Coordination of a histidine residue of the protein‐component S to the cobalt atom in coenzyme B12‐dependent glutamate mutase from Clostridium cochlearium

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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 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 15N‐labelled component S, unlabelled component E, coenzyme B12 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 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.

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