Domain-domain interaction in cytochrome P450BM-3.

The influence of ionic strength on the interactions between individually expressed functional domains of cytochrome P450BM-3 and the domains in the holoenzyme has been analyzed by spectrophotometric and fluorometric techniques. High ionic strength facilitated electron transfer from NADPH to the FMN moiety of the reductase domain (BMR) of P450BM-3 and did not affect the first electron transfer from FMN to the heme in the holoenzyme. The cytochrome c reductase activity of the holoenzyme was higher than that of BMR within the range of ionic strength tested. Two electron reduced FMN, ie incapable of transferring electrons to the heme iron of P450BM-3, was found to be capable of reducing cytochrome c. Fluorometric studies of the domains of P450BM-3 revealed that: 1) fluorescence of FAD is completely quenched in the FAD-binding domain; 2) BMR gives the highest quantum yield which is 2.5 times higher than that of the FMN-binding domain alone; 3) the heme domain (BMP) quenches a half and three-fourths of the fluorescence of the FMN in the linked BMP/FMN-binding domain and in the holoenzyme, respectively; 4) maximal quenching of the flavin fluorescence in the mixtures containing different combinations of the functional domains of P450BM-3 was observed at high ionic strength. The results indicate that the flavins in P450BM-3 are not in close proximity. Moreover, the presence of the FAD domain causes structural changes in the FMN domain resulting in an increase in the polarity of the FMN environment in BMR and may promote the interaction between FMN- and heme-binding domains in P450BM-3. Such domain interaction may facilitate the delivery of electrons from the FMN semiquinone to the heme and prevent the formation of the inactive two electron reduced species of the FMN. Thus, the high turnover number of P450BM-3 and tight coupling of the monooxygenation reaction are provided not only by the mechanism of reduction of the heme by the reductase but also by domain-domain interaction.

NADPH-P-450 reductase: structural and functional comparisons of the eukaryotic and prokaryotic isoforms.

The comparison of the properties of microsomal NADPH-P-450 reductase and the flavoprotein domain of P-450BM-3 (BMR) has revealed a significant difference in the mechanism of reduction of the hemoprotein P-450 by these flavoproteins. Microsomal NADPH-P-450 reductase transfers electrons to the hemoprotein by shuttling between hydroquinone and semiquinone forms of the FMN delivering one electron per cycle. Since the microsomal NADPH-P450 reductase has evolved as a component of multi-enzyme system, this type of mechanism may permit regulation of the steps of the P-450 reaction via variation in the affinity of the reductase for different P-450s, interaction with cytochrome b5, etc. In contrast, in the soluble, bacterial flavocytochrome P-450BM-3, the reductase domain has evolved together with a single unique heme domain. This enzyme was found to utilize the fastest and simplest way to reduce the heme iron, with the FMN moiety of BMR shuttling between the semiquinone and oxidized states. This mechanism of reduction provides the highest turnover number of any P-450 and tight coupling of the monooxygenation reaction. While there are clear differences in the intermediates involved in the reduction of P-450s by these two enzymes, the domain structure and presumably the mode of interaction between the reductase and P-450s has been maintained over evolutionary time.

P450BM-3; a tale of two domains--or is it three?

Over 400 P450s have been identified to date in prokaryotes and eukaryotes, plants and animals, mitochondria and endoplasmic reticulum. These enzymes function in areas such as metabolism and steroidogenesis. The eukaryotic members of this gene superfamily of proteins have proved difficult to study because of the hydrophobic nature of their substrates, their various redox partners, and membrane association. To better understand the structure/function relationship of P450s-what determines substrate specificity and selectivity, what determines redox-partner binding, and which regions are involved in membrane binding-we have compared the three crystallized, soluble bacterial P450s (two class I and one class II) and a model of a steroidogenic, eukaryotic P450 (P450arom), to define which structural elements form a conserved structural fold for P450s, what determines specificity of substrate binding and redox-partner binding, and which regions are potentially involved in membrane association. We believe that there is a conserved structural fold for all P450s that can be used to model those P450s that prove intransigent to structural determination. However, although there appears to be a conserved structural core among P450s, there is sufficient sequence variability that no two P450s are structurally identical. NADPH-P450 reductase transfers electrons from NADPH to P450 during the P450 catalytic cycle. This enzyme has usually been thought of as a simple globular protein; however, sequence analysis has shown that NADPH-P450 reductase is related to two separate flavoprotein families, ferredoxin nucleotide reductase (FNR) and flavodoxin. Recent studies by Wolff and his colleagues have shown that the FAD-binding FNR domain and FMN-binding flavodoxin domain of human NADPH-P450 reductase can be independently expressed in Escherichia coli. The subdomains can be used to reconstitute, however poorly, the monooxygenase activity of the P450 system. We have been utilizing the reductase domain of P450BM-3 to study the mechanism of electron transfer from NADPH to P450 in this complex multidomain protein. We have overexpressed both the FNR subdomain and the flavodoxin subdomain in E. coli and fully reconstituted the cytochrome c reductase activity of this enzyme. Our studies have shown that electron transfer from NADPH through the reductase domain to the P450 requires shuttling of the FMN subdomain between the reductase subdomain and the P450. Studies of the factors that control the molecular recognition and interaction among these three proteins are complicated by the weakness of the association and changes in the strength of the interaction depending on the redox state of each of the components. How these structural and mechanistic studies of a soluble bacterial P450 can be extended to gain a better understanding of the control of membrane-bound eukaryotic P450-dependent redox systems is discussed.

