Mutations of glutamate-84 at the putative potassium-binding site affect camphor binding and oxidation by cytochrome P450(cam)

Cytochrome P450(cam) (CYP101) from Pseudomonas putida is unusual among P450 enzymes in that it exhibits co-operative binding between the substrate cam phor and a potassium ion. This behaviour has been investigated by mutagenes is of Glu84, a surface residue which forms part of the cation-binding site. Substitutions that neutralize or reverse the charge of this side chain are shown to disrupt the co-operativity of potassium and camphor binding by P4 50(cam), and also to influence the catalytic activity. In particular, repla cement of Glu84 by positively charged residues such as lysine results in in creased high-spin haem fractions and camphor turnover activities in the abs ence of potassium, along with decreased camphor dissociation constants. How ever, in the presence of potassium the camphor dissociation constants of th ese mutants are significantly increased compared with the wild-type, althou gh the camphor turnover activities remain marginally higher. In contrast, s ubstitution by aspartate results in tighter binding of both potassium and c amphor, but has little effect on the enzymatic activity. In all cases the r eaction remains essentially 100% coupled and gives 5-exo-hydroxycamphor as the only product. These results suggest that an anionic side chain at the 8 4 position is crucial for the co-operativity of camphor and cation binding, and that the physiological role for potassium binding by cytochrome P450(c am), is to promote camphor binding even at the expense of turnover rate, th us allowing the organism to utilize low environmental concentrations of thi s substrate for growth.