COORDINATION OF A HISTIDINE RESIDUE OF THE PROTEIN-COMPONENT-S TO THE COBALT ATOM IN COENZYME B-12-DEPENDENT GLUTAMATE MUTASE FROM CLOSTRIDIUM-COCHLEARIUM

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 B-12 to the enzyme. The shapes and the superhyperfine splitting of the g(z)-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 N-15-labelled component S, unlabelled component E, coenzyme B-12 and glutamate gave slightly sharper g(z)-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 g(z)-line, whereas the N-15-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 N-15-labelled component S, in which only the histidines were N-14-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 B-12 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.