Role of protein and substrate dynamics in catalysis by Pseudomonas putida cytochrome P450_cam

Abstract

The role of protein structural flexibility and substrate dynamics in catalysis by cytochrome P450 enzymes is an area of current interest. We have addressed these in cytochrome P450_cam (P450_cam) and its Y96A mutant with camphor and its related compounds using fluorescence spectroscopy. Previously [Prasad et al. (2000) FEBS Lett. 477, 157-160], we provided experimental support to dynamic fluctuations in P450_cam, and substrate access into the active site region via the channel next to the flexible F-G helix-loop-helix segment. In the investigation described here, we show that the dynamic fluctuations in the enzyme are substrate dependent as reflected by tryptophan fluorescence quenching experiments. The orientation of tryptophan relative to heme (k²) for W42 obtained from time-resolved tryptophan fluorescence measurements show variation with type of substrate bound to P450_cam suggesting regions distant from heme-binding site are affected by physicochemical and steric characteristics/protein-substrate interactions of P450_cam active site. We monitored substrate dynamics in the active site region of P450_cam by time-resolved substrate anisotropy measurements. The anisotropy decay of substrates bound to P450_cam indicate that mobility of substrates is modulated by physicochemical and steric characteristics/protein-substrate interactions of local active site structure, and provides an understanding of factors controlling observed hydroxylated products for substrate bound P450_cam complexes. The present study shows that P450_cam local and peripheral structural flexibility and heterogeneity along with substrate mobility play an important role in regulating substrate binding orientation during catalysis and accommodating diverse range of substrates within P450_cam heme pocket.