligands; D006418:Heme; D005293:Ferritins; C018032:bacterioferritin; D007501:Iron