Cytochrome P-450 LM2 reduction. Substrate effects on the rate of reductase-LM2 association

The effect of substrate on LM2 reduction was examined using a reconstituted system containing dilauroylphosphatidylcholine, NADPH-cytochrome P-450 reductase, and cytochrome P-450 LM2 in a 160:1.5:1 molar ratio. In general, most substrates increased the rate constants of both the first and second phases of reduction as well as the fraction of LM2 reduced in the first phase. The correlation between the high spin content of the cytochrome and each of these kinetic parameters was weaker than expected if spin state controlled LM2 reduction. Further, substrate was shown to exert a rapid effect on both the high spin content and stimulation of reduction indicating that the low spin to high spin shift cannot be responsible for the slow phase of reduction for this particular isoform. Cytochrome P-450 reduction was also examined in both phospholipid-containing and soluble systems where the LM2 and reductase were not present as a preformed complex. In these systems the reactions were substantially slower than with the standard reconstituted system. Addition of substrate enhanced the rate of reduction, indicating that the rate of association between LM2 and the reductase was increased by substrate addition. The strong correlation between the rate of LM2 reduction in a preformed complex and the logarithm of the rate of LM2 and reductase association implicates the rate of functional complex formation as the factor controlling the slow phase of reduction.     

Inverse relationship between cytochrome P-450 phosphorylation and complexation with cytochrome b5

Cytochrome P-450 LM2 purified from rabbit liver microsomes has been shown to be a substrate for cAMP-dependent protein kinase. Cytochrome b5, in contrast, was a very poor substrate for cAMP-dependent protein kinase, although it stimulated the activity of the kinase toward histone. When purified rabbit cytochrome b5 was mixed with purified LM2, phosphorylation of LM2 by cAMP-dependent protein kinase was inhibited approximately 80-90%. Recently, a functional covalent complex of cytochrome b5 and LM2 was prepared and purified to homogeneity (P.P. Tamburini and J.B. Schenkman (1987) Proc. Natl. Acad. Sci. USA 84, 11-15). When present as a covalent complex with cytochrome b5, the phosphorylation of LM2 in the complex by cAMP-dependent protein kinase was also inhibited about 80-90% relative to an equivalent amount of LM2 alone. On the other hand, when the LM2 was phosphorylated prior to interaction with cytochrome b5, the ability of the latter to perturb the spin equilibrium of LM2 and oxidation of p-nitroanisole by the LM2 was diminished to an extent comparable to the degree of phosphorylation. The results suggest either that the phosphorylation site on LM2 may be within the cytochrome b5 binding site or that phosphorylation and cytochrome b5 cause mutually exclusive conformational changes in LM2. In addition, eight different forms of cytochrome P-450 from the rat (RLM2, RLM3, fRLM4, RLM5, RLM5a, RLM5b, RLM6, and PBRLM5) were examined as potential substrates for cAMP-dependent protein kinase under the same conditions. Maximal phosphorylation of about 20 mol% was obtained with LM2, and about half as much with PBRLM5. The low extent of phosphorylation of LM2 was not due to the prior presence of phosphate on the enzyme since LM2, as isolated, contains less than 0.1 mol phosphate/mol of enzyme. The other forms of cytochrome P-450 tested showed little or no phosphorylation in vitro despite the presence of a cAMP-dependent protein kinase phosphorylation sequence on at least two of them.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions.

The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.     

Phosphorylation of cytochrome P450: regulation by cytochrome b5

Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by cAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maximum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of cAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for cAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259, 441-448) that cytochrome b5 inhibits the phosphorylation of LM2 by cAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b5 is competitive, with a Ki = 0.48 microM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome b5. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome b5 binding domain of the enzymes.     

Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4

The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form, P-420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P-450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P-450 isozymes.     

Inhibition of cytochrome-P450 reductase by polyols has an electrostatic nature.

