Domains of the catalytically self-sufficient cytochrome P-450 BM-3. Genetic construction, overexpression, purification and spectroscopic characterization.

Abstract

1. The gene CYP102 encoding cytochrome P-450 BM-3 and subgenes encoding the cytochrome P-450 and cytochrome P-450 reductase domains have been cloned in Escherichia coli. 2. The protein products of these genes have been overexpressed and purified to homogeneity. 3. The cytochrome P-450 domain is purified in the ferric low-spin state, but is readily converted into the high-spin state by addition of the substrate palmitate (Ks = 1 microM). The cytochrome P-450 reductase domain readily reduces cytochrome c. Mixing the two domains reconstitutes only about one-thousandth of the fatty acid hydroxylase activity associated with the intact cytochrome P-450 BM-3. 4. The X-band e.p.r. spectra of both the cytochrome P-450 domain and intact cytochrome P-450 BM-3 give g-values indicating low-spin ferric haem. The spectra are virtually identical with those of the equivalent form of cytochrome P-450 cam indicating that the haem ligation in cytochrome P-450 BM-3 is identical with that of cytochrome P-450 cam. 5. Resonance Raman spectra of the substrate-free and substrate-bound forms of the cytochrome P-450 domain are given. Spectral differences in comparison with cytochrome P-450 cam may reflect subtle electronic differences between the respective haem environments.