Ferric Cytochrome P-450 Research Articles

Structure of cytochrome P450 2B4 with an acetate ligand and an active site hydrogen bond network similar to oxyferrous P450cam

Superposition of the active site of acetate-bound P4502B4 and oxyferrous P450cam. Bond lengths between the heme iron, the sixth ligand, and the hydroxyl of the conserved threonine are shown. Note that different threonine rotamers form the hydrogen bonds to the acetate and oxygen. • Crystal structure of cytochrome P450 2B4 in complex with acetate • Two water molecules insert in the I-helix of the cytochrome P450 2B4 mutant active site • In cytochrome P450 2B4 acetate binds to the heme and side chain of Thr302. • The acetate bound active site of ferric cytochrome P450 2B4 resembles that of the oxygen bound P450 cam .

Inhibition of hepatic microsomal monooxygenase activity by cinchocaine: mechanistic studies and effects of ionization.

The quinoline-based local anaesthetic cinchocaine (dibucaine) was found to be a mixed-type inhibitor of microsomal aminopyrine N-demethylase and 7-ethoxycoumarin O-deethylase activities from control and phenobarbitone-induced rat liver in-vitro. Cinchocaine also elicited a characteristic type I optical difference spectrum in oxidized liver microsomes (Ks = 24 microM; delta Amax = 3.4 X 10(-3) absorbance units (nmol cytochrome P450)-1) but did not appear to bind to the reduced form of the cytochrome. Additional studies indicated that cinchocaine competitively inhibited the type I spectral binding of substrate (aminopyrine) to ferric cytochrome P450. Studies of monooxygenase inhibition by cinchocaine over a relatively narrow pH range (6.5-8.5) indicated that, as might be expected, the un-ionized form of the drug is associated with inhibitory potency superior to that of the ionized form. Thus 40% inhibition of aminopyrine N-demethylase activity was observed with 100 microM cinchocaine at pH 8.0 and 8.5 (24% and 50% un-ionized drug, respectively), whereas only 16% inhibition was observed at pH 6.5 (1% un-ionized drug). These findings suggest that the inhibitory action of cinchocaine is mediated exclusively via an interaction with ferric cytochrome P450 and that the extent of ionization is a determinant of mixed function oxidase inhibition.

Ferric His93Gly myoglobin cavity mutant and its complexes with thioether and selenolate as heme protein models

The composition of ferric exogenous ligand-free His93Gly sperm whale myoglobin (H93G Mb) at neutral pH has been determined by examination of the spectral properties of the protein over the pH range from 3.0 to 10.5. An apparent pKa value of ~6.6 has been observed for the conversion of a postulated six-coordinate bis-water-bound coordination structure at pH 5.0 to a five-coordinate hydroxide-bound form at pH 10.5. Starting from the exogenous ligand-free ferric H93G protein, ferric mono- and bis-thioether (tetrahydrothiophene, THT)-ligated adducts have been prepared and characterized by UV-visible (UV-vis) absorption and magnetic circular dichroism (MCD) spectroscopy. The mon-THT ferric H93G Mb species has hydroxide as the sixth ligand. The bis-THT derivative is a model for the low-spin ferric heme binding site of native bis-Met-ligated bacterioferritin or streptococcal heme-associated protein (Shp). A novel THT-bound ferryl H93G Mb moiety has been partially formed. The high-spin five-coordinate ferric H93G(selenolate) Mb complex has been prepared using benzeneselenol and characterized by UV-vis and MCD spectroscopy as a model for Se-Cys-ligated ferric cytochrome P450. The results described herein further demonstrate the versatility of the H93G cavity mutant for modeling the coordination structures of novel heme iron protein active sites.

Five- and Six-Coordinate Adducts of Nitrosamines with Ferric Porphyrins: Structural Models for the Type II Interactions of Nitrosamines with Ferric Cytochrome P450

