L358P mutation on cytochrome P450cam simulates structural changes upon putidaredoxin binding: the structural changes trigger electron transfer to oxy-P450cam from electron donors

To investigate the functional and structural characterization of a crucial cytochrome P450cam (P450cam)-putidaredoxin (Pdx) complex, we utilized a mutant whose spectroscopic property corresponds to the properties of the wild type P450cam in the presence of Pdx. The 1H NMR spectrum of the carbonmonoxy adduct of the mutant, the Leu-358 --> Pro mutant (L358P), in the absence of Pdx showed that the ring current-shifted signals arising from d-camphor were upfield-shifted and observed as resolved signals, which are typical for the wild type enzyme in the presence of Pdx. Signals from the beta-proton of the axial cysteine and the gamma-methyl group of Thr-252 were also shifted upfield and down-field, respectively, in the L358P mutant as observed for Pdx-bound wild type P450cam. The close similarity in the NMR spectra suggests that the heme environment of the L358P mutant mimics that of the Pdx-bound enzyme. The functional analysis of the L358P mutant has revealed that the oxygen adduct of the L358P mutant can promote the oxygenation reaction for d-camphor with nonphysiological electron donors such as dithionite and ascorbic acid, showing that oxygenated L358P is "activated" to receive electron from the donor. Based on the structural and functional characterization of the L358P mutant, we conclude that the Pdx-induced structural changes in P450cam would facilitate the electron transfer from the electron donor, and the Pdx binding to P450cam would be a trigger for the electron transfer to oxygenated P450cam.     

Spectral studies of tert-butyl isothiocyanate-inactivated P450 2E1

Inactivation of cytochrome P450 2E1 by tert-butyl isothiocyanate (tBITC) resulted in a loss in the spectrally detectable P450-reduced CO complex. The heme prosthetic group does not appear to become modified, since little loss of the heme was observed in the absolute spectra or the pyridine hemochrome spectra, or in the amount of heme recovered from HPLC analysis of the tBITC-inactivated samples. Prolonged incubations of the inactivated P450 2E1 with dithionite and CO resulted in a recovery of both the CO complex and the enzymatic activity. Inactivated samples that were first reduced with dithionite for 1 h prior to CO exposure recovered their CO spectrum to the same extent as samples not pretreated with dithionite, suggesting that the major defect was an inability of the inactivated sample to bind CO. Spectral binding studies with 4-methylpyrazole indicated that the inactivated P450 2E1 had an impaired ability to bind the substrate. Enzymatic activity could not be restored with iodosobenzene as the alternate oxidant. EPR analysis indicated that approximately 24% of the tBITC-inactivated P450 2E1 was EPR-silent. Of the remaining tBITC-inactivated P450 2E1, approximately 45% exhibited an unusual low-spin EPR signal that was attributed to the displacement of a water molecule at the sixth position of the heme by a tBITC modification to the apoprotein. ESI-LC-MS analysis of the inactivated P450 2E1 showed an increase in the mass of the apoprotein of 115 Da. In combination, the data suggest that tBITC inactivated P450 2E1 by binding to a critical active site amino acid residue(s). This modified amino acid(s) presumably acts as the sixth ligand to the heme, thereby interfering with oxygen binding and substrate binding.     

Effects of polychlorinated biphenyls on cytochrome P450 induction in the chick embryo hepatocyte culture

