Flavocytochrome P-450 BM3 from Bacillus megaterium is a 119 kDa polypeptide whose heme and diflavin domains are fused to produce a catalytically self-sufficient fatty acid monooxygenase. Redox potentiometry studies have been performed with intact flavocytochrome P-450 BM3 and with its component heme, diflavin, FAD, and FMN domains. Results indicate that electron flow occurs from the NADPH donor through FAD, then FMN and on to the heme center where fatty acid substrate is bound and monooxygenation occurs. Prevention of futile cycling of electrons is avoided through an increase in redox potential of more than 100 mV caused by binding of fatty acids to the active site of P-450. Redox potentials are little altered for the component domains with respect to their values in the larger constructs, providing further evidence for the discrete domain organization of this flavocytochrome. The reduction potentials of the 4-electron reduced diflavin domain and 2-electron reduced FAD domain are considerably lower than those for the blue FAD semiquinone species observed during reductive titrations of these enzymes and that of the physiological electron donor (NADPH), indicating that the FAD hydroquinone is thermodynamically unfavorable and does not accumulate under turnover conditions. In contrast, the FMN hydroquinone is thermodynamically more favorable than the semiquinone.

Aromatic amine dehydrogenase (AADH) catalyzes the oxidative deamination of aromatic amines including tyramine and dopamine. AADH is structurally similar to methylamine dehydrogenase (MADH) and possesses the same tryptophan tryptophylquinone (TTQ) prosthetic group. AADH exhibits an alpha 2 beta 2 structure with subunit molecular weights of 39,000 and 18,000 and with a quinone covalently attached to each beta subunit. Neither subunit cross-reacted immunologically with antibodies to the corresponding subunits of MADH, and the N-terminal amino acid sequence of the beta subunit of AADH exhibited no homology with the highly conserved beta subunits of MADH. The absorption spectra for the oxidized, semiquinone, and reduced forms of AADH have been characterized, and extinction coefficients for the absorption maxima of each redox form have been determined. These spectra are very similar to those for MADH, indicating the likelihood of a TTQ cofactor. This was verified by the near identity of the vibrational frequencies and intensities in the resonance Raman spectra for the oxidized forms of AADH and MADH. A stable semiquinone of AADH could be observed during a reductive titration with dithionite, whereas titration with tyramine proceeded directly from the oxidized to the reduced form. AADH was very stable against denaturation by heat and exposure to guanidine. The individual subunits could be separated by gel filtration after incubation in guanidine hydrochloride, and partial reconstitution of activity was observed on recombination of the subunits. Steady-state kinetic analysis of AADH yielded a Vmax of 17 mumol/min/mg and a Km for tyramine of 5.4 microM. Substrate inhibition by tyramine was observed. AADH was irreversibly inhibited by hydrazine, phenylhydrazine, hydroxylamine, semicarbazide, and aminoguanidine. Isonicotinic acid hydrazide (isoniazid) and isonicotinic acid 2-isopropyl hydrazide (iproniazid) were reversible noncompetitive inhibitors of AADH and exhibited K(i) values of 8 and 186 microM, respectively. The similarities and differences between AADH and other amine oxidizing enzymes are also discussed.

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Cytochrome P450BM-3 is a fatty acid monooxygenase that contains the catalytic P450 heme domain covalently attached to a diflavin P450 reductase domain. The function of the linker region connecting the C-terminal end of the heme domain to the N-terminal end of the reductase domain has been studied by deleting parts of the linker and changing the sequence of the linker. Deleting three or six residues or changing an Arg-Lys-Lys stretch in the middle of the linker to Ala-Ala-Ala does not alter the functional properties of either domain. The mutants retain full cytochrome c and ferricyanide reductase activities characteristic of the P450 reductase domain. The heme domain in the mutants retains its ability to bind a fatty acid substrate giving the full low-to-high spin shift and exhibits the normal 450 nm absorption band characteristic of the reduced carbon monoxide complex. However, the six amino acid deletion mutant exhibit nearly undetectable levels of fatty acid hydroxylase activity, the three amino acid deletion mutant about 10% activity, and the three Ala substitution mutant about 50% activity. The mutants also exhibit slower rates of reductase-to-heme electron transfer rates that correlate with the loss in fatty acid hydroxylase activity. These results indicate that the length of the linker and, to a much less extent, the sequence are important for correctly orienting the reductase and heme domains, which apparently is necessary to achieve efficient reductase-to-heme electron transfer rates.