Nitrosamines are well-known for their toxic and carcinogenic properties. The metabolic activation of nitrosamines occurs via interaction with the heme-containing cytochrome P450 enzymes. We report the preparation and structural characterization of a number of nitrosamine adducts of synthetic iron porphyrins. The reactions of the cations [(por)Fe(THF)(2)]ClO(4) (por = TPP, TTP, OEP) with dialkylnitrosamines (R(2)NNO; R(2) = Me(2), Et(2), (cyclo-CH(2))(4), (cyclo-CH(2))(5), (PhCH(2))(2)) in toluene generate the six-coordinate high-spin (S = 5/2) [(por)Fe(ONNR(2))(2)]ClO(4) compounds and a five-coordinate intermediate-spin (S = 3/2) [(OEP)Fe(ONNMe(2))]ClO(4) derivative in 57-72% yields (TPP = 5,10,15,20-tetraphenylporphyrinato dianion, TTP = 5,10,15,20-tetra-p-tolylporphyrinato dianion, OEP = 2,3,7,8,12,13,17,18-octaethylporphyrinato dianion). The N-O and N-N vibrations of the coordinated nitrosamine groups in [(por)Fe(ONNR(2))(2)]ClO(4) occur in the 1239-1271 cm(-1) range. Three of the six-coordinate [(por)Fe(ONNR(2))(2)]ClO(4) compounds and one five-coordinate [(OEP)Fe(ONNMe(2))]ClO(4) compound have been characterized by single crystal X-ray crystallography. All the nitrosamine ligands in these complexes bind to the ferric centers via a sole eta(1)-O binding mode. No arylnitrosamine adducts were obtained from the reactions of the precursor compounds [(por)Fe(THF)(2)]ClO(4) with three arylnitrosamines (Ph(2)NNO, Ph(Me)NNO, Ph(Et)NNO). However, prolonged exposure of [(por)Fe(THF)(2)]ClO(4) to these arylnitrosamines resulted in the formation of the known five-coordinate (por)Fe(NO) derivatives. The latter (por)Fe(NO) compounds were obtained more readily by the reactions of the three arylnitrosamines with the four-coordinate (por)Fe(II) precursors.

Electronic structure and properties of model oxy and carboxy ferrous cytochrome P450: Comparison of semiempirical and ab-initio calculations

Semiempirical and ab-initio LCAO-MO-SCF calculations are reported for model oxy and carboxy ferrous cytochrome P450 with a methyl mercaptide SCH3− as an axial ligand for a number of conformations of the CO ligand and S-Fe-O-O torsion angle. For the semiempirical calculations a newly developed indo method including transition metal complexes is used with both a theoretical and a spectroscopic evaluation of one- and two-electron integrals. The electronic distributions obtained using the theoretical procedure compare well with the ab-initio results. Spectroscopic parametrization gives distributions and orbital energies which include some relaxation effects. The ground state description obtained with this parameterization yields values for the electric field gradient for both the oxy and the carboxy compounds which are in excellent agreement with experiment. Thus the anomalously large electric field gradient observed for the oxy and the extremely small electric field gradient observed for the carboxy P450 are both accounted for and support the validity of the calculated electronic structure.

Spectroscopic investigations of intermediates in the reaction of cytochrome P450 BM3–F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450 BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450 CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450 CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.

Combined Quantum Mechanical/Molecular Mechanical Study on the Pentacoordinated Ferric and Ferrous Cytochrome P450cam Complexes

The pentacoordinated ferric and ferrous cytochrome P450(cam) complexes have been investigated by combined quantum mechanical/molecular mechanical (QM/MM) calculations in the presence of a protein/solvent environment and by QM calculations on the isolated QM regions with use of density functional theory. The B3LYP functional has been found more reliable than the BLYP and BHLYP functionals for estimating the relative state energies. The B3LYP/CHARMM calculations with an all-electron basis set for iron give high-spin ground states for the title complexes, in agreement with experiment. The comparison of the B3LYP/CHARMM results of the entire protein system with the B3LYP calculations on the naked QM regions shows that the amount of stabilization by the protein environment is largest for the intermediate-spin states, followed by the high-spin states of the complexes. The calculation of Mössbauer parameters in the presence of the enzyme environment confirms the double occupation of the d(xz) orbital in the quintet spin state of the ferrous complex, consistent with the computed QM/MM energies in the enzyme environment, while the d(x)2(-)(y)2 orbital is doubly occupied in the gas-phase quintet state.

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Cytochrome b(5) (cyt b(5)) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane alpha-helix at its carboxyl terminus. These two domains are connected by an approximately 15-amino acid linker domain, Ser(90)-Asp(104), which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b(5) to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b(5) to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b(5) except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b(5) to interact with cyt P450 2B4. A model for the membrane-bound cyt b(5)-cyt P450 complex is presented.