Reaction of carbon monoxide and molecular oxygen with P450terp (CYP108) and P450BM-3 (CYP102).

The kinetics and equilibrium of carbon monoxide binding and the reaction of autodecomposition of oxycomplexes of two bacterial P450s--P450BM-3 and P450terp--have been studied and compared with the corresponding properties of P450cam and some of the microsomal P450s. The second-order reaction rate constants for the reaction of P450terp and P450BM-3 with carbon monoxide in the absence of substrate at 5 degrees C were 3.6 x 10(6) and 1.6 x 10(6) M-1 s-1, respectively. The presence of the physiological substrate markedly influenced the rate of carbon monoxide binding with P450terp, decreasing the rate constant by approximately 100-fold, and did not have a significant effect on carbon monoxide binding with P450BM-3. The reaction of carbon monoxide with both cytochromes was monophasic in the absence or in the presence of substrate. The carbon monoxide binding enthalpy change was very small for P450BM-3 (-0.2 kcal mol-1) and more negative for P450terp (-3.2 kcal mol-1). The rate constants of decomposition of oxy-P450terp and oxy-P450BM-P (heme domain of P450BM-3) at 5 degrees C were 7.1 x 10(-4) and 2.5 x 10(-2) s-1, respectively. Raising the temperature to 20 degrees C resulted in 24- and 9-fold increase of decomposition of oxy-complexes of P450terp and P450BM-P, respectively. The kinetic properties of the binding reaction of diatomic gases to P450cam, P450terp, and P450BM-3 are consistent with the structures of their active sites.

Analysis of the myoglobin gene in heart disease.

Analysis of the myoglobin gene from a large number of patients with cardiac disease disclosed a single substantive mutation. However, no evidence of biochemical or physiological dysfunction due to this mutation was detected. We conclude that, within the limits of presently available techniques, myoglobin mutations are unlikely to contribute substantially to the genotypic background of cardiac disease in the general population.

Reconstitution of the fatty acid hydroxylase activity of cytochrome P450BM-3 utilizing its functional domains.

Cytochrome P450BM-3, a catalytically self-sufficient fatty acid monooxygenase from Bacillus megaterium, is a multidomain protein containing heme, FAD, and FMN. Previous attempts to reconstitute the fatty acid monooxygenase activity of intact P450BM-3 utilizing equimolar concentrations of the separate heme (BMP) and reductase (BMR) domains, have been unsuccessful because two-electron reduced FMN, which rapidly accumulates, is incapable of electron transfer to the heme iron. The present study of the reconstitution of the monooxygenase activity of P450BM-3 utilized combinations of the different functional domains of P450BM-3. For this purpose, the FAD/NADPH- and FMN-binding domains of P450BM-3 as well as the combination of the heme- and FMN-binding domains (BMP/FMN) have been expressed and purified. The reconstitution systems, consisting of either BMP/FMN and FAD domains or BMP, FMN, and FAD domains, were still less effective than the holoenzyme, P450BM-3, but were much more effective than a system consisting of BMP and BMR. The maximal rate of oxidation of palmitic acid by the newly developed reconstitution systems is still only approximately 5% of the activity of the holoenzyme. The reconstitution systems produced omega-1, omega-2, and omega-3 monohydroxy palmitic acid, but not the secondary products of palmitic acid hydroxylation observed with the holoenzyme. The physical cause of the inability to reconstitute fully the maximal activity of the holoenzyme as well as the lack of secondary product formation is not presently understood.

The flavoprotein domain of P450BM-3: expression, purification, and properties of the flavin adenine dinucleotide- and flavin mononucleotide-binding subdomains.

