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.     

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.     

Characterization of heme environment and mechanism of peroxide bond cleavage in human prostacyclin synthase

Prostacyclin is a potent mediator of vasodilation and anti-platelet aggregation. It is synthesized from prostaglandin H(2) by prostacyclin synthase (PGIS), a member of Family 8 in the cytochrome P450 superfamily. Unlike most P450s, which require exogenous reducing equivalents and an oxygen molecule for mono-oxygenation, PGIS catalyzes an isomerization with an initial step of endoperoxide bond cleavage of prostaglandin H(2) (PGH(2)). The low abundance of PGIS in natural tissues necessitates heterologous expression for studies of structure/function relationships and reaction mechanism. We report here a high-yield prokaryotic system for expression of enzymatically active human PGIS. The PGIS cDNA is modified by replacing the hydrophobic amino-terminal sequence with the more hydrophilic amino-terminal sequence from P450 2C5 and by adding a four-histidine tag at the carboxyl terminus. The resulting recombinant PGIS associates with host cell membranes and was purified to electrophoretic homogeneity by nickel affinity, hydroxyapatite and CM Sepharose column chromatography. The recombinant PGIS, with a heme:protein ratio of 0.9:1, catalyzes prostacyclin formation at a K(m) of 13.3 muM PGH(2) and a V(max) of 980 per min. The dithionite-reduced PGIS binds CO with an on-rate of 5.6 x 10(5) M(-1) s(-1) and an off-rate of 15 s(-1). The ferrous-CO complex of PGIS is very short-lived and decays at a rate of 0.7 s(-1). Spectral binding assays showed that imidazole binds weakly to PGIS (K(d) approximately 0.5 mM,) but clotrimazole, a bulky and rigid imidazole derivative, binds strongly (K(d) approximately 1 microM). The transient nature of the CO complex and the weak imidazole binding seem to support an earlier proposal that PGIS active site has a limited space, but the tight binding of clotrimazole argues against this view. It appears that the heme distal pocket of PGIS is fairly adaptable to ligands of various structures. UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance spectra indicate that PGIS has a typical low-spin heme with a hydrophobic active site. PGIS catalyzes homolytic scission of the peroxide bond of a test substrate, 10-hydroperoxyoctadeca-8,12-dienoic acid, accompanied by formation of a heme intermediate with a Compound II-like optical spectrum.     

The influence of substrate on the spectral properties of oxyferrous wild-type and T252A cytochrome P450-CAM

To probe whether the nature of the substrate can directly influence the spectral properties of oxyferrous cytochrome P450-CAM, the complex has been investigated in the absence and in the presence of the natural substrate (1R)-camphor (camphor) and of several camphor analogs. The oxyferrous complex of T252A P450-CAM, a mutant lacking the hydroxyl group that forms a hydrogen bond to the heme iron-coordinated dioxygen, has also been studied to gauge the influence of this hydrogen bond. UV-visible absorption and magnetic circular dichroism (MCD) spectra of these oxyferrous adducts prepared and stabilized at -40 degrees C in 60% (v/v) ethylene glycol are generally similar, exhibiting absorption bands at approximately 355, approximately 420, approximately 554, and approximately 585 nm (shoulder) and a characteristic MCD trough at approximately 585 nm. The MCD spectrum of camphor-bound oxyferrous P450-CAM is similar to that of the substrate-free oxyferrous enzyme, but the spectrum of the oxyferrous enzyme differs detectably in the presence of substrate analogs. The spectra of the oxyferrous T252A mutant and wild-type enzyme are overall similar except for Soret band position blue shifts by 2-6 nm for the mutant. 5-Methylenylcamphor (epoxidation substrate) appears to have an anomalous binding mode for the mutant compared with that for the wild-type enzyme. The present results indicate that the structures of the camphor analogs can sensitively influence the physical (spectroscopic) properties of the P450 dioxygen complex and could also affect its reactivity. The ability of substrate to modulate the reactivity of P450 intermediates could be a relevant factor in explaining the remarkable diversity of reactions catalyzed by the enzyme.     

