Characterization of the zinc sites in cobalamin-independent and cobalamin-dependent methionine synthase using zinc and selenium X-ray absorption spectroscopy

X-ray absorption spectroscopy has been used to investigate binding of selen ohomocysteine 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. Sec. 120, 8410-8416] that the Zn sites in both enzymes show an increase in the number of sulfur ligands when homo cysteine binds. The present data provide direct evidence that this change i s due to coordination of the substrate to the Zn. Addition of L-selenohomoc ysteine to either MetE or the N-terminal fragment of MetH, MetH-(2-649), ca uses changes in the zinc X-ray absorption near-edge structure that are rema rkably similar to those observed following the addition of L-homocysteine. Zinc EXAFS spectra show that the addition of L-selenohomocysteine changes t he 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 h ave an approximately tetrahedral geometry. The selenium edge energy for sel enohomocysteine shifts to higher energy when binding to either methionine s ynthase enzyme, suggesting that there is a slight decrease in the effective charge of the selenium. Increases in the Zn-Cys bond distances upon seleno homocysteine binding together with identical magnitudes of the shifts to hi gher energy in the Se XANES spectra of MetE and MetH(2-649) suggest that th e Lewis acidity of the Zn sites in these enzymes appears the same to the su bstrate and is electronically buffered by the Zn-Cys interaction.