Atomic force microscopy detection of molecular complexes in multiprotein P450cam containing monooxygenase system

The application of atomic force microscopy (AFM) technique in proteomic research, identification and visualization of individual molecules and molecular complexes within the P450cam containing monooxygenase system was demonstrated. The method distinguishes between the binary protein complexes and appropriate monomeric proteins and, also, between the binary and ternary complexes. The AFM images of the components of a cytochrome P450cam containing monooxygenase system - cytochrome P450cam (P450cam), putidaredoxin (Pd) and putidaredoxin reductase (PdR) - were obtained on a mica support. The molecules of P450cam, Pd and PdR were found to have typical heights of 2.6 +/- 0.3 nm, 2.0 +/- 0.3 and 2.8 +/- 0.3 nm, respectively. The measured heights of the binary Pd/PdR and P450cam/PdR complexes were 4.9 +/- 0.3 nm and 5.1 +/- 0.3 nm, respectively. The binary P450cam/Pd complexes were found to have a typical height of about (3.9 / 5.7 nm) and the ternary PdR/Pd/P450cam complexes, a typical height of about 9.1 +/- 0.3 nm.     

Optical biosensor study of ternary complex formation in a cytochrome P4502B4 system

The optical biosensor method was used for the revelation of ternary complexes, formed by the full-length NADPH-cytochrome P450 reductase (d-Fp) and cytochromes P4502B4 (d-2B4) and b5 (d-b5) in the course of their interactions within the reconstituted d-2B4-containing system. Based on the lack of competition between d-b5 and d-Fp for the binding sites on immobilized 2B4 (3) as well as on the analysis of data obtained in the three proteins' dissociation reactions, the possibility of formation of ternary complexes through the interactions between membranous hydrophobic fragments of proteins was substantiated. All the complexes obtained were productive.     

Revelation of ternary complexes between redox partners in cytochrome P450-containing monooxygenase systems by the optical biosensor method.

Formation of binary and ternary complexes in the water-soluble cytochrome P450cam (P450cam)-containing as well as in the membrane P4502B4(2B4)- and the mixed P450scc-containing monooxygenase systems was investigated in real time by the 'resonant mirror' optical biosensor method. It was shown that the inter-protein electron transfer occurs not only during complex formation but also upon random collision--as was the case with the d-Fp/d-b5 pair (2B4 system). Binary complexes may be either facilitative to electron transfer (electron-transfer complexes) or prohibitive to it (non-productive complexes). Although the binary PdR/Pd and P450cam/Pd complex formation (within the P450cam-system) as well as the binary AdR/Ad and P450scc/Ad complex formation (within the P450scc-system) does occur, the lifetimes of these complexes formed are several orders of magnitude higher than the time required for realization of a complete hydroxylation cycle. At the same time, the lifetimes of the ternary PdR/Pd/P450cam and AdR/Ad/P450scc complexes are sufficient to permit the realization of a complete hydroxylation cycle in either of these systems. For the membrane P450 2B4 system, the formation of both the binary (Fp/2B4 and 2B4/b5) and ternary (Fp/2B4/b5) complexes was registered. The lifetimes of the binary Fp/2B4 and the ternary Fp/2B4/b5 complexes are sufficient for realization of a complete hydroxylation cycle in each of them.     

The optical biosensor study of the redox partner interaction with the cytochrome P450 2B4-containing monooxygenase system under hydroxylation conditions