P450BM-3 is a self-sufficient fatty acid monooxygenase that can be expressed in Escherichia coli as either the holoenzyme or as the individual hemo- and flavoprotein domains. The flavoprotein domain (BMR) of P450BM-3 is soluble and contains an equimolar ratio of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and is functionally analogous to microsomal nicotinamide adenine dinucleotide phosphate (NADPH)-P450 reductases. These reductases have been proposed to have evolved through a fusion of genes encoding simple flavin-containing electron-transport proteins [Porter, T. D. (1991) Trends Biochem. Sci. 16, 154-158]. The gene encoding BMR has been divided into the coding regions for the FAD/NADPH- and FMN-binding domains. These proteins were overexpressed in E. coli and both domains were found to contain not less than 0.9 +/- 0.05 mol of FAD or FMN/mol of protein. Compared to BMR, the electron-accepting properties of the recombinant flavin domains were mainly conserved. Titration of the FMN domain with sodium dithionite resulted in the conversion of the protein to the fully reduced FMNH2 form without accumulation of intermediate semiquinone forms; however, a similar titration of the FAD domain gave clear evidence for the presence of a neutral, blue flavin semiquinone during the reduction. Titrations of the reduced forms of the domains with artificial electron acceptors indicated that the electron-transferring properties of both the FAD- and FMN domains were also conserved. The rate constants of reoxidation of the fully reduced FAD and FMN domains by molecular oxygen at 20 degrees C were found to be 2.5 and 0.1 min-1, respectively. The cytochrome c reductase activity of BMR could be fully reconstituted with the individual domains. The data presented support the hypothesis that BMR has a discrete multidomain structure.

The FMN-binding domain of cytochrome P450BM-3: resolution, reconstitution, and flavin analogue substitution.

Cytochrome P450BM-3 is a self-sufficient bacterial protein containing three naturally fused domains which bind either heme, FMN, or FAD. Resolution of protein and FMN from the isolated FMN-containing domain of cytochrome P450Betamicro-3 was accomplished using trichloroacetic acid. The apoprotein thus prepared was shown to rebind FMN to regenerate the original holoprotein as indicated by both spectroscopy and activity measurements. To better understand how the protein/flavin interaction might contribute to reactivity, the association process was studied in detail. Fluorescence quenching was used to measure a dissociation constant of the flavin-protein complex of 31 nM, comparable to FMN-containing proteins of similar reactivity and higher than that of flavodoxins. Stopped-flow kinetics were performed, and a multistep binding process was indicated, with an initial k(on) value of 1.72 x 10(5) M(-)(1) s(-)(1). Preparation of the apoprotein allowed substitution of flavin analogues for the native FMN cofactor using 8-chloro-FMN and 8-amino-FMN. Both were found to bind efficiently to the protein with only minor variations in affinity. Reductive titrations established that, as in the native FMN-containing FMN-binding domain, the 8-amino-FMN-substituted domain does not produce a stable one-electron-reduced species during titration with sodium dithionite. The 8-chloro-FMN-substituted domain, however, had sufficiently altered redox properties to form a stable red anionic semiquinone. The 8-chloro-FMN-substituted FMN-binding domain was shown in reconstituted systems to retain most of the cytochrome c reductase activity of the native domain but only a very small amount of palmitic acid hydroxylase activity. The 8-amino-FMN-substituted FMN-binding domain showed no palmitic acid hydroxylase activity and only 30% of the native cytochrome c reductase activity, demonstrating the importance of thermodynamics to the mechanism of this protein.

Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3.

The flavoprotein domain of P450BM-3 (BMR), which is functionally analogous to eukaryotic NADPH-P450 oxidoreductases, contains both FAD and FMN. When BMR is titrated with NADPH or sodium dithionite under anaerobic conditions, addition of 2 electron equivalents per mole of BMR results in the reduction of the high potential flavin (FMN) without the accumulation of semiquinone intermediates. Additional sodium dithionite first produces some neutral, blue flavin semiquinone radical and, finally, fully reduced FADH2. During reduction with NADPH, an absorbance increase characteristic of the formation of a flavin-pyridine nucleotide charge-transfer complex was observed only during the addition of the second mole of NADPH per mole of BMR. On the basis of these results, we conclude that the midpoint reduction potential for the FMN semiquinone/FMNH2 couple is more positive than that for FMN/FMN semiquinone. The kinetics of reduction of BMR with NADPH were studied by stopped-flow spectrophotometry. With a 1:1 ratio of NADPH to BMR, the absorbance changes can be fit to five consecutive first order reactions with rate constants of 350 s-1, 130 s-1, 27 s-1, 2.3 s-1, and 0.05 s-1. These reactions are most probably the following: (a) complex formation between BMR and NADPH; (b) reduction of FAD with formation of the NADP(+)-FADH- charge-transfer complex; (c) transfer of the first electron from FADH- to FMN to form an anionic, red FMN semiquinone leaving the FAD as the neutral, blue semiquinone. Precise identification of intermediates beyond this point is difficult. In the presence of a 10-fold molar excess of NADPH, the absorbance changes and rate constants are somewhat different due to the formation of several additional reduced species of BMR. The rate of the first step increases, confirming that this is the formation of the NADPH-BMR complex. Our results indicate that the kinetic and thermodynamic control of the flavins in BMR is significantly different from that in microsomal P450 reductase. The low potential of the anionic FMN semiquinone can be utilized to reduce the P450 heme. When the anionic semiquinone becomes protonated, its potential becomes more positive and it is readily reduced to FMNH2, which is not capable of reducing P450.