Characterization of the oxygenated intermediate of the thermophilic cytochrome P450 CYP119

Using UV–Vis, resonance Raman, and EPR spectroscopy we have studied the properties of the oxygenated ferrous cytochrome P450 from Sulfolobus Solfataricus, (CYP119). The recently determined crystal structure of CYP119 is compared with other available structures of P450s, and detailed structural and spectroscopic analyses are reported. With several structural similarities to CYP102, such as in-plane iron position and a shorter iron-proximal ligand bond, CYP119 shows low-spin conformation preference in the ferric form and partially in the ferrous form at low temperatures. These structural features can explain the fast autoxidation of the oxyferrous complex of CYP119. Finally, we report the first UV–Vis and EPR spectra of the cryoradiolytically reduced oxygenated intermediate of CYP119. The primary reduced intermediate, a hydroperoxo–ferric complex of CYP119, undergoes a ‘peroxide shunt’ pathway during gradual annealing at 170–195 K and returns to the low-spin ferric form.

Comparisons of Hydroperoxide Isomerase and Monooxygenase Activities of Cytochrome P450 for Conversions of Allylic Hydroperoxides and Alcohols to Epoxyalcohols and Diols: Probing Substrate Reorientation in the Active Site

Ferric cytochrome P450 (P450) catalyzes intramolecular rearrangements of hydroperoxides to diols by heterolytic scission of the O-O bond and insertion of the terminal oxygen atom into the resulting alcohol. The goals of this work were to further characterize the regio- and stereochemistry of P450 isomerase activity using allylic hydroperoxides and to compare these reactions with NADPH-supported monooxygenations of the corresponding alcohols. Microsomes from phenobarbital-treated rats or purified P450 2B1 catalyzed the conversions of several peroxyquinols, including 2-tert-butyl-4-hydroperoxy-4-methylcyclohexa-2,5-dien-1-one (BMPOOH) and its 2,6-dimethyl derivative (TMPOOH), to diols and to alpha,beta-epoxyquinols by predominant (>93%) cis addition of oxygen to the least hindered double bond. Monooxygenation of the 4-hydroxy analogues (quinols) yielded identical cis-epoxyquinols, and both isomerization and monooxygenation reactions exhibited similar enantioselectivities (32-74% enantiomer excess). Regioselectivities were similar for BMPOOH and BMPOH (epoxyquinol:diol ratios of 5.8-7.6), but quite different for TMPOOH and TMPOH (ratios of 0.6 and 6.4, respectively). Bulky peroxyquinols and quinols derived from the A-rings of 17beta-estradiol and estrone were utilized to further compare these reactions. Both estradiol derivatives underwent approximately equal amounts of 6beta-hydroxylation and 1,2-epoxidation. The estrone derivatives also underwent 6beta-hydroxylation, but only estrone quinol yielded a second product consistent with hydroxylation at position 16. The results support several conclusions. (i) Allylic hydroperoxides may be isomerized to alpha,beta-epoxyalcohols by a heterolytic O-O cleavage mechanism with high stereoselectivity. (ii) Hydroperoxide isomerization is an efficient process relative to monooxygenation. (iii) Isomerase substrates remain in proximity to the P450 oxoferryl intermediate and are rapidly captured by the oxidant. Monooxygenase substrates, on the other hand, may bind to ferric P450 in multiple orientations and undergo more extensive substrate reorientation prior to oxidative attack.

Biogenesis of Volatile Aldehydes from Fatty Acid Hydroperoxides: Molecular Cloning of a Hydroperoxide Lyase (CYP74C) with Specificity for both the 9- and 13-Hydroperoxides of Linoleic and Linolenic Acids

A novel member of the plant cytochrome P450 CYP74 family of fatty acid hydroperoxide metabolizing enzymes has been cloned from melon fruit (Cucumis melo). The cDNA is comprised of 1446 nucleotides encoding a protein of 481 amino acids. The homology at the amino acid level to other members of the CYP74 family is 35–50%, the closest relatives being allene oxide synthases. The cDNA was expressed in Escherichia coli, and the corresponding protein was purified by affinity column chromatography. The native enzyme showed a main Soret band at 418 nm, indicative of a low spin ferric cytochrome P450, and a 447-nm peak appeared in the CO-difference spectrum. Using [U-14C]radiolabeled substrate, HPLC, UV, and GC-MS, the products of conversion of 9S-hydroperoxy-linoleic acid were identified as 9-oxo-nonanic acid and 3Z-nonenal. Kinetic analysis of this hydroperoxide lyase showed the highest rate of reaction with 9-hydroperoxy-linolenic acid followed by 9-hydroperoxy-linoleic acid and then the corresponding 13-hydroperoxides. Overall, the newly characterized cytochrome P450 enzyme is a fatty acid hydroperoxide lyase with a preference, but not absolute specificity for the 9-positional hydroperoxides of linoleic and linolenic acids.