The present study was undertaken to examine the nature of the inhibitory action of glycerol on the liver microsomal monooxygenase system. In agreement with earlier observations, glycerol inhibited benzphetamine N-demethylation by liver microsomes of the phenobarbital-treated rabbit. The presence of glycerol in the medium did not affect binding of the substrate to cytochrome P450. Another polyol, ethylene glycol, was equally efficient in inhibiting benzphetamine N-demethylation. Both also inhibited reduction of rabbit cytochrome P450 LM2, cytochrome c and potassium ferricyanide by NADPH-cytochrome-P450 reductase in microsomes. Recently, we showed that the stimulation of electron transfer by increased ionic strength is due to neutralization of electrostatic interaction between NADPH-cytochrome-P450 reductase and its charged redox partners [Voznesensky, A. I. & Schenkman, J. B. (1992) J. Biol. Chem. 267, 14669-14676]. Polyols have an opposite effect to that of salt on ionic properties of a solution. They decrease the dielectric constant, thereby promoting electrostatic interactions between proteins. Addition of polyols decreased the conductivity of the medium. When rates of electron transfer to charged acceptors, cytochrome P450, cytochrome c and potassium ferricyanide, at various salt and polyol concentrations, relative to activities in 200 mM sodium phosphate, were plotted as a function of the conductivity the data for each acceptor fit on the same line. In contrast, neither alteration of ionic strength nor polyol addition affected the rate of electron transfer from NADPH-cytochrome-P450 reductase to an uncharged acceptor 1,4-benzoquinone. The data obtained is consistent with our earlier suggestion that charge repulsion limits redox interactions between rabbit cytochrome P450 LM2 and its reductase at low ionic strength, and suggest that the observed action of polyols is the result of enhancement of electrostatic interactions that inhibits electron transfer between NADPH-cytochrome-P450 reductase and its charged redox partners. In congruence with the hypothesis, the Km of rabbit cytochrome P450 LM2 for NADPH-cytochrome-P450 reductase was increased almost one order of magnitude by elevating the glycerol content from 5% to 25% (by vol.) without a change in Vmax.     

Complex formation in vesicle-reconstituted mitochondrial cytochrome P450 systems (CYP11A1 and CYP11B1) as evidenced by rotational diffusion experiments using EPR and ST-EPR.

Rotational diffusion measurements using EPR and saturation transfer EPR were applied to analyze complex formation between the electron-transfer components of the mitochondrial steroid-hydroxylating cytochrome P450 systems (CYP11A1 and CYP11B1) in phosphatidylcholine/phosphatidylethanolamine/cardiolipin vesicles prepared by octyl glucoside dialysis/adsorption. Octyl glucoside reconstitution of P450SCC results in large vesicles, which have an advantage over small vesicles in that vesicle tumbling does not contribute to measured rotational diffusion rates. Immobilization of spin-labeled adrenodoxin by both P450SCC and adrenodoxin reductase indicates equimolar complexation between P450SCC and adrenodoxin as well as between adrenodoxin reductase and adrenodoxin. Combination of rotational diffusion and antibody cross-linking confirmed the complexation of adrenodoxin with P450SCC and for the first time provided direct evidence of a complex between P450SCC and P45011beta in the membrane. In contrast, no evidence was found for the existence of adrenodoxin reductase-P450SCC complexes or a ternary complex of all three proteins. Thus, these experiments confirm the shuttle mechanism of electron transfer to vesicle-reconstituted P450SCC and P45011beta.     

Differences in the spectral interactions between NADPH-cytochrome P-450 reductase and a series of cytochrome P-450 enzymes

The interaction between NADPH-cytochrome P-450 reductase and a series of cytochrome P-450 isozymes was investigated using UV-visible spectrophotometry. In the absence of substrate the interactions between the reductase and RLM3, RLM5, and RLM5a were tight, exhibiting sub-micromolar dissociation constants and resulted in type I spectra of varying magnitude from which the following increases in the proportion of high spin hemoprotein were calculated; RLM3 (7%), RLM5 (36%), RLM5a (6%), LM2 (29%), RLM2 (0%). Preincubation of LM2 with its type I substrate benzphetamine increased the affinity of the cytochrome for the reductase. Using initial estimates of the P-450 spin states in the absence of reductase in conjunction with the spectral binding data and equations relating these parameters to the microequilibria for the association of reductase with high or low spin P-450, RLM3, RLM5, RLM5a and LM2 were shown to bind significantly more tightly to high spin P-450. The relevance of this data to the understanding of spin state influence on P-450 reduction is discussed.     

Photochemical properties of a riboflavins/cytochrome P450 2B4 complex

The present study demonstrates the possible use of a non-covalent complex of riboflavins with cytochrome P450 2B4 (artificial flavocytochrome P450 2B4) for photo-induced intermolecular electron transfer between the isoalloxazine cycle of flavins and the ferric heme group of cytochrome P450 2B4. Riboflavin was used as a light-induced electron donor for the transfer of electrons to cytochrome P450. The quantitative measurement of the photocurrent, generated by photoreduction of non-covalent flavocytochrome P450 2B4, was carried out. In the presence of typical substrates for cytochrome P450 2B4 the decrease of cathodic photocurrent occurred, generated not only by riboflavin itself but also by a riboflavin/cytochrome P450 complex. It was demonstrated that flavocytochromes might serve as molecular amplifiers of a photocurrent, generated upon flavins' reduction. Introduction of flavin residues into the cytochrome P450 molecule transformed this haemoprotein into a photoreceptor and a photodiode and, in addition, into a photosensitive and photo-activated enzyme.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5

Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome P-450 was substantially increased. Cytochrome b5 caused 3- and 7-fold increases in the binding affinities of RLM5 and LM2 for benzphetamine, respectively, and benzphetamine decreased the apparent Kd for cytochrome b5 binding. Upon formation of the ternary complex between cytochromes P-450, b5, and benzphetamine the percentage of cytochrome P-450 in the high spin state was increased from 28 to 74 (RLM5) and from 9 to 85 (LM2). Cytochrome b5 caused 13- and 7-fold increases in the rate of RLM5- and LM2-dependent p-nitroanisole demethylation, respectively. Amino-modified (ethyl acetimidate or acetic anhydride) cytochrome b5 produced results similar to those obtained above with native cytochrome b5. In contrast, modification of as few as 5 mol of carboxyl groups/mol of amidinated cytochrome b5 resulted in both a substantial loss of the spectrally observed interactions with either cytochrome P-450 LM2 or cytochrome P-450 RLM5, and in a loss of the cytochrome b5-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase. In further studies, native and fully acetylated cytochromes b5 reoxidized carbonmonoxy ferrous LM2 at least 20 times faster than amidinated, carboxyl-modified cytochrome b5 derivatives. In contrast, amidination, or acetylation of amino groups, or amidination of amino groups plus methylamidination of the carboxyl groups did not appreciably slow the rate of reduction of the cytochrome b5 by NADPH-cytochrome P-450 reductase. Collectively, the results provide strong evidence for an essential role of cytochrome b5 carboxyl groups in functional interactions with RLM5 and LM2.     

Spin state control of cytochrome P-450 reduction and catalytic activity in a reconstituted P-450 LM2 system as induced by a series of benzphetamine analogues

Reconstituted liposomal cytochrome P-450 LM2 was reacted with a series of benzphetamine analogues as substrates. Based on the thermodynamical model of Ristau et al. (Biochim. Biophys. Acta, 536 (1978) 226-234) the free enthalpy of substrate binding to the high spin form of the enzyme was shown to correlate with the total high spin content of the respective enzyme substrate complex. Reduction and substrate N-demethylation rates as well have been evidenced to linearly correlate with the substrate-induced spin shift delta alpha and moreover with the spin content alpha. The data obtained provide further experimental support for the spin state regulation of the reduction and conversion rate of cytochrome P-450 LM2.     

Mechanistic studies of cytochrome P450 2B1 inactivation by xanthates

Xanthates have previously been shown to inactivate the phenobarbital-inducible rat cytochrome P450 2B1 as well as its human homologue P450 2B6. The inactivation was mechanism-based and the loss in enzymatic activity was due to covalent binding of a reactive xanthate intermediate to the P450 2B1 apoprotein. In this report, we investigated various mechanistic events to elucidate the individual step(s) in the P450 catalytic cycle that are compromised due to the inactivation by xanthates. Different xanthates displayed typical type I binding spectra and the spectral binding constants were in the low-millimolar range. A dramatic loss in 7-ethoxy-4-(trifluoromethyl)coumarin activity was observed when P450 2B1 was incubated with five different xanthates in the presence of NADPH. With the exception of the C14 xanthate, virtually no loss of absorbance at 418 or 450 nm in the reduced-CO complex was observed. Long-chain xanthates were able to affect the rate of the first electron transfer in the P450 catalytic cycle by stabilizing the heme in its low-spin state. n-Octyl xanthate (C8) metabolism led to very little observable oxy-ferro intermediate complex formation. The alternate oxidant tert-butyl hydroperoxide was able to support the inactivation reaction of C8 in the absence of reductase or NADPH. The rates of reduction of native, C8-exposed, and C8-inactivated P450 2B1 were measured. The C8-inactivated P450 had a 62% lower rate of reduction in the absence or presence of benzphetamine compared to the native enzyme. Product formation of the three enzyme preparations was quantified with benzphetamine as the substrate. The C8-inactivated P450 2B1 exhibited a much lower rate of NADPH consumption and formation of formaldehyde. However, the ratio of H2O2 to formaldehyde production increased from 1:1 for the native enzyme to 2.8:1 for the inactivated P450. Together these observations indicate that the covalent modification of P450 2B1 by a reactive intermediate of xanthates reduces the rate of the first electron transfer by the reductase and also leads to uncoupling of electron transfer from product formation by diverting a greater proportion of the electrons to H2O2 formation.     

Direct observation of a novel perturbed oxyferrous catalytic intermediate during reduced putidaredoxin-initiated turnover of cytochrome P-450-CAM: probing the effector role of putidaredoxin in catalysis

The single turnover of (1R)(+)-camphor-bound oxyferrous cytochrome P450-CAM with one equivalent of dithionite-reduced putidaredoxin (Pdx) was monitored for the appearance of transient intermediates at 3 degrees C by double mixing rapid scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting versus reduced Pdx: a perturbed oxyferrous derivative, a species that was a mixture of high and low spin Fe(III), and high spin ferric camphor-bound enzyme. The rates of the first two steps, approximately 140 and approximately 85 s(-1), were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a preformed reduced P450-CAM.Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity (i.e. during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substrate-free P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first but not the second electron.     

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.     

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.     

Determination of the rate of reduction of oxyferrous cytochrome P450 2B4 by 5-deazariboflavin adenine dinucleotide T491V cytochrome P450 reductase

The use of 5-deazaFAD T491V cytochrome P450 reductase has made it possible to directly measure the rate of electron transfer to microsomal oxyferrous cytochrome (cyt) P450 2B4. In this reductase the FMN moiety can be reduced to the hydroquinone, FMNH(2), while the 5-deazaFAD moiety remains oxidized [Zhang, H., et al. (2003) Biochemistry 42, 6804-6813]. The rate of electron transfer from 5-deazaFAD cyt P450 reductase to oxyferrous cyt P450 was determined by rapidly mixing the ferrous cyt P450-2-electron-reduced 5-deazaFAD T491V reductase complex with oxygen in the presence of substrate. The 5-deazaFAD T491V reductase which can only donate a single electron reduces the oxyferrous cyt P450 and oxidizes to the air-stable semiquinone, with rate constants of 8.4 and 0.37 s(-1) at 15 degrees C. Surprisingly, oxyferrous cyt P450 turns over more slowly with a rate constant of 0.09 s(-1), which is the rate of catalysis under steady-state conditions at 15 degrees C (k(cat) = 0.08 s(-1)). In contrast, the rate constant for electron transfer from ferrous cyt b(5) to oxyferrous cyt P450 is 10 s(-1) with oxyferrous cyt P450 and cyt b(5) simultaneously undergoing spectral changes. Quantitative analyses by LC-MS/MS revealed that the product, norbenzphetamine, was formed with a coupling efficiency of 52% with cyt b(5) and 32% with 5-deazaFAD T491V reductase. Collectively, these results suggest that during catalysis a relatively stable reduced oxyferrous intermediate of cyt P450 is formed in the presence of cyt P450 reductase but not cyt b(5) and that the rate-limiting step in catalysis follows introduction of the second electron.     

Chemical modification of cytochrome P-450 LM4. Identification of functionally linked tyrosine residues

Cytochrome P-450 LM4 (RH, reduced flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes was chemically modified with tetranitromethane. Nitration of two tyrosine residues inhibits the p-nitrophenetole O-deethylase activity of the enzyme by about 80%. Sequencing the 3-nitrotyrosine-containing peptides after HPLC tryptic peptide mapping reveals that mainly Tyr-243 and Tyr-271 are nitrated, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent. Nitration of tyrosine residues affects the complex formation with p-nitrophenetole, alpha-naphthoflavone and metyrapone as indicated by an increased affinity towards p-nitrophenetole and by a decreased affinity for the latter compounds. Furthermore, nitration interferes with the electron transfer from NADPH-cytochrome P-450-reductase to cytochrome P-450 LM4 resulting in a slowed down reduction reaction. The results suggest that Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are functionally involved in the interaction with NADPH-cytochrome P-450 reductase.     

A shunted system of electron transport from NAD(P)H to cholesterol-hydroxylating cytochrome P-450 in adrenal cortex mitochondria

The two main approaches presently used for cytochrome P-450scc modelling are as follows: i) the use of chemical compounds carrying activated oxygen species, e. g., peracids, organic hydroperoxides, iodosobenzene, etc., ii) the use of electrochemical reduction in the presence of redox-active compounds. In the present work, a new model system for simulation of steroidogenic electron transfer is proposed, which reduces cytochrome P-450 scc by NADPH in the absence of adrenodoxin reductase and adrenodoxin. Phenazine methosulfate is used as an electron carrier. More than 95% of cytochrome P-450scc is reduced in a model system. The reduction kinetics is characterized by a lag phase, thus indicating complex formation between cytochrome P-450scc and phenazine methosulfate or formation of intermediate reducing equivalents. NADH may also serve as an electron donor for cytochrome P-450scc. Phenazine methosulfate can reduce microsomal cytochrome P-450 LM2 and b5, but not cytochrome P-450 LM4. Superoxide dismutase does not affect the reduction, thus indicating that O9.- is not involved in the reduction process. The mechanism of hemoprotein reduction and the nature of intermediates which can be formed in the model system is proposed.     

Cysteinyl-tRNA formation: the last puzzle of aminoacyl-tRNA synthesis

A 1H nuclear magnetic resonance study of the complex of cytochrome P450cam-putidaredoxin has been performed. Isocyanide is bound to cytochrome P450cam in order to increase the stability of the protein both in the reduced and the oxidized state. Diprotein complex formation was detected through variation of the heme methyl proton resonances which have been assigned in the two redox states. The electron transfer rate at equilibrium was determinated by magnetization transfer experiments. The observed rate of oxidation of reduced cytochrome P450 by the oxidized putidaredoxin is 27 (+/- 7) per s.