title: Coordination of a histidine residue of the protein-component S to the cobalt atom in coenzyme B12-dependent glutamate mutase from Clostridium cochlearium.
abstract: Electron paramagnetic resonance (EPR) spectroscopy of glutamate mutase from Clostridium cochlearium was performed in order to test the idea, that a histidine residue of component S replaces the dimethylbenzimidazole ligand of the Co-atom during binding of coenzyme B12 to the enzyme. The shapes and the superhyperfine splitting of the gz-lines of the Co(II) EPR spectra were used as indicators of the interaction of the axial base nitrogen with the Co-atom. A mixture of completely 15N-labelled component S, unlabelled component E, coenzyme B12 and glutamate gave slightly sharper gz-lines than that with unlabelled component S. A more dramatic change was observed in the Co(II) spectrum of the inactivated enzyme containing tightly bound cob(II)alamin, in which unlabelled component S caused a threefold superhyperfine-splitting of the gz-line, whereas the 15N-labelled protein only caused a twofold splitting, as expected for a direct interaction of a nitrogen of the enzyme with the Co-atom. By using a sample of 15N-labelled component S, in which only the histidines were 14N-labelled, the EPR spectra showed no difference to those with unlabelled component S. The experiments indeed demonstrate a replacement of the dimethylbenzimidazole ligand in coenzyme B12 by a histidine when bound to glutamate mutase. The most likely candidate is H16, which is conserved among the carbon skeleton rearranging mutases and methionine synthase.
authors.affiliation: Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Marburg, Germany.
authors.forename: O
authors.initials: O
authors.lastname: Zelder
authors.forename: B
authors.initials: B
authors.lastname: Beatrix
authors.forename: F
authors.initials: F
authors.lastname: Kroll
authors.forename: W
authors.initials: W
authors.lastname: Buckel
mesh_terms: D000591:Amino Acid Isomerases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D001562:Benzimidazoles; D003013:Clostridium / Q000201:enzymology*; D003035:Cobalt / Q000737:chemistry*; D003038:Cobamides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D019751:Intramolecular Transferases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
chemical_list: D000591:Amino Acid Isomerases; D001426:Bacterial Proteins; D001562:Benzimidazoles; D003035:Cobalt; D003038:Cobamides; D006639:Histidine; D019751:Intramolecular Transferases; D011994:Recombinant Proteins; C021570:methylaspartate mutase; C000913:cobamamide; C000731191:component S, glutamate mutase protein, Bacteria; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)