The interactions between cytochrome P450 2B4 (d-2B4), NADPH:cytochrome P450 reductase and cytochrome b5 have been investigated in the monomeric reconstituted P450 2B4-containing monooxygenase system in the presence of a substrate (7-pentoxyresorufin) and an electron donor, NADPH. Each partner was immobilized via its amino groups on the carboxymethyldextran biochip surface of the optical biosensor IAsys+. Such mode immobilization was not accompanied by any loss of activities of the immobilized proteins. The formation of binary d-Fp/d-2B4 complexes was registered. The association/dissociation rate constants (k_on/k_off) were (0.013 ± 0.005) × 10⁶ M^?1 s^?1/0.05 ± 0.02 s^?1, and dissociation constant (K_D) was (0.26 ± 0.13) × 10^?6 M. Comparison of k_on, k_off and K_D values for d-Fp/d-2B4 complexes formed under hydroxylation (O-dealkylation) with corresponding constants obtained for the oxidized proteins of (0.10 ± 0.03) × 10⁶ M^?1 s^?1/(0.14 ± 0.06) s^?1, and (0.71 ± 0.37) × 10^?6 M, respectively shows that the decrease in k_on and an insignificant decrease in K_D are associated with the increase of complex lifetime during transition from the oxidized to hydroxylation conditions. Complex formation between d-Fp and d-b5 was not registered in both hydroxylation conditions and in the case of oxidized forms of these proteins. In both cases formation of the ternary d-Fp/d-2B4/d-b5 complexes occurred.     

Optical biosensor investigation of interactions of biomembrane and water-soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers

A phospholipid-containing biochip was created by covalently immobilizing phospholipids on the optical biosensor's aminosilane cuvette and employed to monitor the interactions of the membrane and water-soluble proteins in cytochrome P450-containing monooxygenase systems with planary layers of dilauroylphosphatidylethanolamine (DLPE) and distearoylphosphatidylethanolamine (DSPE), differing in acyl chain length. It was shown that the full-length membrane proteins-cytochrome P4502B4 (d-2B4), cytochrome b5 (d-b5) and NADPH-cytochrome P450 reductase (d-Fp)-readily incorporated into the phospholipids. The incorporation was largely due to hydrophobic interactions of membranous protein fragments with the phospholipid layer. However, electrostatic forces were also but not always involved in the incorporation process. They promoted d-Fp incorporation but had no effect on d-b5 incorporation. In low ionic strength buffer, no incorporation of these two proteins into the DSPE lipid layer was observable. Incorporation of d-b5 into the DLPE layer was abruptly increased at temperatures exceeding phospholipid phase transition point. Incorporation of d-2B4 was dependent on its aggregation state and decreased with increasing protein aggregability. Water-soluble proteins either would not interact with the phospholipid layer (adrenodoxin) or would bind to the layer at the cost of only electrostatic (albumin) or both electrostatic and hydrophobic (P450cam) interactions.     

Mass spectrometry identification of cytochrome P450 2B4 interaction sites for NADPH: Cytochrome P450 reductase

The interaction sites for protein partners, cytochrome P450 2B4 (d-2B4) and NADPH: cytochrome P450 reductase (d-Fp), have been identified. These proteins form complexes during their functioning. Nonspecific covalent cross-linking of the d-2B4 complexes with d-Fp in the Emulgen 913 monomerized system was achieved by 4,4′- dithiobis-phenyl azide. Covalently cross-linked peptides of this complex were identified by ESI-MS/MS. Several binding sites have been identified for these proteins. Based on these sites a model for intermolecular interaction between these proteins has been proposed. This model includes 5 contact sites on d-2B4 for d-Fp (stabilized by the cross-linker); these include the following pairs of corresponding peptides of d-2B4 and d-Fp: 1) d-2B4_324–336 and d-Fp_570–585; 2) d-2B4_423–433 and d-Fp_102–109; 3) d-2B4_327–336 and d Fp_452–464; 4) d-2B4_192–197 and d-Fp_456–464; 5) d-2B4_134–139 and d-Fp_406–425. In the two last cases d-Fp peptides are located in the interdomain cleft and stabilize the protein-protein complex via the cross-linker and so the d 2B4/d-Fp complex formation by these sites may involve amino acid residues of the peptides d-Fp_456–464 and d-Fp_406–425, which surround the interdomain cleft.     