The effects of pure synthetic polychlorinated biphenyl (PCB) congeners on the induction of cytochrome P450 and associated activities were examined in cultured chick embryo hepatocytes. Dose-response effects for the induction of total cytochrome P450 ethoxyresorufin-O-deethylase (EROD) activity, and benzphetamine demethylase (BPDM) activity were studied using 10 selected tetra- to hexachlorinated PCB congeners. These studies revealed that PCBs caused effects in the chick hepatocyte culture different from previously observed effects in rat liver. Based on their effects in chick hepatocytes, the PCBs could be categorized into two groups. The first group (consisting of 3,3',4,4'-PCB, 3,3',4,4',5-PCB, 3,3',4,4',5,5'-PCB, 2',3,3',4,5-PCB, 2,3,3',4,4',5'-PCB, and 2,3,4,4',5-PCB) induced total cytochrome P450 2.4- to 2.9-fold and EROD activity from 1-2 pmol/min/mg protein to 162-247. There was marked variation in potency, but all these congeners had a maximal inducing dose above which cytochrome P450 concentrations and EROD activities declined. BPDM activities were increased only slightly (1.2- to 1.6-fold) at the maximal cytochrome P450 inducing dose. The second group of congeners (consisting of 2,2',4,5,5'-PCB. 2,2',4,4',5,5'-PCB, and 2,2',3,4,4',6-PCB) induced total cytochrome P450 concentrations 4.0-fold and BPDM activities 2.2- to 2.6-fold with greatest activity occurring at the highest doses which could be added (10-50 microM). However, EROD activities were also increased by these congeners to 60-112 pmol/min/mg protein with declining activities seen at the highest PCB doses (i.e., resembling EROD induction patterns of the first group). The EROD induction patterns with these latter PCB congeners are noteworthy since these PCBs do not induce EROD activity in the rat. For both groups of PCB congeners, EROD induction was associated with increased accumulation of uroporphyrin in cultures exposed to exogenous 5-aminolevulinate. Studies investigating the reason for the depression of cytochrome P450 concentrations and/or EROD activities by high doses of the PCBs revealed that with the first group there was slightly decreased total protein synthesis, decreased total cell heme concentrations, and decreased accumulation of radiolabeled heme synthesized from 5-[14C]aminolevulinate. These changes might represent nonspecific toxic effects of the first group of PCBs. However, since these changes were not seen with the second group of PCBs, it is unlikely that either inhibition of heme synthesis or toxicity cause the depression of EROD activity with high PCB doses.     

Clay-bridged electron transfer between cytochrome p450(cam) and electrode

We demonstrate a very fast heterogeneous redox reaction of substrate-free cytochrome P450(cam) on a glassy carbon electrode modified with sodium montmorillonite. The linear relationship of the peak current in the cyclic voltammogram with the scan rate indicates a reversible one-electron transfer surface process. The electron transfer rate is in the range from 5 to 152 s(-1) with scan rates from 0.4 to 12 V/s, respectively. These values are comparable to rates reported for the natural electron transfer from putidaredoxin to P450(cam). The formal potential of adsorbed P450(cam) is -139 mV (vs NHE) and therefore positively shifted by 164 mV compared to the potential of substrate-free P450(cam) in solution. UV-VIS and FTIR spectra do not indicate an influence of the clay colloidal particles on the heme and the secondary structure of P450(cam) in solution. However, P450(cam) adsorbed on the surface of the clay-modified electrode may undergo partial dehydration resulting in the shift of the formal potential.     

Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: effects of di-ortho substitution

All of the 13 possible polychlorinated biphenyl (PCB) isomers and congeners substituted at both para positions, at least two meta positions (but not necessarily on the same ring) and at two ortho positions have been synthesized and tested as rat hepatic microsomal enzyme inducers. The effects of these compounds were evaluated by measuring microsomal benzo-[a]pyrene (B[a]P) hydroxylase, 4-chlorobiphenyl (4-CBP) hydroxylase, 4-dimethylaminoantipyrine (DMAP) N-demethylase and NADPH-cytochrome c reductase activities, the cytochrome b5 content and the relative peak intensities and spectral shifts of the carbon monoxide(CO)- and ethylisocyanide(EIC)-difference spectra of ferrocytochrome P-450. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC) and PB plus MC (coadministered). At dose levels of 150 mumol . kg-1, all of the PCB congeners, except 2,3',4,4',5',6-hexachlorobiphenyl, significantly enhanced hepatic microsomal cytochrome P-450 content, B[a] P hydroxylase and/or DMAP N-demethylase activities compared to the control (corn oil-treated) animals. Only 5 of these compounds, namely 2,3,4,4',5,6-hexa-, 2,2',3,3',4,4'-hexa-, 2,2',3',4,4',5-hexa-, 2,3,3',4,4',6-hexa-and 2,2',3,3',4,4',5-heptachlorobiphenyl, enhanced microsomal B[a]P hydroxylase, 4-CBP hydroxylase, NADPH-cytochrome c reductase and DMAP N-demethylase activities in a manner consistent with a mixed pattern of induction. The results suggest that PCB isomers and congeners substituted at both para positions, at least two meta positions, at two ortho positions and containing a 2,3-4-trichloro substitution pattern on one ring are mixed-type inducers; in addition the effects of 2,3,4,4',5,6-hexachlorobiphenyl were also consistent with a mixed pattern of induction.     

