Kinetics of cytochrome P-450 reduction: evidence for faster reduction of the high-spin ferric state

Results are presented that support our hypothesis [Backes, W. L., Sligar, S. G., & Schenkman, J. B. (1980) Biochem. Biophys. Res. Commun. 97, 860-867] that the multiphasic reduction kinetics of cytochrome P-450 are, in part, due to the spin equilibrium of the ferric hemoprotein. The disappearance of the high-spin charge-transfer band at 650 nm during reduction of the hemoprotein by NADPH was fast, exhibiting a rate constant greater than that of the fast phase of reduction measured by formation of the carbon monoxide adduct. In contrast, the disappearance of the ferric low-spin form of the cytochrome was at a considerably slower rate. A mathematical expression of the fractional content of high-spin cytochrome P-450 was obtained by comparing the ratio of the initial rate of change in the fraction of total oxidized cytochrome remaining to the initial rate of change in the fraction of high-spin ferric P-450 remaining. Results supporting the model were obtained by using both microsomes and purified cytochrome P-450 RLM5. The calculation from experimental data yielded results that were similar to those obtained by different extrapolation methods used for estimation of the amount of high-spin cytochrome P-450, supporting further the proposed relationship between the spin equilibrium and the reduction kinetics of this hemoprotein.     

Evaluation of 3-azidiamantane as photoaffinity probe of cytochrome P450.

3-azidiamantane (DIA-N2) has been shown to be a photolabile carbene-generating probe interacting specifically with cytochrome P450 (P450) active centre. To evaluate the modification of P450 by the probe, radiolabelled [9-3H]-3-azidiamantane was prepared by reductive dehalogenation of its precursor, 3-oxo-9-bromodiamantane ethylene ketal. The synthesis was optimized as the proper precursor and reaction conditions were concerned to produce 96% pure product (overall yield 59%). An incorporation efficacy of the probe photoactivated at 366 nm was examined with two different proteins, BSA and rat phenobarbital-inducible P450 2B1, both having hydrophobic binding sites. Under photolysis the photoaffinity probe generated short-lived (> 90%) intermediates binding immediately to the protein. The yield of photoactivated DIA-N2 incorporation was 12% and 11% for BSA and P450, respectively. The presence of reduced glutathione, a scavenger of reactive intermediates, did not affect the probe incorporation markedly. On the other hand, scavengers entering the P450 active centre, methanol and dithiothreitol, reduced the protein labelling by 36% and 42%, respectively. Similarly, at DIA-N2, aminopyrine (substrates), and metyrapone (inhibitor) 50 times molar excess over the probe, prevented its binding by about 40%. In addition, when photoaffinity labelling was carried out with microsomal preparation, the substrate with a high affinity for the P450 2B1, diamantane, (at 20 times molar excess to the probe) caused 47% inhibition of the P450 covalent labelling. These results, suggesting a high specificity of the probe binding, show that it can be applied as a photoaffinity probe for cytochrome P450 2B1 active centre studies.     

Comparative EPR study on high-spin ferric porphine complexes and cytochrome P-450 having rhombic character.

Comparative EPR studies were made on two high-spin Fe(III) porphine model systems and mammalian liver microsomal cytochromes P-450, all of which exhibit approximately the same degrees of rhombicity in their EPR spectra. Comparison of g values and linewidths as a function of temperature, and of the microwave power saturation demonstrated that EPR characteristics of P-450 are more similar to the Fe(III) porphines having the thiolate axial ligand than in the other model systems, the mixed crystals of Fe(III) porphine with the corresponding free base porphine, in which no thiolate ligand is involved. There is, however, a discrepancy between P-450 and the model thiolates with respect to the size of the zero-field parameter D. These observations indicate that P-450 heme has essential structural features in common with thiolates but the Fe-S bond of P-450 may be modified from its normal orientation in model thiolates, probably as a result of the constraints imposed by the protein structure.     