Productive and non-productive complexes in cytochrome P450-containing systems

The equilibrium dissociation constants K_D, the complex association / dissociation rate constants (k _on /k _off) and lifetimes of the complexes of redox partners were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450scc, and P450 2B4) under hydroxylation conditions. The Q parameter representing the ratio of protein-protein complex lifetime (τ _lT ) to time required for a single hydroxylation cycle (τ_turnover) was introduced for estimation of productivity of complexes formed within the systems studied. The Q parameter was insignificantly changed upon transition from the oxidation to hydroxylation conditions. Lifetimes (τ _lT ) for the binary complexes formed within the P450cam and the P450scc systems obligatory requiring an intermediate electron transfer protein between the reductase and cytochrome P450 could not realize hydroxylation reactions for substrates with known τ_turnover and so they were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such intermediate electron-transfer protein, appeared to be productive. Formation of ternary complexes was demonstrated under hydroxylation conditions in all three systems. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were productive. In the case of the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.     

Fluorescent assay for riboflavin binding to cytochrome P450 2B4

The interactions between the hemoprotein cytochrome P450 2B4 (CYP 2B4) and riboflavin - a low molecular weight component of the flavoprotein NADPH-dependent cytochrome P450 reductase - were investigated by fluorescence spectroscopy. Riboflavin fluorescence quenching by cytochrome P450 2B4 was used to probe the ligand-enzyme binding (lambda(ex)=385 nm, lambda(em)=520 nm). Fluorescence titration experiments showed formation of a complex between cytochrome P450 2B4 and riboflavin with an apparent dissociation constant value, K(d)=8.8+/-1 microM. The fluorescence intensity of riboflavin was decreased with increasing the cytochrome P450 2B4 concentration, indicating the transfer of resonance excitation energy from riboflavin (energy donor) to the cytochrome P450 2B4 heme (energy acceptor). The data obtained are suggestive of the existence of riboflavin binding site(s) on the hemeprotein molecule.     

Study of interaction of cytochrome P450 2B4 with riboflavin by fluorescence spectrometry

Fluorescence quenching of riboflavin by cytochrome P450 2B4 was used to probe the ligand--enzyme binding interaction ((lambda ex = 385 nm, lambda em = 520 nm). Riboflavin is a component of a flavoprotein NADPH dependent cytochrome P450 reductase, an essential electron carrier during cytochrome P450 catalysis. Fluorescence titration measurements revealed that cytochrome P450 2B4 and riboflavin formed a complex with an apparent Kd = 8.8 +/- 1 microM. The fluorescence intensity of riboflavin decreased upon the addition of cytochrome P450 2B4, which may be caused by the resonance excitation energy transfer from the fluorescent donor riboflavin to the cytochrome P450 2B4 heme acceptor. These data suggest that there may exist specific sites of binding of riboflavin with the protein globule of cytochrome P450 2B4.     

Global analysis of protein-protein interactions reveals multiple CYP2E1-reductase complexes

Although a single binary functional complex between cytochrome P450 (P450 or CYP for a specific isoform) and cytochrome P450 reductase (CPR) has been generally accepted in the literature, this simple model failed to explain the experimentally observed catalytic activity of recombinant CYP2E1 in dependence on the total concentration of the added CPR-K56Q mutant. Our rejection of the simplest 1:1 binding model was based on two independent lines of experimental evidence. First, under the assumption of the 1:1 binding model, separate analyses of titration curves obtained while varying either P450 or CPR concentrations individually produced contradictory results. Second, an asymmetric Job plot suggested the existence of higher order molecular complexes. To identify the most probable complexation mechanism, we generated a comprehensive data set where the concentrations of both P450 and P450 were varied simultaneously, rather than one at a time. The resulting two-dimensional data were globally fit to 32 candidate mechanistic models, involving the formation of binary, ternary, and quaternary P450.CPR complexes, in the absence or presence or P450 and CPR homodimers. Of the 32 candidate models (mechanisms), two models were approximately equally successful in explaining our experimental data. The first plausible model involves the binary complex P450.CPR, the quaternary complex (P450)2.(CPR)2, and the homodimer (P450)2. The second plausible model additionally involves a weakly bound ternary complex (P450)2.CPR. Importantly, only the binary complex P450.CPR seems catalytically active in either of the two most probable mechanisms.     