Direct electron transfer between cytochrome P450scc and gold nanoparticles on screen-printed rhodium-graphite electrodes

This paper is concerned with an investigation of electron transfer between cytochrome P450scc (CYP11A1) and gold nanoparticles immobilised on rhodium-graphite electrodes. Thin films of gold nanoparticles were deposited onto the rhodium-graphite electrodes by drop casting. Cytochrome P450scc was deposited onto both gold nanoparticle modified and bare rhodium-graphite electrodes. Cyclic voltammetry indicated enhanced activity of the enzyme at the gold nanoparticle modified surface. The role of the nanoparticles in mediating electron transfer to the cytochrome P450scc was verified using ac impedance spectroscopy. Equivalent circuit analysis of the impedance spectra was performed and the values of the individual components estimated. On addition of aliquots of cholesterol to the electrolyte bioelectrocatalytic reduction currents were obtained. The sensitivity of the nanoparticle modified biosensor to cholesterol was 0.13 microA microM-1 in a detection range between 10 and 70 microM of cholesterol. This confirms that gold nanoparticles enhance electron transfer to the P450scc when present on the rhodium-graphite electrodes.     

Effect of alcohols on binding of camphor to cytochrome P450cam: spectroscopic and stopped flow transient kinetic studies

Addition of alcohols to cytochrome P450cam (CYP101) was shown to release the substrate camphor from the heme pocket of the enzyme. The release of the substrate was found to be caused both due to increased solubility of the substrate in solution in presence of alcohol and due to change in the tertiary structure of the active site of the enzyme. The far-UV CD and near-UV CD spectra reveal that addition of alcohols to cytochrome P450cam cause a small change in the secondary structural elements but a significant change in the tertiary structural organization of this enzyme. The CD spectra at the heme region at various concentrations of alcohols indicate a substantial change in the tertiary structural organization around the heme moiety too. The equilibrium constant associated with the binding of camphor to Cyt P450cam is strongly dependent on the concentration of alcohols and the corresponding free energy associated with the binding is found to scale linearly with the concentration of alcohols. Kinetic experiments on binding of camphor to Cyt P450cam show that both k(on) and k(off) rate constants are strongly affected by addition of alcohols suggesting that alcohol expel camphor out of the heme cavity of Cyt P450cam by affecting tertiary structure of Cyt P450cam as well as by modifying the solubility properties of camphor in aqueous medium.     

Polychlorinated biphenyl isomers and congeners as inducers of both 3-methylcholanthrene- and phenobarbitone-type microsomal enzyme activity

Highly purified synthetic polychlorinated biphenyls substituted in the meta and para positions of both phenyl rings and at one ortho position were administered to male Wistar rats and the effects of these compounds on the microsomal drug-metabolising enzymes were evaluated. The in vivo effects of these compounds were determined by measuring the microsomal benzo[a]pyrene hydroxylase, dimethylaminoantipyrine N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b5 content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450 : CO and ethylisocyanide binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC), 2,2',4,4'-tetrachlorobiphenyl (TCBP-II) (a PB-type inducer), 3,3',4,4'-tetrachlorobiphenyl (TCBP-I) (an MC-type inducer), PB plus MC (coadministered) and TCBP-II + TCBP-I (coadministered) to the test animals. At dosage levels of 30 and 150 mumol . kg-1, pretreatment with 2,3,3',4,4'-pentachlorobiphenyl (PCBP-II), 2,3',4,4',5-pentachlorobiphenyl (PCBP-I), 2,3,3',4,4',5-hexachlorobiphenyl (HCBP-II) and 2,3,3',4,4',5-hexachlorobiphenyl (HCBP-III) gave hepatic microsomes with enzymic and spectral properties consistent with a mixed pattern of induction. These polychlorinated biphenyl (PCB) isomers and congeners have been identified as either major or minor components of the commercial PCB mixtures and must contribute to their activity as MC-type inducers. The only PCB isomer in this series which was not a mixed type inducer was 2,3',4,4',5,5'-hexachlorobiphenyl (HCBP-I) which appeared to be a PB-type inducer. This contrasted to the mixed-type activity observed for 2,3',4,4',5,5'-hexabromobiphenyl which was isolated from a commercial polybrominated biphenyl (PBB) mixture.     

Elucidation of the differences between the 430- and 455-nm absorbing forms of P450-isocyanide adducts by resonance Raman spectroscopy.