4-cyanopyridine, a versatile spectroscopic probe for cytochrome P450 BM3

The nitrogenous pi -acceptor ligand 4-cyanopyridine (4CNPy) exhibits reversible ligation to ferrous heme in the flavocytochrome P450 BM3 (Kd=1.8 microm for wild type P450 BM3) via its pyridine ring nitrogen. The reduced P450-4CNPy adduct displays unusual spectral properties that provide a useful spectroscopic handle to probe particular aspects of this P450. 4CNPy is competitively displaced upon substrate binding, allowing a convenient route to the determination of substrate dissociation constants for ferrous P450 highlighting an increase in P450 substrate affinity on heme reduction. For wild type P450 BM3, Kd(red)(laurate)=82.4 microm (cf. Kd(ox)=364 microm). In addition, an unusual spectral feature in the red region of the absorption spectrum of the reduced P450-4CNPy adduct is observed that can be assigned as a metal-to-ligand charge transfer (MLCT). It was discovered that the energy of this MLCT varies linearly with respect to the P450 heme reduction potential. By studying the energy of this MLCT for a series of BM3 active site mutants with differing reduction potential (Em), the relationship EMLCT + (3.53 x = Em 17,005 cm)(-1) was derived. The use of this ligand thus provides a quick and accurate method for predicting the heme reduction potentials of a series of P450 BM3 mutations using visible spectroscopy, without the requirement for redox potentiometry.     

Monooxygenation by a thermophilic cytochrome P450 via direct electron donation from NADH

The catalysis of cytochrome P450s requires two-electron donation for the activation of an oxygen molecule. Here, we report the enzymatic catalysis of cytochrome P450, CYP119A2 (P450st), from a thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7, with NAD(P)H as an electron donor and no redox partners and the crystallographic analysis of P450st at high resolution. P450st can catalyse styrene epoxidation with either NADH or NADPH as an electron donor. The P450st reaction with NADH exhibited a sequential mechanism. X-ray crystallography at a resolution of 1.94 ? revealed a sufficiently large heme pocket for NAD(P)H binding and a novel contiguous channel from the active site to bulk solvent in the distal heme pocket. The narrow channel may transfer protons or water to the heme pocket even when a bulky compound, such as NAD(P)H, binds in the pocket. In addition, the F/G loop region (Leu151-Glu156), located around the substrate channel, was deleted in the mutant and constructed to improve the accessibility of NAD(P)H to the heme pocket. Kinetic properties of the Δ151-156 mutant were compared with those of the wild-type P450st. The K(m) value of the mutant was about 2 times lower than that of the wild-type. The results indicated that NAD(P)H could provide the electrons for P450st within the heme pocket.     

Structural evidence for direct hydride transfer from NADH to cytochrome P450nor

Nitric oxide reductase cytochrome P450nor catalyzes an unusual reaction, direct electron transfer from NAD(P)H to bound heme. Here, we succeeded in determining the crystal structure of P450nor in a complex with an NADH analogue, nicotinic acid adenine dinucleotide, which provides conclusive evidence for the mechanism of the unprecedented electron transfer. Comparison of the structure with those of dinucleotide-free forms revealed a global conformational change accompanied by intriguing local movements caused by the binding of the pyridine nucleotide. Arg64 and Arg174 fix the pyrophosphate moiety upon the dinucleotide binding. Stereo-selective hydride transfer from NADH to NO-bound heme was suggested from the structure, the nicotinic acid ring being fixed near the heme by the conserved Thr residue in the I-helix and the upward-shifted propionate side-chain of the heme. A proton channel near the NADH channel is formed upon the dinucleotide binding, which should direct continuous transfer of the hydride and proton. A salt-bridge network (Glu71-Arg64-Asp88) was shown to be crucial for a high catalytic turnover.     

Comparative EPR study on high-spin ferric porphine complexes and cytochrome P-450 having rhombic character

Comparative EPR studies were made on two high-spin Fe(III) porphine model systems and mammalian liver microsomal cytochromes P-450, all of which exhibit approximately the same degrees of rhombicity in their EPR spectra. Comparison of g values and linewidths as a function of temperature, and of the microwave power saturation demonstrated that EPR characteristics of P-450 are more similar to the Fe(III) porphines having the thiolate axial ligand than in the other model systems, the mixed crystals of Fe(III) porphine with the corresponding free base porphine, in which no thiolate ligand is involved.There is, however, a discrepancy between P-450 and the model thiolates with respect to the size of the zero-field parameter D. These observations indicate that P-450 heme has essential structural features in common with thiolates but the Fe-S bond of P-450 may be modified from its normal orientation in model thiolates, probably as a result of the constraints imposed by the protein stucture.     