Nitric oxide binding to ferric cytochrome P450: a computational study.

The interaction between nitric oxide (NO) and the active site of ferric cytochrome P450 was studied by means of density functional theory (DFT), at the generalized gradient approximation level, and of the SAM1 semiempirical method. The electrostatic effects of the protein environment were included in our DFT scheme by using a hybrid quantum classical approach. The active-site model consisted of an iron(III) porphyrin, the adjacent cysteine residue, and one coordinated water molecule. For this system, spin populations and relative energies for selected spin states were computed. Interestingly, the unpaired electron density, the HOMO, and the LUMO were found to be highly localized on the iron and in an appreciable extent on the sulfur coordinated to the metal. This provides central information about the reactivity of nitric oxide with the active site. Since the substitution of a molecule of H2O by NO has been proposed as being responsible for the inhibition of the cytochrome in the presence of nitric oxide, we have analyzed the thermodynamic feasibility of the ligand exchange process. The structure of the nitrosylated active site was partially optimized using SAM1. A low-spin ground state was obtained for the nitrosyl complex, with a linear Fe-N-O angle. The trends found in Fe-N-O angles and Fe-N lengths of the higher energy spin states provided a notable insight into the electronic configuration of the complex within the framework of the Enemark and Feltham formalism. In relation to the protein environment, it was assessed that the electrostatic field has significant effects on several computed properties. However, in both vacuum and protein environments, the ligand exchange reaction turned out to be exergonic and the relative orders of spin states of the relevant species were the same.

The Role of the Distal and Proximal Protein Environments in Controlling the Ferric Spin State and in Stabilizing Thiolate Ligation in Heme Systems: Thiolate Adducts of the Myoglobin H93G Cavity Mutant

Recently, heme protein cavity mutants have been engineered in which the proximal coordinating amino acid has been replaced by a smaller, noncoordinating residue leaving a cavity that can be filled by exogenous axial ligands. This approach was pioneered by Barrick (Biochemistry 1994, 33, 6546−6554) with H93G sperm whale myoglobin where the coordinating histidine is replaced by glycine and the proximal cavity is filled with imidazole. In the present study, models for cysteine thiolate-ligated ferric cytochrome P450 have been prepared using H93G myoglobin containing thiolate ligands in the cavity. Despite the availability of water to serve as a distal ligand as occurs in both ferric wild type myoglobin and ferric H93G myoglobin with imidazole in the cavity, the ferric H93G thiolate complexes spectroscopically resemble five-coordinate high-spin substrate-bound ferric P450, which contains a thiolate proximal ligand and lacks a water distal ligand. Thus, the distal protein environment plays a crucial role in controlling whether a five-coordinate thiolate-ligated ferric heme binds water or not. Two advantages pertain to the present thiolate-ligated heme protein model relative to purely synthetic thiolate-ligated ferric porphyrins: (a) aliphatic thiols can form complexes without reduction of the ferric iron and (b) mixed ligand complexes that are stable at ambient temperatures can be prepared with a neutral ligand such as imidazole trans to thiolate. However, when anionic ligands are added to the ferric thiolate adduct in an attempt to prepare mixed ligand complexes with two anionic ligands, the thiolate ligand is displaced (or lost) without formation of a stable mixed ligand derivative. Further, reduction of the ferric-thiolate complex leads to loss of the thiolate ligand even in the presence of CO. The data presented for the thiolate adducts of ferric H93G myoglobin are analyzed in the context of the spectrally related H93C myoglobin mutants. The inability of the thiolate adducts of H93G myoglobin to accommodate a second anionic ligand in the ferric state or to remain thiolate-ligated in the ferrous state is likely due to the lack of (a) correctly positioned hydrogen bond donating groups and (b) a properly oriented helix dipole to stabilize the thiolate ligand as occurs in the proximal protein environment of P450. The present results illustrate the important role of the distal and proximal heme environments in controlling the ferric spin state and in stabilizing thiolate ligation in heme systems, respectively.

