title: Bis-methionine ligation to heme iron in mutants of cytochrome b562. 2. Characterization by NMR of heme-ligand interactions.
abstract: Previous work has shown that, in variants of cytochrome b562 containing the H102M mutation, methionine residues provide both axial ligands to the heme iron. NMR spectroscopic studies of such bis-methionine-coordinated cytochrome have not previously been feasible, since the only other cytochrome with such a ligand arrangement, bacterioferritin, is too large to be studied by current NMR methods. The present work provides the first NMR characterization of 6-coordinate, bis-methionine-ligated heme centers in both ferrous and ferric oxidation states. We have used one and two dimensional, homonuclear NMR spectroscopy to assign the proton resonances of the heme group and ligand side chains in the reduced, cytochrome b562 variants, H102M and covR98C/H102M. The latter protein has heme covalently attached to the protein, and our results prove that the covalent linkage is a c-type thioether bond formed between the cysteine at residue 98 and the heme 2-vinyl group. Spectra of the ferrous H102M variant are consistent with the presence of two species differing in the orientation of the heme in the protein. We have interpreted results from NOESY experiments on the ferrous covR98C/H102M protein in terms of the conformation of the two methionine side chains, and we present a model for the structure of the heme ligand arrangement. The Met7 side chain adopts an extended conformation almost identical to that observed in the wild type protein with R stereochemistry at the chiral sulfur ligand. The Met102 side chain has a different, buckled side chain conformation and has S stereochemistry at the chiral center. Our NMR derived model is consistent with the spectroscopic data presented in the previous paper. Studies on the ferric forms of these proteins confirm that the double variant at low pH has a "stable" bis-methionine ligation arrangement, but that it is a thermal mixture of species with differing spin states. No hyperfine coupled proton resonances can be identified in spectra of the high-spin forms of either of these proteins.
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: S M
authors.initials: SM
authors.lastname: Freund
mesh_terms: D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013696:Temperature
chemical_list: D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D008024:Ligands; D006418:Heme; C017850:cytochrome b562, E coli; D008715:Methionine; D007501:Iron