A highly sensitive fluorogenic probe for cytochrome P450 activity in live cells

A derivative of rhodamine 110 has been designed and assessed as a probe for cytochrome P450 activity. This probe is the first to utilize a 'trimethyl lock' that is triggered by cleavage of an ether bond. In vitro, fluorescence was manifested by the CYP1A1 isozyme with k(cat)/K(M)=8.8x10(3)M(-1)s(-1) and K(M)=0.09microM. In cellulo, the probe revealed the induction of cytochrome P450 activity by the carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin, and its repression by the chemoprotectant resveratrol.     

Ontogenesis of rabbit liver cytochrome P450. Evidence for a cytochrome P450-IIE (3a)-related form prevailing during the post-natal period

The liver hydroxylating system, mainly composed of cytochromes P450, is not highly active during foetal life. If develops after birth and reaches the adult level several weeks post-partum. We have studied the ontogenesis of rabbit cytochrome P450 during the post-natal period. Total P450 as well as isozymes 2, 3b, 3c, 4 and 6 were measured. The evolution of these proteins with ageing, together with qualitative modification of an electrophoretic profile, produced evidence of an early developing P450 prevailing from one week to three weeks after birth. We isolated and characterized a cytochrome, called P450 2y, from two-week liver microsomes. It is closely related to P450 3a, an adult form of rabbit P450 induced by ethanol. They have similar molecular masses, the same lambda max of CO-reduced spectrum and exhibit immunological cross-reactivity. However, we cannot conclude that the two proteins are identical from N-terminal amino acid analysis or the two-dimensional gel electrophoresis pattern. These results, as well as the recent evidence of two different genes coding for the P450 3a family, strengthen the idea that P450 2y and 3a are distinct proteins. P450 2y seems to be an early developing form abundant soon after birth, while P450 3a is a delayed form appearing like most P450 isozymes during the fourth post-natal week. Besides the quantitative development during perinatal life, there is an important qualitative modification of liver cytochrome P450 content.     

Purification of cytochrome P450 BM-3 as a monooxygenase

After investigating two anion-exchange resins, the purification factor and activity yields of P450 BM-3 were higher with Resource Q than with DEAE-Sepharose FF. Screening of HIC media showed that Source 15ISO was the most suitable for purification of P450 BM-3. An effective isolation and purification procedure of P450 BM-3 was developed and included three steps: 35%-70% saturation (NH(4))(2)SO(4) precipitation, Source 15ISO hydrophobic interaction chromatograph and Sephacryl S-200 gel filtration chromatography. Using this protocol, the purification factor and P450 BM-3 activity recovery was 13.5 and 13.7%, respectively.     

Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4

Experiments demonstrating that cytochrome (cyt) b5 inhibits the activity of cytochrome P450 2B4 (cyt P450 2B4) at higher concentrations suggested that cyt b5 was occupying the cyt P450 reductase-binding site on cyt P450 2B4 and preventing the reduction of ferric cyt P450 (Zhang, H., Im, S.-C., and Waskell, L. (2007) J. Biol. Chem. 282, 29766-29776). In this work experiments were undertaken with manganese-containing cyt b5 (Mn-cyt b5) to test this hypothesis. Because Mn-cyt b5 does not undergo oxidation state changes under our experimental conditions, interpretation of the experimental results was unambiguous. The rate of electron transfer from cyt P450 reductase to ferric cyt P450 2B4 was decreased by Mn-cyt b5 in a concentration-dependent manner. Moreover, reduction of cyt P450 2B4 by cyt P450 reductase was incomplete in the presence of Mn-cyt b5. At a Mn-cyt b(5):cyt P450 2B4:cyt P450 reductase molar ratio of 5:1:1, the rate of reduction of ferric cyt P450 was decreased by 10-fold, and only 30% of the cyt P450 was reduced, whereas 70% remained oxidized. It could be demonstrated that Mn-cyt b5 had its effect by acting on cyt P450, not the reductase, because the reduction of cyt c by cyt P450 reductase in the presence of Mn-cyt b5 was not effected. Furthermore, under steady-state conditions in the cyt P450 reconstituted system, Mn-cyt b5, which lacks the ability to reduce oxyferrous cyt P450 2B4, was unable to stimulate the activity of cyt P450. Mn-cyt b5 only inhibited the cyt P450 2B4 activity. In conjunction with site-directed mutagenesis studies and experiments that strongly suggested that cyt b5 competed with cyt P450 reductase for binding to cyt P450, the current investigation demonstrates unequivocally that cyt b5 inhibits the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to cyt P450, a prerequisite for electron transfer from cyt P450 reductase to cyt P450 and catalysis.     

