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-azidoadenosine monophosphate) and TNP-8N(3)-ATP to probe the ATP binding site(s) of cytochrome c. Irradiation of cytochrome c with close to stoichiometric amounts of TNP-8N(3)-AMP at low ionic strength derivatized approximately 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 suggested that the main peaks C and D resulted from more specific nucleotide binding, Thermolysin digestion and TNP-peptide purification and sequencing 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 interaction of the adenyl ring with Lys-86 and Lys-87 and to a lesser extent with Lys-72, The results are compatible with previous findings that 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, 359-364), Molecular modeling with energy-minimized docking of ATP between the 60s helix and the 80s stretch with the gamma-phosphate constrained to 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 distance of Glu-69.