Identification of the metal-binding sites of restriction endonucleases by Fe2+-mediated oxidative cleavage

Fenton chemistry [Fenton (1894) J. Chem. Sec. 65, 899-910] techniques were employed to identify the residues involved in metal binding located at the active sites of restriction endonucleases. This process uses transition met als to catalytically oxidize the peptide linkage that is in close proximity to the amino acid residues involved in metal ligation. Fe2+ was used as th e redox-active transition metal. It was expected that Fe2+ would bind to th e endonucleases at the Mg2+-binding site [Liaw et al. (1993) Biochemistry 3 2, 7999-4003; Ermacora et al. (1992) Proc Natl. Acad. Sci. U.S.A. 89, 6383- 6387; Soundar and Colman (1993) J. Biol. Chem. 268, 5264-5271; Wei et al. ( 1994) Biochemistry 33, 7931-7936;Ettner et al. (1995) Biochemistry 34, 22-3 1; Hlavaty and Nowak (1997) Biochemistry 36, 15515-15525). Fe2+-mediated ox idation was successfully performed on TaqI endonulease, suggesting that thi s approach could be applied to a wide array of endonucleases [Cao and Baran y (1998) J. Biol. Chem. 273, 33002-33010]. The restriction endonucleases Ba mHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, BsoBI, EcoRI, EcoRV, MspI, and HinP1I were subjected to oxidizing conditions in the presence of Fe2+ and a scorbate. All proteins were inactivated upon treatment with Fe2+ and ascorb ate. BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, and BsoBI were specifica lly cleaved upon treatment with Fe2+/ascorbate. The site of Fe2+/ascorbate- induced protein cleavage for each enzyme was determined. The Fe2+-mediated oxidative cleavage of BamHI occurs between residues Glu77 and Lys78. G1u77 has been shown by structural and mutational studies to be involved in both metal ligation and catalysis [Newman et al. (1995) Science 269, 656-663; Vi adiu and Aggarwal (1998) Nat. Struct. Biol. 5, 910-916; Xu and Schildkraut (1991) J. Biol. Chem. 266, 4425-4429]. The sites of Fe2+/ascorbate-induced cleavage for PvuII, FokI, BglI, and BsoBI agree with the metal-binding site s identified in their corresponding three-dimensional structures or from mu tational studies [Cheng et al. (1994) EMBO J. 13, 3297-3935; Wah et al. (19 97) Nature 388, 97-100; Newman et al. (1998) EMBO J. 17, 5466-5476; Ruan et al. (1997) Gene 188, 35-39]. The metal-binding residues of BglII, SfiI, an d BssSI are proposed based on amino acid sequencing of their Fe2+/ascorbate -generated cleavage fragments. These results suggest that Fenton chemistry may be a useful methodology in identifying amino acids involved in metal bi nding in endonucleases.