Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme

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Abstract
Cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) has been isolated from Escherichia coli B in homogenous form. The enzyme is isolated in an inactive form with the visible absorbance properties of cob(II)alamin. The inactive enzyme exhibits an electron paramagnetic resonance (EPR) spectrum at 38 K that is characteristic of cob(II)alamin at acid pH, where the protonated dimethylbenzimidazole substituent is not coordinated with the cobalt nucleus (base-off cobalamin). An additional, variable component of the EPR spectrum of the inactive enzyme has the characteristics of a cob(III)alamin-superoxide complex. Previous work by others [Taylor, R. T., and Weissbach, H. (1969) Arch. Biochem. Biophys. 129, 745-766, Fujii, K., and Huennekens, F. M. (1979) in Biochemical Aspects of Nutrition (Yagi, K., Ed.) pp 173-183, Japan Scientific Societies, Tokyo] has demonstrated that the enzyme can be activated by reductive methylation using adenosylmethionine as the methyl donor. We present data indicating that the conversion of inactive to methylated enzyme is correlated with the disappearance of the EPR spectrum as expected for the conversion of paramagnetic cob(II)alamin to diamagnetic methylcobalamin. When the methyl group is transferred from the methylated enzyme to homocysteine under aerobic conditions, cob(II)alamin/cob(III)alamin-superoxide enzyme is regenerated as indicated by the return of the EPR spectrum. Our enzyme preparations contain copper in .apprx.1:1 stiochiometry with cobalt as determined by atomic absorption spectroscopy. This copper is EPR silent in the enzyme as isolated and in methylated enzyme, but it can be detected as a cupric-EDTA complex following aerobic denaturation of the enzyme in the presence of mersalyl, 6 M urea, and EDTA.