Alkylisocyanide adducts of microsomal P450 exist in two interconvertible forms, each giving the Soret maximum around 430 or 455 nm. This is demonstrated with a rabbit liver P450 2B4. Resonance Raman spectra of the 430- and 455-nm forms were examined for typical P450s of the two types as well as for P450 2B4 because the 430-nm form of P450 2B4 is liable to change into P420. P450cam and P450nor were selected as a model of the 430- and 455-nm forms, respectively. For the n-butyl isocyanide (CNBu) adduct, the Fe(II)-CNBu stretching band was observed for the first time at 480/467 cm(-1) for P450cam and at 471/459 cm(-1) for P450nor with their (12)CNBu/(13)CNBu derivatives. For P450cam, but not P450nor, other (13)C isotope-sensitive bands were observed at 412/402, 844/835, and 940/926 cm(-1). The C-N stretching mode was identified by Fourier transform IR spectroscopy at 2116/2080 cm(-1) for P450cam and at 2148/2108 cm(-1) for P450nor for the (12)C/(13)C derivatives. These findings suggest that the binding geometry of isocyanide differs between the two forms-bent and linear structures for P450cam-CNBu and P450nor-CNBu, respectively. In contrast, in the ferric state, the Raman (13)C isotopic frequency shifts, and the IR C-N stretching frequencies (2213/2170 and 2215/2172 cm(-1)) were similar between P450cam and P450nor, suggesting similar bent structures for both.     

Structural characterization of n-butyl-isocyanide complexes of cytochromes P450nor and P450cam

Alkyl-isocyanides are able to bind to both ferric and ferrous iron of the heme in cytochrome P450, and the resulting complexes exhibit characteristic optical absorption spectra. While the ferric complex gives a single Soret band at 430 nm, the ferrous complex shows double Soret bands at 430 and 450 nm. The ratio of intensities of the double Soret bands in the ferrous isocyanide complex of P450 varies, as a function of pH, ionic strength, and the origin of the enzyme. To understand the structural origin of these characteristic spectral features, we examined the crystallographic and spectrophotometric properties of the isocyanide complexes of Pseudomonas putida cytochrome P450cam and Fusarium oxysporum cytochorme P450nor, since ferrous isocyanide complex of P450cam gives a single Soret band at 453 nm, while that of P450nor gives one at 427 nm. Corresponding to the optical spectra, we observed C-N stretching of a ferrous iron-bound isocyanide at 2145 and 2116 cm(-1) for P450nor and P450cam, respectively. The crystal structures of the ferric and ferrous n-butyl isocyanide complexes of P450cam and P450nor were determined. The coordination structure of the fifth Cys thiolate was indistinguishable for the two P450s, but the coordination geometry of the isocyanide was different for the case of P450cam [d(Fe-C) = 1.86 A, angleFe-C-N = 159 degrees ] versus P450nor [d(Fe-C) = 1.85 A, angleFe-C-N = 175 degrees ]. Another difference in the structures was the chemical environment of the heme pocket. In the case of P450cam, the iron-bound isocyanide is surrounded by some hydrophobic side chains, while, for P450nor, it is surrounded by polar groups including several water molecules. On the basis of these observations, we proposed that the steric factors and/or the polarity of the environment surrounding the iron-bound isocyanide significantly effect on the resonance structure of the heme(Fe)-isocyanide moiety and that differences in these two factors are responsible for the spectral characteristics for P450s.     

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.     

Steady-state kinetic investigation of cytochrome P450cam: interaction with redox partners and reaction with molecular oxygen

Cytochrome P450cam (CYP101) is a prokaryotic monooxygenase that requires two proteins, putidaredoxin reductase (PdR) and putidaredoxin (Pdx), to supply electrons from NADH. This study addresses the mechanism by which electrons are transported from PdR to P450cam through Pdx and used to activate O(2) at the heme of P450cam. It is shown that k(cat)/Km(O2) is independent of the PdR concentration and hyperbolically dependent on Pdx. The phenomenon of saturation of reaction rates with either P450cam or PdR at high ratios of one enzyme to the other is investigated and shown to be consistent with a change in the rate limiting step. Either the reduction of Pdx by PdR (high P450) or the reduction of P450 by Pdx (high PdR) determines the rate. These data support a mechanism where Pdx acts as a shuttle for transport of electrons from PdR to P450cam, effectively ruling out the formation of a kinetically significant PdR/Pdx/P450cam complex.     