Activity profile of glutathione-dependent enzymes and respiratory chain complexes in rats supplemented with antioxidants and treated with carcinogens

A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted. The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized. The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated. It was found that Pd/P450cam complex formation and decay obeys biphasic kinetics in contrast to the monophasic one for complexes formed by other redox partners within the system. Evidence for PdR/P450cam complex formation was obtained. It was found that, in contrast to Pd, which binds only to its redox partners, PdR and P450cam were able to form PdR/PdR and P450cam/P450cam complexes. A ternary PdR/Pd/P450cam complex was also registered. Its lifetime was sufficient to permit up to 60 turnovers to occur. The binding of Pd to P450cam and to PdR within the ternary complex occurred at distinct sites, with Pd serving as a bridge between the two proteins.     

Catalytic activity of isolated cytochromes P-450d and P-450c

Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined.     

Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios 相似文献   

Effect of homomeric P450-P450 complexes on P450 function

Previous studies have shown that the presence of one P450 enzyme can affect the function of another. The goal of the present study was to determine if P450 enzymes are capable of forming homomeric complexes that affect P450 function. To address this problem, the catalytic activities of several P450s were examined in reconstituted systems containing NADPH-POR (cytochrome P450 reductase) and a single P450. CYP2B4 (cytochrome P450 2B4)-, CYP2E1 (cytochrome P450 2E1)- and CYP1A2 (cytochrome P450 1A2)-mediated activities were measured as a function of POR concentration using reconstituted systems containing different concentrations of P450. Although CYP2B4-dependent activities could be explained by a simple Michaelis-Menten interaction between POR and CYP2B4, both CYP2E1 and CYP1A2 activities generally produced a sigmoidal response as a function of [POR]. Interestingly, the non-Michaelis behaviour of CYP1A2 could be converted into a simple mass-action response by increasing the ionic strength of the buffer. Next, physical interactions between CYP1A2 enzymes were demonstrated in reconstituted systems by chemical cross-linking and in cellular systems by BRET (bioluminescence resonance energy transfer). Cross-linking data were consistent with the kinetic responses in that both were similarly modulated by increasing the ionic strength of the surrounding solution. Taken together, these results show that CYP1A2 forms CYP1A2-CYP1A2 complexes that exhibit altered catalytic activity.     

Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase

Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.     

The cytochrome P450 2B4-NADPH cytochrome P450 reductase electron transfer complex is not formed by charge-pairing.

Attempts to covalently link NADPH-cytochrome P450 reductase to cytochrome P450 2B4 using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylisopropyl)carbodiimide, were unsuccessful, despite the fact that under the same conditions about 30% of P450 2B4 could be covalently linked with cytochrome b5 in a functionally active complex (Tamburini, P. P., and Schenkman, J. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 11-15). This suggested that the functional electron transfer complex between P450 2B4 and reductase is not stabilized by electrostatic forces. Raising the ionic strength of the medium is disruptive to salt bridges and was used to further test whether P450 2B4 and the reductase form charge-pairing complexes. Instead of inhibiting electron transfer, high ionic strength increased the apparent fast phase rate constant and the fraction of P450 2B4 reduced in the fast phase. The possibility that electron transfer between NADPH-cytochrome P450 reductase and P450 2B4 is diminished by charge repulsion was examined. Consistent with this hypothesis, the Km of P450 2B4 for reductase was decreased 26-fold by increasing the ionic strength from 10 to 100 mM sodium phosphate without affecting the Vmax. The rate of benzphetamine N-demethylation also was increased by elevation of the ionic strength. Electron transfer from the reductase to other charged redox acceptors, e.g. cytochrome c and ferricyanide, was also stimulated by increased ionic strength. However, no similar stimulation was observed with the uncharged acceptor 1,4-benzoquinone. Polylysine, a polypeptide that binds to anionic sites, enhanced electron transfer from NADPH to ferricyanide and the apparent fast phase of reduction of cytochrome P450. The results are consistent with the hypothesis that charges on NADPH-cytochrome P450 reductase and cytochrome P450 decrease the stability of the electron transfer complex.     

