title: Bis-methionine ligation to heme iron in mutants of cytochrome b562. 1. Spectroscopic and electrochemical characterization of the electronic properties.
abstract: We have generated mutants of cytochrome b562 in which the histidine ligand to the heme iron (His102) has been replaced by a methionine. The resulting proteins can have bis-methionine coordination to the heme iron, but the stability of this arrangement is dependent on oxidation state and solution pH. We have used optical, MCD, and EPR spectroscopies to study the nature of the heme coordination environment under a variety of conditions. Optical spectra of the reduced state of the single variant, H102M, are consistent with bis-methionine ligation. In its oxidized state, this protein is high-spin under all conditions studied, and the spectroscopic properties are consistent with only one of the methionine ligands being coordinated. We cannot identify what, if anything, provides the other axial ligand. A double variant, R98C/H102M (in which the heme is covalently attached to the protein through a c-type thioether linkage), is also bis-methionine coordinated in the ferrous state, but has significantly different properties in the oxidized state. With a pKa of 7.1 at 20 degrees C, the protein converts from a low-spin, 6-coordinate heme protein at low pH, to a high-spin species, similar to the high-spin species observed for the single variant. Our spectroscopic data prove that the low-spin species is bis-methionine coordinated. The reduction potential of this bis-methionine species has been measured using direct electrochemical techniques and is +440 mV at pH 4.8. The electrochemistry of these proteins is complicated by coupled coordination-state changes. Proof that the ferrous state is bis-methionine coordinated is provided by NMR results presented in the following paper.
authors.affiliation: Centre for Protein Engineering, MRC Centre, Cambridge, U.K. pxb@mrc-lmb.cam.ac.uk
authors.forename: P D
authors.initials: PD
authors.lastname: Barker
authors.forename: E P
authors.initials: EP
authors.lastname: Nerou
authors.forename: M R
authors.initials: MR
authors.lastname: Cheesman
authors.forename: A J
authors.initials: AJ
authors.lastname: Thomson
authors.forename: P
authors.initials: P
authors.lastname: de Oliveira
authors.forename: H A
authors.initials: HA
authors.lastname: Hill
mesh_terms: D001426:Bacterial Proteins; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins; D005293:Ferritins / Q000737:chemistry; D006418:Heme / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D008715:Methionine / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D001426:Bacterial Proteins; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D008024:Ligands; D011994:Recombinant Proteins; D006418:Heme; D005293:Ferritins; C018032:bacterioferritin; C017850:cytochrome b562, E coli; D008715:Methionine; D007501:Iron