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J Biol Chem, Vol. 273, Issue 40, 26248-26255, October 2, 1998

Purification of Soluble Cytochrome b₅ as a Component of the Reductive Activation of Porcine Methionine Synthase

From the Biochemistry Department, University of Nebraska, Lincoln, Nebraska 68588-0664

In mammals, methionine synthase plays a central role in the detoxification of the rogue metabolite homocysteine. It catalyzes a transmethylation reaction in which a methyl group is transferred from methyltetrahydrofolate to homocysteine to generate tetrahydrofolate and methionine. The vitamin B₁₂ cofactor cobalamin plays a direct role in this reaction by alternately accepting and donating the methyl group that is in transit from one substrate (methyltetrahydrofolate) to another (homocysteine). The reactivity of the cofactor intermediate cob(I)alamin renders the enzyme susceptible to oxidative damage. The oxidized enzyme may be returned to the catalytic turnover cycle via a reductive methylation reaction that requires S-adenosylmethionine as a methyl group donor, and a source of electrons. In this study, we have characterized an NADPH-dependent pathway for the reductive activation of porcine methionine synthase. Two proteins are required for the transfer of electrons from NADPH, one of which is microsomal and the other cytoplasmic. The cytoplasmic protein has been purified to homogeneity and is soluble cytochrome b₅. It supports methionine synthase activity in the presence of NADPH and the microsomal component in a saturable manner. In addition, purified microsomal cytochrome P450 reductase and soluble cytochrome b₅ reconstitute the activity of the porcine methionine synthase. Identification of soluble cytochrome b₅ as a member of the reductive activation system for methionine synthase describes a function for this protein in non-erythrocyte cells. In erythrocytes, soluble cytochrome b₅ functions in methemoglobin reduction. In addition, it identifies an additional locus in which genetic polymorphisms may play a role in the etiology of hyperhomocysteinemia, which is correlated with cardiovascular diseases.

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