A rabbit liver constitutive form of cytochrome P450 responsible for amphetamine deamination

A cytochrome P450 isozyme responsible for amphetamine deamination was purified from hepatic microsomes of untreated rabbits. The purification procedures consisted of a set of column chromatographies with omega-aminooctyl-Sepharose 4B, DEAE-cellulose, CM-Sephadex C-50, and hydroxyapatite. The deamination activity was determined by measuring the formation of phenylacetone after derivatization to the p-nitrobenzyloxim by HPLC. This isozyme, which was designated P450APD, showed a monomeric molecular weight of 51,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited an absorption maximum of reduced CO complex at 451 nm. On the basis of the specificity toward testosterone metabolism and the N-terminal amino acid sequence, P450APD was attributed to a member of P450 class IIC subfamily, which is identical or closely related to LM3b (D. R. Koop and M. J. Coon (1979) Biochem, Biophys, Res. Commun. 91, 1075-1081), form 3b (E. F. Johnson (1980) J. Biol. Chem. 255, 304-309), and other similar preparations. Antibody against the P450APD inhibited about 80% of the amphetamine deamination activity in rabbit hepatic microsomes as well as in the reconstitution system of this P450. The present results support that P450APD is the major P450 isozyme responsible for amphetamine deamination in rabbit liver.     

Proton nmr spectroscopy of high-spin ferrous cytochrome P-450 models

Alkyl mercaptide complexes of both synthetic and natural-derivative iron(II) porphyrins have been characterized in DMSO solution by proton nmr spectroscopy. A single mercaptide ligand binds to form a high-spin iron(II) complex as determined by solution magnetic measurements and the nmr isotropic shift pattern. Ligand exchange is slow on the nmr time scale unlike corresponding 2-methyl imidazole exchange rates which are very rapid. Further comparison of mercaptide and 2-methyl imidazole adducts reveals a downfield bias in isotropic shift values for the mercaptide species, which may be explained by different signs in the dipolar shift term for the two complexes. This apparent magnetic anisotropy of the mercaptide complex is in the same direction, although smaller, than that observed for bacterial cytochrome P-450. Isotropic shift values of at least 250 ppm for methylene resonances of the coordinated mercaptide support a very efficient unpaired spin delocalization for this axial ligand.     

The high-spin/low-spin equilibrium in cytochrome P-450--a new method for determination of the high-spin content.

A new method for determination of the population of the high-spin state (high-spin content) in ferric cytochrome P-450 is presented. Based on curve fitting the electronic absorption spectra with a linear combination of gaussian bands analytical functions for the pure high-spin and pure low-spin states were constructed. These functions were used to fit the high-spin/low-spin mixed spectra. A good fit of the absorption spectra of six different cytochrome P-450 proteins in the presence and absence of substrates was found, indicating a similar pi-electron structure of the porphyrin and a similar chemical nature of the nearest coordination sphere of the iron in all cytochrome P-450 proteins.     

Identification of cytochrome P450IA2 as a human autoantigen

Autoantibodies occurring in a patient with idiopathic autoimmune type chronic active hepatitis (CAH) were found to react with purified rabbit cytochrome P450IA2 and to a much lesser extent with P450IA1. Both cytochrome P450s are known to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rabbit, and the expression of the microsomal protein recognized by the patient serum was induced in adult rabbit livers after treatment with TCDD. This protein is only weakly detected in liver microsomes from neonatal rabbits exposed to TCDD in utero, which is consistent with the age-dependent induction of P450IA2 by TCDD. The serum specifically reacted with a protein of similar size in microsomes prepared from COS-1 cells transfected with an expression vector containing the full length human P450IA2 cDNA. This reactivity was not detected in the cells transfected with the vector alone, indicating that the antibody recognizes human P450IA2. In addition, the serum extensively inhibited 7-ethoxyresorufin O-deethylation catalyzed by isolated human liver microsomes. This catalytic activity is associated with class IA P450s in other species. A screen of sera from patients with various hepatic and nonhepatic diseases indicates that the autoantibody to P450IA2 occurs rarely in CAH. Cytochrome P450IA2 becomes the third P450 identified as an autoantigen in inflammatory liver diseases.     

