Domains of the catalytically self-sufficient cytochrome P-450 BM-3. Genetic construction, overexpression, purification and spectroscopic characterization.
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.
Miles
J S
JS
Department of Biochemistry, University of Glasgow, U.K.
Munro
A W
AW
Rospendowski
B N
BN
Smith
W E
WE
McKnight
J
J
Thomson
A J
AJ
D001410:Bacillus megaterium / Q000201:enzymology*; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D005798:Genes, Bacterial; D006418:Heme / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases