AFFINITY ISOLATION AND CHARACTERIZATION OF CYTOCHROME-P450-102 (BM-3)FROM BARBITURATE-INDUCED BACILLUS-MEGATERIUM

Cytochrome P450 102 (BM-3) is a catalytically self-sufficient enzyme f rom Bacillus megaterium that is presently accepted as an important mod el of the mammalian microsomal P450 monooxygenase system. We have deve loped a novel affinity approach to purify P450 102 in a single chromat ographic step and have studied the spectroscopic, catalytic, nucleotid e binding, and crystallization properties of the highly purified enzym e. B. megaterium ATCC 14581 was grown to high cell density, and P450 1 02 was purified rapidly and in high yield by chromatography on adenosi ne-2'5'-diphosphate agarose from crude cell-free extract. The cytochro me bound to the column with remarkable avidity, in contrast to the sig nificantly weaker binding observed for NADPH-cytochrome P450 reductase . Chromatographic behavior also showed that the cytochrome bound NADP-type nucleotides more tightly than any other cellular polypeptide. Th e purified protein was electrophoretically homogeneous and had essenti ally theoretical contents of FAD, FMN, and heme. Optical spectra showe d the expected heme and flavin absorption bands, and three previously undescribed charge-transfer-type absorptions were characterized. Molar extinction coefficients in the oxidized, fully reduced, and ferrous c arbonyl states have been determined; notable is the large soret extinc tion in the ferrous carbonyl state (epsilon449nm - 143,500 M-1 cm-1). Final preparations were active in the oxidation of a wide variety of s ubstrates. Of the C-14 alkyl compounds studied, tetradecyltrimethylamm onium bromide showed the highest substrate-dependent oxidation of NADP H, followed by myristate and myristyl alcohol; however, myristate exhi bited the lowest K(m) value. Activities were tightly coupled to NADPH oxidation (>97%). Phenobarbital, benzphetamine, cocaine, cyclohexane, methanol, ethanol, retinoic acid, benzoate, heptaflouro-butyrate, and 7-ethoxycoumarin were not substrates. NADP+ titrations showed, as expe cted, that the coenzyme was bound very tightly, with an average K(d) o f 580 nM. Our preparations of P450 102 are of sufficient purity and st ability that crystals of the native holoenzyme have been grown. (C) 19 94 Academic Press, Inc.