New perspectives on the conformational equilibrium regulating multi-phasic reduction of cytochrome P450 2B4 by cytochrome P450 reductase

The pre-steady-state reduction of cytochrome P450 (P450) 2B4 by P450 reductase (reductase) was modeled by assuming that an equilibrium between three catalytic conformers of P450 regulates the multi-phasic reduction of the enzyme. This model was compared to a model of reduction involving a minimum number of phases. Based on several criteria, the former model seems to provide an improved fit to the reduction data. Substrates were divided into two groups based on their effects at different concentrations of reductase. Surprisingly, in the presence of some substrates (group 1) but not others (group 2), the rate of reduction was actually slower with an excess of reductase than with equimolar reductase and P450. Presumably, oxidized reductase binds differently to P450 than reduced reductase. A schematic model based on two sites of interaction between reductase and P450 2B4 is offered to explain the unusual reduction kinetics with the two different groups of substrates.     

The B' helix determines cytochrome P450nor specificity for the electron donors NADH and NADPH

Nitric oxide reductase (Nor) cytochrome P450nor (P450nor) is unique because it is catalytically self-sufficient, receiving electrons directly from NADH or NADPH. However, little is known about the direct binding of NADH to cytochrome. Here, we report that oxidized pyridine nucleotides (NAD(+) and NADP(+)) and an analogue induce a spectral perturbation in bound heme when mixed with P450nor. The P450nor isoforms are classified according to electron donor specificity for NADH or NADPH. One type (Fnor, a P450nor of Fusarium oxysporum) utilizes only NADH. We found that NAD(+) induced a type I spectral change in Fnor, whereas NADP(+) induced a reverse type I spectral change, although the K(d) values for both were comparable. In contrast, NADP(+) as well as NAD(+) caused a type I spectral change in Tnor, a P450nor isozyme from Trichosporon cutaneum that utilizes both NADH and NADPH as electron donors. The B' helix region of Tnor ((73)SAGGKAAA(80)) contains some Ala and Gly residues, whereas the sequence is replaced at a few sites with more bulky amino acid residues in Fnor ((73)SASGKQAA(80)). A single mutation (S75G) significantly improved the NADPH- dependent Nor activity of Fnor, and the overall activity was accelerated via the NADPH-enhanced reduction step. These results showed that pyridine nucleotide cofactors can bind P450nor and that only a few residues in the B' helix region determine cofactor specificity. We further showed that a poor electron donor (NADPH) could also bind Fnor, but an appropriate configuration for electron transfer is blocked by steric hindrance mainly by Ser(75) against the 2'-phosphate moiety. The present results along with previous observations together revealed a novel motif for cofactor binding.     

Resolution of two substrate-binding sites in an engineered cytochrome P450eryF bearing a fluorescent probe

To elucidate the mechanisms of cooperativity of cytochrome P450eryF an SH-reactive fluorescent probe was introduced close to the substrate-binding site. Cys-154, the only accessible cysteine, was eliminated by site-directed mutagenesis, and a novel cysteine was substituted for Ser-93 in the B'/C loop. S93C, C154A, C154S, S93C/C154A, and S93C/S154C were characterized in terms of affinity for 1-pyrenebutanol (1-PB), cooperativity, and ionic-strength dependence of the 1-PB-induced spin shift. S93C/C154S retains the key functional properties of the wild-type, and modification by three different SH-reactive probes had little effect on the characteristics of the enzyme. The labeled proteins exhibited fluorescence resonance energy transfer from 1-PB to the label, which allowed us to resolve two substrate-binding events, and to determine the corresponding KD values (KD1 = 1.2 +/- 0.2 microM, KD2 = 9.4 +/- 0.8 microM). Using these values for analysis of the substrate-induced spin transition, we demonstrate that the interactions of P450eryF with 1-PB are consistent with a sequential binding mechanism, where substrate interactions at a higher-affinity site cause a conformational transition crucial for the binding of the second substrate molecule and subsequent spin shift. This transition is apparently associated with an important rearrangement of the system of salt links in the proximity of Cys-154.     

Electrochemical reduction of cytochrome P450 as an approach to the construction of biosensors and bioreactors

In the present review an attempt was made to present an up-to-date amount of the data on electrochemical reduction of the hemoprotein cytochrome P450. The concept and potentialities of enzyme electrodes--transducers--as the main element for construction of electrochemical biosensors were discussed. Different types of electrodes for bioelectrochemistry were analysed. New nanotechnological approaches to cytochrome P450 immobilisation were reported. It was shown that nanobiotechnology in electrochemistry has potential application in manufacturing biosensors and bioreactors for clinical medicine and pharmacology.