Definition of a Nucleotide Binding Site on Cytochrome c by Photoaffinity Labeling

Abstract

We have used TNP-8N3-AMP (2'(3')-O-(2,4,6-trinitrophenyl)-8-azidoadenosine monophosphate) and TNP-8N3-ATP to probe the ATP binding site(s) of cytochrome c. Irradiation of cytochrome c with close to stoichiometric amounts of TNP-8N3-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-8N3-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.