COORDINATION OF A HISTIDINE RESIDUE OF THE PROTEIN-COMPONENT-S TO THECOBALT 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 dimethylbenzimid azole ligand of the Co-atom during binding of coenzyme B-12 to the enz yme. 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 t he axial base nitrogen with the Co-atom. A mixture of completely N-15- labelled component S, unlabelled component E, coenzyme B-12 and glutam ate gave slightly sharper g(z)-lines than that with unlabelled compone nt S. A more dramatic change was observed in the Co(II) spectrum of th e 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 sh owed no difference to those with unlabelled component S. The experimen ts indeed demonstrate a replacement of the dimethylbenzimidazole ligan d in coenzyme B-12 by a histidine when bound to glutamate mutase. The most likely candidate is H16, which is conserved among the carbon skel eton rearranging mutases and methionine synthase.