Biomonitoring of polybrominated diphenyl ether (PBDE) pollution: a field study

Polybrominated diphenyl ethers (PBDEs) and cytochrome P450 enzyme activities were investigated in European eels (Anguilla anguilla) collected from seven sites in a coastal lagoon in the north-western Mediterranean Sea, Orbetello lagoon (Italy). Twelve PBDE congeners were measured in muscle and two CYP1A enzyme activities, 7-ethoxyresorufin-O-deethylase (EROD) and benzo(a)pyrene monooxygenase (BP(a)PMO), were investigated in liver microsomal fraction in order to obtain insights into the health of the lagoon environment. PBDE muscle levels were low and the most abundant congeners were 2,2',4,4'-tetrabromodiphenylether (BDE-47), 2,2',4,4',5,5'-hexaBDE (BDE-153) and 2,2',4,5'-tetraBDE (BDE-49). EROD and B(a)PMO activities were also low and no differences were observed between eels from different sites. Multivariate analysis (PCA) did not indicate correlations between PBDEs and either P450 activities.     

Calculation of the electronic structure and spectra of model cytochrome P450 compound I

The electronic structure and spectra of the oxyferryl (Fe=O) compound I P450 heme species, the transient putative active intermediate of cytochrome P450s, have been calculated employing a full protoporphyrin IX heme model representation. The principal aim of this work was to compare the computed spectra of this species with the observed transient spectra attributed to it. Computations were made using both nonlocal density functional theory (DFT) and semiempirical INDO/CI methods to characterize the electronic structure of the compound I P450 species. Both methods resulted in a similar antiferromagnetic doublet as the ground state with a ferromagnetic quartet excited state partner, slightly higher in energy. The INDO/ROHF/CI semiempirical method was used to calculate the spectrum of the protoporphyrin IX P450 compound I heme species in its lowest energy antiferromagnetic doublet state at the DFT optimized geometry. As a reference, the spectrum of the ferric resting form of the protoporphyrin IX P450 heme species was also calculated. The computed shifts in the Soret and Q bands of compound I relative to the resting state were both in good agreement with the corresponding experimentally observed shifts in the transient spectra of cytochrome P450cam (Biochem. Biophys. Res. Commun. 201 (1994) 1464) and chloroperoxidase (Biochem. Biophys. Res. Commun. 94 (1980) 1123) both ascribed to their common compound I heme site. This consistency provides additional, independent support for the assignment of compound I as the origin of the reported observed transient spectra.     

Electron-conformational interactions at the active site of reduced bacterial cytochrome P450cam induced by a substrate and analysis of the electron structure of heme]

Magnetic circular dichroism (MCD) spectra in the Soret region (360-480 nm) of camphor-free and camphor-bound reduced bacterial cytochrome P450cam from Pseudomonas putida were recorded and analysed in the temperature range from 2 K to 290 K. The temperature dependences of the MCD intensity are qualitatively changed by binding of substrate to the enzyme. In the absence of camphor the linear increase of the MCD intensity with 1/T at T < 4.2 K gives evidence for degeneracy or near degeneracy of the ground electronic state. In the presence of substrate the degeneracy is removed and temperature profiles show saturation behaviour at T < 4.2 K and wavelength dependence of their high-temperature parts. The temperature profiles for the long-wavelength region of the Soret band have a maximum approximately at 15 K, whereas the MCD intensity increases in a monotonous manner up to saturation in the short-wavelength region. The wavelength dependence of temperature profiles gives evidence for the co-existence of two different forms of substrate-bound reduced P450cam. The following conclusions were obtained from a theoretical analysis of the temperature profiles. In the absence of substrate there are very small if any rhombic distortions at the heme iron, and a parameter D of axial zero-field splitting is negative (D = -8.3 cm-1 and -6.2 cm-1 for P450cam and P450LM2, respectively). In the presence of substrate the two forms of reduced P450cam have positive parameters D but of different values (D1 = 12 cm-1 and D2 = 28 cm-1), and there are large rhombic distortions at the heme iron. More than two-fold difference between the D values made it possible to isolate temperature-dependent contributions of the two enzyme forms from the total MCD spectra and to simulate the alterations of the MCD spectra with temperature for reduced P450cam in the presence of substrate. Taking into account the drastic effect of substrate binding on the ground electronic state of reduced P450cam one can suggest that substrate binding induces the transition of enzyme from an inactive to an active state.     

Combined QM/MM calculations of active-site vibrations in binding process of P450cam to putidaredoxin

Combined QM/MM calculations of the active-site of cytochrome P450cam have been performed before and after the binding of P450cam to putidaredoxin. The calculations were carried out for both a 5-coordinated and a 6-coordinated active-site of cytochrome P450cam, with either a water molecule or a carbon monoxide molecule as a 6th distal ligand. An experimentally observed increase in the Fe-S stretching frequency that occurs after cytochrome P450cam binds to putidaredoxin, has been reproduced in our study. Experimentally observed changes in the Fe-C and C-O vibration frequencies that occur after binding of both proteins, have also been reproduced in our study. The computed increase of the Fe-S and Fe-C stretching frequencies is correlated with a corresponding decrease of the Fe-S and Fe-C interatomic distances. According to our calculations, for the active-site with carbon monoxide in the triplet electronic state, the binding process increases the spin densities on the iron and sulfur atoms, which changes the Fe-C and C-O stretching frequencies in opposite directions, in agreement with experimental data.     

