| Abstract: | When Bacillus megaterium ATCC 14581 is grown in the presence of barbiturates, a cytochrome P-450-dependent fatty acid monooxygenase (Mr 120000) is induced (Kim, B.-H. and Fulco, A.J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). Gel filtration chromatography of a crude monooxygenase preparation from pentobarbital-induced B. megaterium indicated that not all of the induced cytochrome P-450 present in the extract was accounted for by this high-molecular-weight component. Further purification revealed the presence of two additional but smaller cytochrome P-450 species. The minor component, designated cytochrome P-450BM-2, had a molecular mass of about 46 kDa, but has not yet been completely purified or further characterized. The major component, designated cytochrome P-450BM-1, was obtained in pure form, exhibited fatty acid monooxygenase activity in the presence of iodosylbenzenediacetate, and has been extensively characterized. Its Mr of 38000 makes it the smallest cytochrome P-450 yet purified to homogeneity. Although it is a soluble protein, a complete amino acid analysis indicated that it contains 42% hydrophobic residues. By the dansyl chloride procedure the NH2-terminal amino acid is proline; the penultimate NH2-terminal residue is alanine. The absolute absorption spectra of cytochrome P-450BM-1 show maxima in the same general regions as do P-450 cytochromes from mammalian or other bacterial sources, but they differ in detail. The oxidized form of P-450BM-1 has absorption maxima at 414, 533 and 567 nm, while the reduced form has peaks at 410 and 540 nm. The absorption maxima for the CO-reduced form of P-450BM-1 are found at 415, 448 and 550 nm. Antisera from rabbits immunized with pure P-450BM-1 strongly reacted with and precipitated this P-450, but showed no detectable affinity for either the 46 kDa P-450 or the 120 kDa fatty acid monooxygenase. |
