Characterization of the Zinc Sites in Cobalamin-Independent and Cobalamin-Dependent Methionine Synthase Using Zinc and Selenium X-ray Absorption Spectroscopy ² Katrina Peariso, ‡ Zhaohui S. Zhou, § April E. Smith, | Rowena G. Matthews,* ,§ and James E. Penner-Hahn* ,‡ Department of Chemistry, Biophysics Research DiVision and Department of Biological Chemistry, and Interdepartmental Program in Medicinal Chemistry, The UniVersity of Michigan, Ann Arbor, Michigan 48109-1055 ReceiVed July 24, 2000; ReVised Manuscript ReceiVed NoVember 2, 2000 ABSTRACT: X-ray absorption spectroscopy has been used to investigate binding of selenohomocysteine to cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthase enzymes of Escherichia coli. We have shown previously [Peariso et al. (1998) J. Am. Chem. Soc. 120, 8410-8416] that the Zn sites in both enzymes show an increase in the number of sulfur ligands when homocysteine binds. The present data provide direct evidence that this change is due to coordination of the substrate to the Zn. Addition of L-selenohomocysteine to either MetE or the N-terminal fragment of MetH, MetH- (2-649), causes changes in the zinc X-ray absorption near-edge structure that are remarkably similar to those observed following the addition of L-homocysteine. Zinc EXAFS spectra show that the addition of L-selenohomocysteine changes the coordination environment of the zinc in MetE from 2S + 2(N/O) to 2S + 1(N/O) + 1Se and in MetH(2-649) from 3S + 1(N/O) to 3S + 1Se. The Zn-S, Zn-Se, and Se-S bond distances determined from the zinc and selenium EXAFS data indicate that the zinc sites in substrate- bound MetE and MetH(2-649) both have an approximately tetrahedral geometry. The selenium edge energy for selenohomocysteine shifts to higher energy when binding to either methionine synthase enzyme, suggesting that there is a slight decrease in the effective charge of the selenium. Increases in the Zn-Cys bond distances upon selenohomocysteine binding together with identical magnitudes of the shifts to higher energy in the Se XANES spectra of MetE and MetH(2-649) suggest that the Lewis acidity of the Zn sites in these enzymes appears the same to the substrate and is electronically buffered by the Zn-Cys interaction. Catalytic roles for zinc are well established in a variety of metalloenzymes. In most of these, the zinc is ligated by nitrogen- and oxygen-containing amino acid side chains and has an open ligation site occupied by a water molecule (1). This water molecule can be ionized or polarized to provide hydroxide ions at physiological pH, or it can be displaced by substrate with subsequent Lewis acid activation of the substrate by the zinc ion. Recent studies have discovered a new class of catalytic zinc metalloproteins in which the zinc is ligated primarily by cysteine thiolates (2). These metallo- proteins catalyze the transfer of an alkyl group to a nucleophilic thiolate substrate. It has been suggested that coordination of the substrate thiol to the zinc ion lowers the pK a of the thiol, thereby increasing the concentration of thiolate at physiological pH (3). However, the detailed mechanism of the alkyl transfer has yet to be determined. Two recently characterized zinc-containing enzymes that catalyze alkyl transfers to thiol substrates are the cobalamin- independent (MetE) 1 and cobalamin-dependent (MetH) me- thionine synthases from E. coli. These enzymes catalyze methyl transfer from methyltetrahydrofolate (CH 3 -H 4 folate) to homocysteine to complete the de novo biosynthesis of methionine (4). While the two enzymes have no sequence similarity and have different requirements for catalytic activity (5), both enzymes contain 1 equiv of zinc per mole of protein (6). Moreover, both enzymes require zinc for binding of homocysteine and for catalysis of methyl transfer to the homocysteine thiolate. An N-terminal fragment of MetH, MetH(2-649), includes both substrate domains, but lacks the cobalamin-binding and activation modules. This fragment retains the ability to catalyze methyl transfer to exogenous cob(I)alamin from CH 3 -H 4 folate. MetH(2-649) ² This research was supported in part by grants from the National Institutes of Health (GM-24908 to R.G.M. and GM-38047 to J.E.P.- H.). * Correspondence should be addressed to J.E.P.-H. at the Department of Chemistry, The University of Michigan, 930 N. University Ave., Ann Arbor, MI 48109-1055. FAX (734)647-4865; Phone (734)764- 7324; E-mail jeph@umich.edu. Correspondence should be addressed to R.G.M. at the Biophysics Research Division, The University of Michigan, 930 N. University Ave., Ann Arbor, MI 48109-1055. FAX (734)764-3323; Phone (734)764-9459; E-mail rmatthew@umich.edu. ‡ Department of Chemistry. § Biophysics Research Division and Department of Biological Chemistry. | Interdepartmental Program in Medicinal Chemistry. 1 Abbreviations: MetE, cobalamin-independent methionine synthase; MetH, cobalamin-dependent methionine synthase; CH3-H4folate, meth- yltetrahydrofolate; IPTG, -D-thiogalactopyranoside; TCEP, tris(car- boxyethyl)phosphine; DEAE-Sepharose, diethylaminoethyl-Sepharose; DTT, dithiothreitol; SDS-PAGE, sodium dodecyl sulfate-polyacryl- amide gel electrophoresis; SeHcy, L-selenohomocysteine; Hcy, L- homocysteine. 987 Biochemistry 2001, 40, 987-993 10.1021/bi001711c CCC: $20.00 © 2001 American Chemical Society Published on Web 12/30/2000