Coordination of vanadyl(IV) cation in complexes of S-adenosylmethionine synthetase: multifrequency electron spin echo...

S-Adenosylmethionine (AdoMet) synthetase requires two freely dissociable divalent cations for activity, and its activity is greatly stimulated by certain monovalent cations (K+, Tl+). Omission of the native Mg2+ cations prevents enzyme-catalyzed reactions, although the substrates and products still bind. Vanadyl (oxovanadium, VO2+) serves as a convenient paramagnetic probe of the substrate-independent, divalent cation binding site in the enzyme. In the present study, multifrequency electron spin echo envelope modulation (ESEEM) is employed to explore the cation's coordination sphere in several functionally relevant complexes. In the substrate complex enzyme.VO2+.ATP.methionine.K+, an equatorially coordinating 14N ligand is found, with Aiso = 4.3 MHz. Selective 15N labeling of the epsilon-amino nitrogens of all lysine residues in the protein reveals that lysine is the source of the ligand. A lysine 14N ligand is also observed in the intermediate complex enzyme.VO2+.AdoMet.PPPi.K+, with Aiso = 4.8 MHz, and in the initial product complex enzyme.VO2+.AdoMet.PPi.K+. In the subsequent product, enzyme.VO2+.AdoMet.K+ (formed upon dissociation of PPi), the methionyl amino nitrogen of AdoMet coordinates VO2+ (Aiso = 5.3 MHz), and the lysine ligand is lost. In each complex, the monovalent cation activator can be changed from K+ to Tl+ or Na+ with no effect on the ESEEM spectra. Combination of the ESEEM data from this study with previous CW data [Markham, G. D. (1984) Biochemistry 23, 470-478] leads to identification of three of the equatorial ligands to VO2+ and places constraints upon the identity of the fourth ligand, in both the substrate and product complexes. A hypothetical outline of changes in the metal coordination scheme during the reaction is presented, based upon these results.