Oxidation and electronic state dependence of proton transfer in the enzymatic cycle of cytochrome P450eryF

Hydrogen bond networks, consisting of hydrogen bonded waters anchored by polar/acidic amino acid sidechains, are often present in the vicinity of the oxygen binding clefts of P450s. Density functional and quantum dynamics calculations of a O(2) binding cleft network model of cytochrome P450eryF(CYP107A1) indicate that such structural motifs facilitate ultrafast proton transfer from network waters to the dioxygen of the reduced oxyferrous species via a multiple proton translocation mechanism with barriers of 7-10 kcal/mol on its doublet ground state, and that the energies of the proton transfer reactant and constrained proton transfer products have an electronic and oxidation state dependence [J. Am. Chem. Soc. 124 (2002) 1430]. In the present study, the origin of the oxidation state dependence is shown to have its roots in differential proton affinities while the electronic state dependence of the reduced oxyferrous heme has its origins in subtle differences in network topologies near the transition state of the initial proton transfer event. Relaxed potential surface scans and unconstrained proton transfer product optimizations indicate that the proton transfer product in both the singlet oxyferrous heme and the reduced oxyferrous heme species in a quartet state are not viable stable (bound) states relative to the reactant form. While the proton affinity of H(3)O(+) is sufficient for it to protonate both the oxyferrous and the reduced oxyferrous heme species, hydrogen bond network stabilized water is only capable of protonating the reduced oxyferrous form. This interpretation is substantiated by study of the NO bound reduced ferrous heme of P450nor, which is isoelectronic with the oxyferrous heme and has a similar proton affinity. Density functional calculations on a more extensive O(2) binding cleft model support the multiple proton translocation mechanism of transfer but indicates that the significant negative charge density on the bound dioxygen of the reduced oxyferrous heme species, in its doublet ground state, polarizes the associated hydrogen bond network sufficiently so as to result in short, strong, low-barrier hydrogen bonds. The computed O-H-O bond distances are less than 2.55 A and have a near degeneracy of the proton transfer reactant and initial (sudden) proton transfer products. These low-barrier hydrogen bond features, in addition to the finding of a (zero point uncorrected) barrier of 1.3 kcal/mol, indicate that proton transfer from water to the distal oxygen should be rapid, facile and may not require large curvature tunneling as originally suggested by use of a smaller model. An initial assessment of protonation of the reduced oxyferrous heme distal oxygen by a model of 6-deoxyerythronolide B (6-DEB) indicates it to be low barrier (3.8 kcal/mol) and exothermic (-2.9 kcal/mol). The combined results indicate the plausibility of simultaneous diprotonation of the distal oxygen of the reduced oxyferrous heme, leading to O-O bond scission, using the combined water network and 6-DEB substrate protonation agents.     

Low-temperature optical absorption spectra suggest a redox role for tetrahydrobiopterin in both steps of nitric oxide synthase catalysis

To investigate the role of tetrahydrobiopterin (BH4) in the catalytic mechanism of nitric oxide synthase (NOS), we analyzed the spectral changes following addition of oxygen to the reduced oxygenase domain of endothelial nitric oxide synthase (NOS) in the presence of different pteridines at -30 degrees C. In the presence of N(G)-hydroxy-L-arginine (NOHLA) and BH4 or 5-methyl-BH4, both of which support NO synthesis, the first observable species were mixtures of high-spin ferric NOS (395 nm), ferric NO-heme (439 nm), and the oxyferrous complex (417 nm). With Arg, no clear intermediates could be observed under the same conditions. In the presence of the BH4-competitive inhibitor 7,8-dihydrobiopterin (BH2), intermediates with maxima at 417 and 425 nm were formed in the presence of Arg and NOHLA, respectively. In the presence of 4-amino-BH4, the maxima of the intermediates with Arg and NOHLA were at 431 and 423 nm, respectively. We ascribe all four spectra to oxyferrous heme complexes. The intermediates observed in this study slowly decayed to the high-spin ferric state at -30 degrees C, except for those formed in the presence of 4-amino-BH4, which required warming to room temperature for regeneration of high-spin ferric NOS; with Arg, regeneration remained incomplete. From these observations, we draw several conclusions. (1) BH4 is required for reductive oxygen activation, probably as a transient one-electron donor, not only in the reaction with Arg but also with NOHLA; (2) in the absence of redox-active pterins, reductive oxygen activation does not occur, which results in accumulation of the oxyferrous complex; (3) the spectral properties of the oxyferrous complex are affected by the presence and identity of the substrate; (4) the slow and incomplete formation of high-spin ferric heme with 4-amino-BH4 suggests a structural cause for inhibition of NOS activity by this pteridine.     

