DEFINITION OF A NUCLEOTIDE-BINDING SITE ON CYTOCHROME-C BY PHOTOAFFINITY-LABELING

We have used TNP-8N(3)-AMP (2'(3')-O-(2,4,6-trinitrophenyl)-8-azidoade nosine monophosphate) and TNP-8N(3)-ATP to probe the ATP binding site( s) of cytochrome c. Irradiation of cytochrome c with close to stoichio metric amounts of TNP-8N(3)-AMP at low ionic strength derivatized appr oximately half of the protein, with the mono-derivatized species being associated with four peaks (B, 6%; C, 17%; D, 24%; E, 4%) eluted from a cation exchange column, Irradiation in the presence of ATP suggeste d that the main peaks C and D resulted from more specific nucleotide b inding, Thermolysin digestion and TNP-peptide purification and sequenc ing revealed that peak C was associated with derivatization of mainly Lys-86 and to a lesser extent Lys-72 and peak D with mainly Lys-87 and less so with Lys-72, Minor peaks B and E could not be identified, TNP -8N(3)-ATP photolabeling produced similar results, showing favored int eraction of the adenyl ring with Lys-86 and Lys-87 and to a lesser ext ent with Lys-72, The results are compatible with previous findings tha t suggest that the principal locus of ATP binding is at nearby Arg-91 (Corthesy, B. E., and Wallace, C, J. A. (1986) Biochem, J. 236, 359-36 4), Molecular modeling with energy-minimized docking of ATP between th e 60s helix and the 80s stretch with the gamma-phosphate constrained t o interact with Arg-91, places the 8 position close to Lys-86 and Lys- 87 in the anti conformation about the glycosidic bond and to Lys-72 in the syn conformation, and the ribose hydroxyls within H-bonding dista nce of Glu-69.