Characterization of the zinc binding site in methionine synthase enzymes of Escherichia coli:: The role of zinc in the methylation of homocysteine

X-ray absorption spectroscopy, using the analytical methodology described in the previous paper, has been used to determine the ligation of the essential zinc ions in cobalamin-dependent (MetH) and cobalamin-independent (MetE) methionine synthases from Escherichia coli and to probe directly the changes in zinc ligation that occur upon addition of the thiol substrate, homocysteine, to each enzyme. Extended X-ray absorption fine structure (EXAFS) spectra of native MetE and a truncated fragment of MetH containing the substrate-binding sites are consistent with ZnS2(N/O)(2) and ZnS3(N/O) ligation, respectively. Previous mutagenesis studies of the homocysteine binding region of MetH had identified two putative thiolate zinc ligands, Cys310 and Cys311. Since the EXAFS spectra indicate that the zinc is coordinated to three sulfur ligands derived from the protein, a third conserved cysteine, Cys247, was mutated to alanine, resulting in a MetH fragment that binds only 0.09 equiv of zinc per mol of protein and exhibits no methylcobalamin-homocysteine methyltransferase activity. Upon addition of L-homocysteine, the X-ray absorption near edge structure changes for both enzymes, and the EXAFS spectra show changes consistent with the coordination of a sulfur, giving a ZnS3(N/O) site for MetE and a ZnS4 site for MetH. Only the L-homocysteine enantiomer causes these effects; the addition of D-homocysteine to MetH(2-649) gives no detectable changes in the EXAFS or the near edge regions. These results are consistent with a mechanism in which the homocysteine is ligated to zinc. Homocysteine is then able to initiate nucleophilic attack on the methyl group needed for methionine formation, with the methyl group bound either to methylcobalamin in MetH or to a polyglutamate derivative of methyltetrahydrofolate in MetE.