Enhanced decomposition of oxyferrous cytochrome P450CIA1 (P450cam) by the chemopreventive agent 3-t-butyl-4-hydroxyanisole

The efficacy of 2(3)-t-butyl-4-hydroxyanisole (BHA) and other chemicals as chemopreventive agents against chemically induced cancer or toxicity may involve direct modulation of cytochrome P450 activity. Direct interaction of BHA with cytochrome P450 was investigated using substrate-bound, oxyferrous cytochrome P450CIA1 either in a reconstituted system containing cytochrome P450CIA1, putidaredoxin, and putidaredoxin reductase with NADH as electron donor or in the absence of physiological electron donors. In the reconstituted system, BHA caused a concentration-dependent decrease in the production of 5-exo-hydroxycamphor and a substoichiometric increase in hydrogen peroxide production. However, BHA did not appreciably inhibit either NADH oxidation or oxygen utilization under conditions optimal for accumulation of oxyferrous cytochrome P450CIA1 during steady-state metabolism of camphor. In the absence of electron donor, BHA enhanced decomposition of the ternary oxyferrous substrate complex of cytochrome P450CIA1 without the formation of any apparent spectral intermediate(s). The rate of decomposition of the oxyferrous complex was pseudo-first order and was dependent upon the concentration of BHA present. Enhanced decomposition of the complex was not attributable to catalytic turnover of cytochrome P450CIA1 (i.e., acquisition of a second electron from an indeterminate source) since no appreciable metabolism of either camphor or BHA was observed. The enhanced decomposition was accompanied by a substoichiometric increase in hydrogen peroxide production, suggesting that BHA may facilitate four-electron reduction of molecular oxygen to water. These results indicate that BHA inhibits cytochrome P450 function, presumably by enhancing autoxidation of the substrate-bound oxyferrous complex.     

Structures of prostacyclin synthase and its complexes with substrate analog and inhibitor reveal a ligand-specific heme conformation change

Prostacyclin synthase (PGIS) is a cytochrome P450 (P450) enzyme that catalyzes production of prostacyclin from prostaglandin H(2). PGIS is unusual in that it catalyzes an isomerization rather than a monooxygenation, which is typical of P450 enzymes. To understand the structural basis for prostacyclin biosynthesis in greater detail, we have determined the crystal structures of ligand-free, inhibitor (minoxidil)-bound and substrate analog U51605-bound PGIS. These structures demonstrate a stereo-specific substrate binding and suggest features of the enzyme that facilitate isomerization. Unlike most microsomal P450s, where large substrate-induced conformational changes take place at the distal side of the heme, conformational changes in PGIS are observed at the proximal side and in the heme itself. The conserved and extensive heme propionate-protein interactions seen in all other P450s, which are largely absent in the ligand-free PGIS, are recovered upon U51605 binding accompanied by water exclusion from the active site. In contrast, when minoxidil binds, the propionate-protein interactions are not recovered and water molecules are largely retained. These findings suggest that PGIS represents a divergent evolution of the P450 family, in which a heme barrier has evolved to ensure strict binding specificity for prostaglandin H(2), leading to a radical-mediated isomerization with high product fidelity. The U51605-bound structure also provides a view of the substrate entrance and product exit channels.     

Oxygen binding to dithionite-reduced chloroperoxidase

Both the kinetics of ferric chloroperoxidase reduction by dithionite and the binding of molecular oxygen to ferrous chloroperoxidase have been studied. The oxyferrous chloroperoxidase decays spontaneously to the ferric enzyme. In addition the corresponding rapid-scan spectra have been recorded. The reduction reaction is caused by SO-.2 with a rate constant of (7.7 +/- 1.0) X 10(4) M-1 S-1. Oxygen binding occurs with a rate constant of (5.5 +/- 1.0) X 10(5) M-1 S-1 over the pH range 3.5-6. Oxyferrous chloroperoxidase has a Soret absorption peak at 428 nm and two partially resolved peaks at 555 nm and 588 nm. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous chloroperoxidase at 419, 545, 555 and 580 nm; between oxyferrous and ferric chloroperoxidase at 419, 487, 540, 609 and 682 nm.     

Features of intermediary steps around the 688-nm substance in the heme oxygenase reaction

