Isolation and sequencing of an active-site peptide from spinach ferredoxin-NADP + oxidoreductase after affinity labeling with periodate-oxidized NADP

Spinach ferredoxin-NADP + oxidoreductase was inactivated by treatment with 2′,3′-dialdehyde NADP + (periodate-oxidized NADP +), which selectively modifies a lysine residue at the nucleotide-binding domain of the enzyme. The identity of the derivatized residue was ascertained by thin-layer chromatography of the protein hydrolysate. Reductase that had been labeled with periodate-oxidized NADP + and NaB 3H 4 was treated with trypsin, and samples of the tryptic digest were subjected to reverse-phase high-performance liquid chromatography. The radioactivity profiles showed modification of one specific peptide. The primary structure of this peptide was found to be Gly-Glu-Lys ∗-Met-Tyr-Ile-Gln-Thr-Arg, where Lys ∗ represents the derivatized lysine. The sequence obtained corresponds to residues 242–250 in the primary structure of spinach ferredoxin-NADP + reductase recently reported [Karplus et al. (1984) Biochemistry 23, 6576–6583].