OPTICALLY DETECTED MAGNETIC-RESONANCE STUDY OF THE INTERACTION OF AN ARSENIC(III) DERIVATIVE OF CACODYLIC ACID WITH ECORI METHYL TRANSFERASE

The interaction of the enzyme Escherichia coli RI methyl transferase (methylase) with an arsenic(III) derivative of cacodylic acid has been investigated by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy in zero applied magnetic field. The reactive derivative (CH3)2AsSR is formed by the reduction of cacodylate by a thiol. The As(III) derivative binds to the enzyme by mercaptide exchange with a cysteine (Cys) residue located close to a tryptophan (Trp) site. The arsenical binding selectively induces an external heavy-atom effect, perturbing the nearby Trp residue in the enzyme. Zero-field splittings (ZFS) and total decay rate constants of the individual triplet-state sublevels of the Trp residue in the presence and absence of perturbation by As(III) have been determined. The perturbed Trp shows a large reduction in the overall decay lifetime compared with unperturbed Trp residue, exhibiting a high selectivity for the T(x) sublevel. This selectivity suggests that the As atom lies in the xz plane of the principal magnetic axis system of Trp, but not directly along the z (out-of-plane) axis. The accessibility of this enzyme binding site to the arsenical is decreased upon forming a ternary complex of methylase with sinefungin and a DNA oligomer, d[GCGAA(BrU)(BrU)CGC], containing two 5-bromouracil (BrU) bases in place of thymine within the hexadeoxynucleotide recognition sequence. This result indicates that the arsenical binding site in methylase which produces the Trp heavy-atom effect is protected from this ligand by ternary complex formation or the enzyme undergoes a conformation change, removing the Cys from the Trp site. This protection is also observed in fluorescence quenching experiments. The As(III) reagent, upon binding to methylase, quenches the Trp fluorescence by 46%. When the ternary complex is formed, the quenching of Trp fluorescence is only 17%. A binding constant for the arsenical to the high-affinity enzyme site was obtained, which is at least 27 times that of binding to a free sulfhydryl residue. The addition of a 1:1 molar ratio of the arsenical to methylase did not affect the activity of the enzyme, but incubation with excess arsenical quenches the activity, suggesting that the high-affinity Cys residue is not involved in the DNA methylation process. In the ternary complex methylase-sinefungin-DNA, no heavy-atom perturbation of the two Trp residues in the enzyme by BrU was observed, demonstrating that Trp residues are not involved in close-range interactions with the two heavy-atom-derivatized nucleic acid bases. A similar result was observed previously with the analogous E. coli RI endonuclease-decanucleotide complex [John, N.-I., Casas-Finet, J. R., Maki, A. H., & Modrich, P. (1988) Biochim. Biophys. Acta 949, 189-194].