The Role of Conformational Flexibility in Inhibitor Binding and Substrate Recognition for Cyp119

Abstract

Conformational flexibility of several cytochromes P450 has been observed in response to substrate and ligand binding, and thus may play an important role in catalysis. CYP119, a bacterial thermophilic protein from cytochrome P450 superfamily has been observed in three different conformations with different inhibitors bound using X-ray crystallography, but the significance of these states in solution and in the function of the enzyme is not well known. According to the crystal structures, much of the diversity in conformational states arises in F and G helices, which fold around the substrate binding site at the distal heme face. Double electron electron resonance (DEER) was used to measure the average distances and the distance distributions between spin-labels for populated conformational states in solution. Pairs of spin-labels were introduced by coupling to engineered cysteines on the protein surface, and the effects of labeling on ligand dissociation constants (Kd) and enzyme function were characterized. DEER results from three different mutants of CYP119 indicated that 4-Phenylimidazole binding results in a conformational state that is distinct substrate-free and Imidazole-bound forms. The DEER distance changes between the two conformational states were determined as 10A for two of the mutants and 12A for the other one. Data will also be presented for the complex between CYP119 its proposed substrate, lauric acid. These results will be discussed in terms of models for substrate recognition and gated functional behavior in these enzymes.