Laser flash induced electron transfer in P450cam monooxygenase: putidaredoxin reductase-putidaredoxin interaction.

The P450cam monooxygenase from Pseudomonas putida consists of three redox proteins: NADH-putidaredoxin reductase (Pdr), putidaredoxin (Pdx), and cytochrome P450cam. The redox properties of the FAD-containing Pdr and the mechanism of Pdr-Pdx complex formation are the least studied aspects of this system. We have utilized laser flash photolysis techniques to produce the one-electron-reduced species of Pdr, to characterize its spectral and electron-transferring properties, and to investigate the mechanism of its interaction with Pdx. Upon flash-induced reduction by 5-deazariboflavin semiquinone, the flavoprotein forms a blue neutral FAD semiquinone (FADH(*)). The FAD semiquinone was unstable and partially disproportionated into fully oxidized and fully reduced flavin. The rate of FADH(*) decay was dependent on ionic strength and NAD(+). In the mixture of Pdr and Pdx, where the flavoprotein was present in excess, electron transfer (ET) from FADH(*) to the iron-sulfur cluster was observed. The Pdr-to-Pdx ET rates were maximal at an ionic strength of 0.35 where a kinetic dissociation constant (K(d)) for the transient Pdr-Pdx complex and a limiting k(obs) value were equal to 5 microM and 226 s(-1), respectively. This indicates that FADH(*) is a kinetically significant intermediate in the turnover of P450cam monooxygenase. Transient kinetics as a function of ionic strength suggest that, in contrast to the Pdx-P450cam redox couple where complex formation is predominantly electrostatic, the Pdx-Pdr association is driven by nonelectrostatic interactions.

Structure of a cytochrome P450-redox partner electron-transfer complex.

The crystal structure of the complex between the heme- and FMN-binding domains of bacterial cytochrome P450BM-3, a prototype for the complex between eukaryotic microsomal P450s and P450 reductase, has been determined at 2.03 A resolution. The flavodoxin-like flavin domain is positioned at the proximal face of the heme domain with the FMN 4.0 and 18.4 A from the peptide that precedes the heme-binding loop and the heme iron, respectively. The heme-binding peptide represents the most efficient and coupled through-bond electron pathway to the heme iron. Substantial differences between the FMN-binding domains of P450BM-3 and microsomal P450 reductase, observed around the flavin-binding sites, are responsible for different redox properties of the FMN, which, in turn, control electron flow to the P450.

The FMN to heme electron transfer in cytochrome P450BM-3. Effect of chemical modification of cysteines engineered at the FMN-heme domain interaction site.

The crystal structure of the complex between the heme and FMN-containing domains of Bacillus megaterium cytochrome P450BM-3 (Sevrioukova, I. F., Li, H., Zhang, H., Peterson, J. A., and Poulos, T. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1863-1868) indicates that the proximal side of the heme domain molecule is the docking site for the FMN domain and that the Pro(382)-Gln(387) peptide may provide an electron transfer (ET) path from the FMN to the heme iron. In order to evaluate whether ET complexes formed in solution by the heme and FMN domains are structurally relevant to that seen in the crystal structure, we utilized site-directed mutagenesis to introduce Cys residues at positions 104 and 387, which are sites of close contact between the domains in the crystal structure and at position 372 as a control. Cys residues were modified with a bulky sulfhydryl reagent, 1-dimethylaminonaphthalene-5-sulfonate-L-cystine (dansylcystine (DC)), to prevent the FMN domain from binding at the site seen in the crystal structure. The DC modification of Cys(372) and Cys(387) resulted in a 2-fold decrease in the rates of interdomain ET in the reconstituted system consisting of the separate heme and FMN domains and had no effect on heme reduction in the intact heme/FMN-binding fragment of P450BM-3. DC modification of Cys(104) caused a 10-20-fold decrease in the interdomain ET reaction rate in both the reconstituted system and the intact heme/FMN domain. This indicates that the proximal side of the heme domain molecule represents the FMN domain binding site in both the crystallized and solution complexes, with the area around residue 104 being the most critical for the redox partner docking.