Peroxide-supported in-vitro cytochrome P450 activities in Haemonchus contortus

The potential for cytochrome P450 from Haemonchus contortus to operate in the oxygen-poor intestinal environment was investigated by examining the ability of the cytochrome to act in vitro as a peroxygenase in utilising cumene hydroperoxide for substrate oxidations not requiring molecular oxygen. Peroxygenase and NADPH-supported monooxygenase activities were measured in microsomes prepared from L3 and adult nematodes. Both cumene hydroperoxide- and NADPH-supported ethoxycoumarin O-deethylase and aldrin epoxidase activities were detected in larval microsomes. Adult microsomes showed low levels of cumene hydroperoxide-supported ethoxycoumarin O-deethylase, as well as NADPH- and cumene hydroperoxide-supported aldrin epoxidase activities. The use of inhibitors in ethoxycoumarin O-deethylase assays with larval microsomes indicated that the peroxygenase pathway does not proceed via ferrous cytochrome P450 (no inhibition by carbon monoxide), did not require molecular oxygen, and did not depend on electron flow from cytochrome P450 reductase. Larval activity was inhibited by typical cytochrome P450 inhibitors (piperonyl butoxide, SKF-525A, chloramphenicol, metyrapone, n-octylamine) and was unaffected by the peroxidase inhibitor salicylhydroxamic acid. In contrast, adult microsomal cumene hydroperoxide-supported ethoxycoumarin O-deethylase activity was significantly inhibited by both cytochrome P450 inhibitors and salicylhydroxamic acid. Adult microsomes also contained potassium ferrocyanide peroxidase activity utilising cumene hydroperoxide. This activity showed a similar pattern of inhibition by both cytochrome P450 and peroxidase inhibitors. Whilst the ability of larval H. contortus cytochrome P450 to act as a peroxygenase in vitro was demonstrated, the inhibition results with adult microsomes showing both cytochrome P450 and peroxidase activities require further investigation to clarify the nature of the adult microsomal cumene hydroperoxide-supported O-deethylase activity.

Structure and Spectra of Ferrous Dioxygen and Reduced Ferrous Dioxygen Model Cytochrome P450

The optimized geometries of the stable ferrous dioxygen and transient reduced ferrous dioxygen forms of a methylmercaptate porphine model of the cytochrome P450 heme system were calculated using nonlocal density functional theoretical (DFT) methods. The optimized geometry of the ferrous dioxygen form is in good agreement with the structure of a model compound of this species and the calculated diamagnetic singlet ground state is consistent with the reported lack of ESR spectrum of this species. The calculated ground state of the reduced ferrous dioxygen species is a low-spin (doublet) state, in agreement with the reported ESR signature of this species in P450cam. The dioxygen ligand in the reduced form is shown to preferentially bind to the heme iron in an asymmetric “end-on” geometry. The most pronounced structural effects of the reduction of the ferrous dioxygen species are the elongation of the Fe−O and Fe−S bonds. This bond lengthening is due to the addition of an electron into a molecular orbital of ...

Reaction of cytochrome P450 with cumene hydroperoxide: ESR spin-trapping evidence for the homolytic scission of the peroxide O-O bond by ferric cytochrome P450 1A2.

ESR spin trapping was used to investigate the reaction of rabbit cytochrome P450 (P450) 1A2 with cumene hydroperoxide. Cumene hydroperoxide-derived peroxyl, alkoxyl, and carbon-centered radicals were formed and trapped during the reaction. The relative contributions of each radical adduct to the composite ESR spectrum were influenced by the concentration of the spin trap. Computer simulation of the experimental data obtained at various 5,5-dimethyl-1-pyrroline N-oxide (DMPO) concentrations was used to quantitate the contributions of each radical adduct to the composite ESR spectrum. The alkoxyl radical was the initial radical produced during the reaction. Experiments with 2-methyl-2-nitrosopropane identified the carbon-centered adducts as those of the methyl radical, hydroxymethyl radical, and a secondary carbon-centered radical. The reaction did not require NADPH-cytochrome P450 reductase or NADPH. It is concluded that the reaction involves the initial homolytic scission of the peroxide O-O bond to produce the cumoxyl radical. Methyl radicals were produced from the beta-scission of the cumoxyl radical. The peroxyl adduct was not observed in the absence of molecular oxygen. We conclude that the DMPO peroxyl radical adduct detected in the presence of oxygen was due to the methylperoxyl radical formed by the reaction of the methyl radical with oxygen. At a higher P450 concentration, a protein-derived radical adduct was also detected.