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.     

Epoxidation of styrene by human cyt P450 1A2 by thin film electrolysis and peroxide activation compared to solution reactions

Films of human cytochrome P450 1A2 (cyt P450 1A2) and polystyrene sulfonate were constructed on carbon cloth electrodes using layer-by-layer alternate absorption and evaluated for electrochemical- and H(2)O(2)-driven enzyme-catalyzed oxidation of styrene to styrene oxide. At -0.6 V vs. saturated calomel reference electrode in an electrochemical cell, epoxidation of styrene was mediated by initial catalytic reduction of dioxygen to H(2)O(2) which activates the enzyme for the catalytic oxidation. Slightly larger turnover rates for cyt P450 1A2 were found for the electrolytic and H(2)O(2) (10 mM) driven reactions compared to conventional enzymatic reactions using cyt P450s, reductases, and electron donors for cytochromes P450 1A2. Cyt P450(cam) gave comparable turnover rates in film electrolysis and solution reactions. Results demonstrate that cyt P450 1A2 catalyzes styrene epoxidation faster than cyt P450(cam), and suggests the usefulness of this thin-film electrolytic method for relative turnover rate studies of cyt P450s.     

Crystal structure of OxyB,a cytochrome P450 implicated in an oxidative phenol coupling reaction during vancomycin biosynthesis

Gene-inactivation studies point to the involvement of OxyB in catalyzing the first oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. The oxyB gene has been cloned and sequenced from the vancomycin producer Amycolatopsis orientalis, and the hemoprotein has been produced in Escherichia coli, crystallized, and its structure determined to 1.7-A resolution. OxyB gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite or by spinach ferredoxin and ferredoxin reductase, the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. Addition of putative heptapeptide substrates to resting OxyB produced type I changes to the UV spectrum, but no turnover was observed in the presence of ferredoxin and ferredoxin reductase, showing that either the peptides or the reduction system, or both, are insufficient to support a full catalytic cycle. OxyB exhibits the typical P450-fold, with helix L containing the signature sequence FGHGXHXCLG and Cys(347) being the proximal axial thiolate ligand of the heme iron. The structural similarity of OxyB is highest to P450nor, P450terp, CYP119, and P450eryF. In OxyB, the F and G helices are rotated out of the active site compared with P450nor, resulting in a much more open active site, consistent with the larger size of the presumed heptapeptide substrate.     

Putidaredoxin-to-cytochrome P450cam electron transfer: differences between the two reductive steps required for catalysis

Cytochrome P450cam (P450cam) is the terminal monooxygenase in a three-component camphor-hydroxylating system from Pseudomonas putida. The reaction cycle requires two distinct electron transfer (ET) processes from the [2Fe-2S] containing putidaredoxin (Pdx) to P450cam. Even though the mechanism of interaction and ET between the two proteins has been under investigation for over 30 years, the second reductive step and the effector role of Pdx are not fully understood. We utilized mutagenesis, kinetic, and computer modeling approaches to better understand differences between the two Pdx-to-P450cam ET events. Our results indicate that interacting residues and the ET pathways in the complexes formed between reduced Pdx (Pdx(r)) and the ferric and ferrous dioxygen-bound forms of P450cam (oxy-P450cam) are different. Pdx Asp38 and Trp106 were found to be key players in both reductive steps. Compared to the wild-type Pdx, the D38A, W106A, and delta106 mutants exhibited considerably higher Kd values for ferric P450cam and retained ca. 20% of the first electron transferring ability. In contrast, the binding affinity of the mutants for oxy-P450cam was not substantially altered while the second ET rates were <1%. On the basis of the kinetic and modeling data we conclude that (i) P450cam-Pdx interaction is highly specific in part because it is guided/controlled by the redox state of both partners; (ii) there are alternative ET routes from Pdx(r) to ferric P450cam and a unique pathway to oxy-P450cam involving Asp38; (iii) Pdx Trp106 is a key structural element that couples the second ET event to product formation possibly via its "push" effect on the heme-binding loop.