The formation of binary and ternary complexes of cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex. The implication in ACTH function.

Binary and ternary complexes of bovine adrenocortical mitochondrial cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex are formed in the presence of cholesterol and Emulgen 913. Both cholesterol and Emulgen 913 are required for the binding of cytochrome P-450scc with adrenodoxin. Since phospholipids are able to replace Emulgen 913 in this reaction, in vivo phospholipids of the mitochondrial inner membrane appear to play the function of the detergent. The dissociation constants of the cytochrome.adrenodoxin complex are 0.3 to 0.4 microM at 130 microM dimyristoylphosphatidylcholine and 0.9 microM at 120 microM Emulgen 913, whereas the dissociation constant for the ternary complex of cytochrome P-450scc with adrenodoxin reductase and adrenodoxin is 4.0 microM at 150 microM Emulgen 913. The stoichiometry of binary and ternary complexes reveals the 1:1 and 1:1:1 molar ratios, respectively, judging from chemical analyses after the fractionation of the complexes by gel filtration. Emulgen 913, Tween 20, ethylene glycol, myristoyllysophosphatidylcholine, dimyristoylphosphatidylcholine, and phosphatidylethanolamine show the enhanced activity of cholesterol side chain cleavage reaction with cytochrome P-450scc, adrenodoxin, adrenodoxin reductase, and NADPH. These results, in conjunction with earlier experiments, lead us to the proposal on the structure of the hydroxylase complex in the membrane and to the hypothesis on the regulation of the enzymatic activity by the availability of substrate cholesterol to the cytochrome. Hence, we propose a mobile P-450scc hypothesis for the response of the mitochondrion to adrenocorticotropic hormone stimuli.     

Active site topologies of bacterial cytochromes P450101 (P450cam), P450108 (P450terp), and P450102 (P450BM-3). In situ rearrangement of their phenyl-iron complexes.

The reactions of cytochromes P450101 (P450cam), P450108 (P450terp), and P450102 (P450BM-3) with phenyldiazene result in the formation of phenyl-iron complexes with absorption maxima at 474-478 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 decreases the 474-478 nm absorbance and shifts the phenyl group from the iron to the porphyrin nitrogens. Acidification and extraction of the prosthetic group from each of the ferricyanide-treated enzymes yields a different mixture of the four possible N-phenylprotoporphyrin IX regioisomers. The ratios of the regioisomers with the phenyl ring on pyrrole rings B, A, C, and D (in order of elution from the high performance liquid chromatography column) are, respectively: cytochrome P450cam, 0:0:1:4; P450terp, 0:0:0:1; and P450BM-3, 2:10:2:1. The isomer ratio for recombinant cytochrome P450BM-3 without the cytochrome P450 reductase domain (2:9:2:1) shows that the reductase domain does not detectably perturb the active site topology of cytochrome P450BM-3. Potassium ions modulate the intensity of the spectrum of the phenyl-iron complex of cytochrome P450cam, but do not alter the N-phenyl isomer ratio. Computer graphics analysis of the crystal structure of the cytochrome P450cam phenyl-iron complex indicates that the active site of cytochrome P450cam is open above pyrrole ring D and, to a small extent, pyrrole ring C, in complete agreement with the observed N-phenylprotoporphyrin IX regioisomer pattern. The regioisomer ratios indicate that the active site of cytochrome P450terp is only open above pyrrole ring D, whereas that of cytochrome P450BM-3 is open to some extent above all the pyrrole rings but particularly above pyrrole ring A. The bacterial enzymes thus have topologies distinct from each other and from those of the mammalian enzymes so far investigated, which have active sites that are open to a comparable extent above pyrrole rings A and D.