The Mechanism of Microsomal Azoreduction: Predictions Based on Electronic Aspects of Structure-Activity Relationships

The mechanism of microsomal azoreductase is regulated by the overall Hammett sigma substituent values on each ring. A substrate dye must exhibit an overall Hammett sigma substituent value either equal or more negative than -0.37 on either ring. Dyes with Hammett sigma substituent constants less negative than -0.37 will not be reduced by microsomal cytochrome P450. Microsomal reduction of azo dyes containing only electron-donating substituents on either ring is insensitive to both oxygen and carbon monoxide. The required Hammett sigma substituent value on the opposite benzene (prime) ring for I-substrates is therefore, sigma' P < or = 0. Reduction of azo dyes containing electron-withdrawing group on opposite (prime) is sensitive to both oxygen and carbon monoxide. The required Hammett sigma substituent value on the opposite benzene (prime) ring for S-substrates is, consequently, sigma' P > 0 (Table 3). Redox Potentials. Anaerobic cyclic voltammograms of azobenzene derivatives verify the following points: A nonsubstrate azo dye will not exhibit a positive potential. (Several nonsubstrate hydrazobenzenes exhibited positive potentials, but in a low range 0.41-0.48 V. Consequently, cyclic voltammetry can distinguish between nonsubstrate azobenzenes and their nonsubstrate half-reduced hydrazo analogs.) A substrate azo dye will exhibit a positive potential in the range +1.00 to +1.50 V. I-substrate: Both negative potentials are stable in air. S-substrate: The first negative potential will immediately quench upon exposure to air. I-substrates exhibit on average potentials which are approximately 0.6 V more negative than those for S-substrates. A comparison between the oxidative and the reductive pathway of microsomal cytochrome P450 indicates a similarity in the first two steps in the reaction cycle, for example, substrate binding and uptake of the first electron by the cytochrome [76, 109, 110]. Upon reduction of the iron, ferrous cytochrome P450 may bind oxygen or carbon monoxide in a competitive manner in the oxidative cycle or may directly transfer the electrons to the substrate in a stepwise fashion in the reductive cycle [76]. Estabrook et al. [111] suggested that carbon monoxide insensitivity can occur when the formation of ferrous cytochrome P450 substrate complex is rate limiting for the overall reaction. Structure-activity relationships of azo compounds depend on (1) the electron transport component and (2) the oxidation-reduction potential of the dye, which determines its ability to accept electrons from cytochrome P-450. Nesnow et al. examined a group of 36 aryl azo dyes for their ability to be reduced by rat liver microsomal azoreductase.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of monovalent cations on cytochrome P-450 camphor evidence for preferential binding of potassium

Binding of monovalent cations of increasing ionic radius to ferric cytochrome P-450 cam was measured. Potassium has the highest affinity for the cation binding site observed in the X-ray crystallographic structure with K d cat = 12 mM, compared with the smaller cation lithium, ( K d cat = 37 mM) and the larger cation cesium ( K d cat = 20 mM). Coupling between cation binding and camphor binding is established by the observation of a linear relationship between the corresponding binding free energies. Potassium binding favours a conformational change of tyrosine 96 which increases the affinity of the protein for camphor and fully dehydrates the active site.

Spectral Intermediate in the Reaction of Ferrous Cytochrome P450 cam with Superoxide Anion

The addition of KO 2 to ferrous cytochrome P450 cam in the presence of d-camphor under anaerobic condition resulted in the formation of exo- and endo-5-hydroxycamphor. The product formation was inhibited by the addition of superoxide dismutase, not catalase. The ferrous form of P450 cam reacts with O − 2 to form an intermediate species of the enzyme, of which spectrum is closely similar to that of the oxygenated form of P450 cam. Subsequently, the intermediate was converted to native ferric P450 cam. In contrast, ferrous form of horseradish peroxidase reacted with O − 2 to form Compound I of the enzyme without detection of intermediates.

High pressure induced inactivation of ferrous cytochrome P-450 LM2 (11B4) CO complex: Evidence for the presence of two conformers in the oligomer

The effect of high pressure on the spectral properties of cytochrome P-450 LM2(Fe 2+)-CO complex was studied. The application of high pressure was shown to induce the conversion of cytochrome P-450 to P-420. In the solution when P-450 was oligomeric only about 65% of the total converted to P-420. The remaining portion of cytochrome P-450 was stable at pressures up to 6 kbar. When P-450 was incorporated into membranes or when it was succinylated, the proportion of the pressure sensitive fraction was slightly higher (about 75%). Dissocation of P-450 oligomers into monomers was made by addition of 0.2% Triton N-101. Monomers were the most sensitive to pressure; they could be completely converted to P-420. These results have been interpreted as evidence for the existence of two different conformers of P-450 LM2, which differ in pressure stability. Splitting between these two states appears to be a result of the oligomeric organization of cytochrome P-450 in solution and in the membrane.