The covalent attachment of the photosensitizer Ru (bpy) 32+ to a substrate constitutes a powerful new method for probing the steric and electronic properties of buried enzyme active sites. Particularly important targets are oxygenases (cytochromes P450) involved in drug metabolism and many disease states. The crystal structure of a P450: Ru-adamantyl complex reveals that the substrate moiety gains access to the active center via a deep channel and rests above the heme much like the natural substrate camphor. This structure also identifies significant P450 conformational changes associated with substrate binding for the first time: the channel opens via a 6-Å loop movement. Turnover studies show that activity is not significantly diminished in the P450: Ru-adamantyl complex. Preliminary light-activated substrate turnover experiments also show promise. The binding of Ru-substrates to P450 was measured by time-resolved luminescence and UV-vis assays. These molecules specifically recognize submicromolar cytochrome P450cam in the presence of other heme proteins. In the P450: Ru-substrate conjugates, energy transfer to the heme dramatically accelerates the Ru-luminescence decay. Quantifying the fraction of quenched Ru2+* provides an accurate method for determining dissociation constants. In addition, for the P450: Ru-adamantyl complex, Forster analysis of the energy-transfer kinetics yields a Ru-Fe distance (21 Å) virtually the same as that measured in the crystal structure. Similar analysis for other Ru-substrates of varying length shows a narrow range of Ru-Fe distances, indicating favorable association of the Ru (bpy) 32+ moiety with the protein. The binding of the Λ and Δ enantiomers of Ru-C9-Ad to P450 was measured, and the results, KD (Δ/Λ) ~2, suggest that the bipyridyl ligands interact with aromatic residues at the mouth of the substrate channel. Thus, enantiospecific interactions may be exploited in the design of enzyme-metallosubstrate conjugates. Oxidative and reductive quenching of P450: Ru- (CH2) n-substrate2+* conjugates in solution triggers the injection of holes and electrons from ruthenium to the heme on submillisecond time scales. In order to accelerate electron transfer, conjugated (perfluorobiphenyl-bridged) Ru-probes were synthesized which bind P450 strongly (KD< 1 μM). Photoexcitation of conjugated Ru-imidazole-P450 complexes reduces the heme on submicrosecond time scales, opening new avenues for the study of short-lived enzyme intermediates.
Ivan Julian Dmochowski (Thu,) studied this question.