The degradation of protoheme in the heme oxygenase reaction involves three oxidation steps: from protoheme to hydroxyheme, from hydroxyheme to a 688-nm substance, a protein-bound intermediate, and from the 688-nm substance to a biliverdin-iron complex. The 688-nm substance has a ferrous iron and it readily binds carbon monoxide to form a CO-complex, called the 638-nm substance (Yoshida, T., Noguchi, M., & Kikuchi, G. (1980) J. Biochem. 88, 557-563). The ferric 688-nm substance was prepared from the 638-nm substance by the addition of potassium ferricyanide together with aspiration to eliminate CO. The ferric 688-nm substance did not show any distinct absorption maximum in the red region of the absorption spectrum. The ferric 688-nm substance was readily reduced on the addition of the NADPH-cytochrome P-450 reductase system, but the ferric 688-nm substance could also be reduced spontaneously though at a very low rate. The ferrous 688-nm substance free from excess reducing agents was prepared by passing the 638-nm substance through a column of Sephadex G-25. The ferrous 688-nm substance was degraded to a biliverdin-iron complex much more rapidly in the presence of the NADPH-cytochrome P-450 reductase system than in its absence, indicating that a reducing equivalent is essential for the initiation of heme degradation even when starting from the ferrous 688-nm substance. Cyanide was found to bind to the ferrous 688-nm substance to form a stable compound; the cyanide compound formed could revert to neither the ferrous 688-nm substance nor the 638-nm substance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of oxygen binding to ferrous myeloperoxidase

Myeloperoxidase (MPO), which is involved in host defence and inflammation, is a unique peroxidase in having a globin-like standard reduction potential of the ferric/ferrous couple. Intravacuolar and exogenous MPO released from stimulated neutrophils has been shown to exist in the oxyferrous form, called compound III. To investigate the reactivity of ferrous MPO with molecular oxygen, a stopped-flow kinetic analysis was performed. In the absence of dioxygen, ferrous MPO decays to ferric MPO (0.04 s(-1) at pH 8 versus 1.4 s(-1) at pH 5). At pH 7.0 and 25 degrees C, compound III formation (i.e., binding of dioxygen to ferrous MPO) occurs with a rate constant of (1.1+/-0.1) x 10(4)M(-1)s(-1). The rate doubles at pH 5.0 and oxygen binding is reversible. At pH 7.0, the dissociation equilibrium constant of the oxyferrous form is (173+/-12)microM. The rate constant of dioxygen dissociation from compound III is much higher than conversion of compound III to ferric MPO (which is not affected by the oxygen concentration). This allows an efficient transition of compound III to redox intermediates which actually participate in the peroxidase or halogenation cycle of MPO.     

High-pressure investigations of cytochrome P-450 spin and substrate binding equilibria

The effects of high pressure (1-2000 bar) on the spin state and substrate binding equilibria in cytochrome P-450 have been determined. The high-spin (S = 5/2) to low spin (S = 1/2) transition of the ferric hemoprotein was monitored by uv-visible spectroscopy at various substrate concentrations. Increasing hydrostatic pressure on a sample of substrate-bound cytochrome P-450 resulted in a decrease in the high-spin fraction as monitored by a Soret maxima at 391 nm and an increase in the low-spin 417-nm region of the spectrum. These pressure-induced optical changes were totally reversible for all pressures below 800 bar and were found to correspond to simple substrate dissociation from the enzyme. High levels of the normally metabolized substrate, d-camphor, corresponding to a 99.9% saturation of the hemoprotein active site (50 mM Tris-Cl, 100 mM KCl, pH 7.2) completely prevented the pressure-induced high-spin to low-spin transition that is observed at less than saturating substrate concentrations. A gradual increase in the formation of the inactive P-420 form of the cytochrome was noted if the pressure of the sample was increased above 800 bar. These pressure-linked spectral changes were used to determine the microscopic volume change accompanying substrate binding, which was found to be -47.0 +/- 2 ml/mol (pH 7.2) which represents a substantial change for a ligand dissociation reaction. The observed volume change for camphor binding decreases to -30.6 +/- 2 ml/mol at pH 6.0, suggesting the involvement of a linked proton equilibrium. Various substrate analogs of camphor induce varying degrees of low-spin to high-spin shift upon binding to ferric cytochrome P-450 (3). The volume changes for the dissociation of these substrates were very similar to those obtained with camphor. The conformational changes associated with a shift from high- to low-spin ferric iron appear to be small in comparison to the overall macroscopic changes in volume accompanying substrate binding to the enzyme.     

Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme

We describe herein for the first time the formation and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) holoenzyme and heme domain (BMP) at -55 degrees C using a 70/30 (v/v) glycerol/buffer cryosolvent. The choice of buffer is a crucial factor with Tris [tris(hydroxymethyl)aminomethane] buffer being significantly more effective than phosphate. The oxyferrous complexes have been characterized with magnetic circular dichroism spectroscopy and the resulting spectra compared to those of the more well-characterized oxyferrous cytochrome P450-CAM. The formation of a stable substrate-bound oxyferrous CYP BM3 holoenzyme, despite the fact that it has the necessary reducing equivalents for turnover, indicates that electron transfer from the flavin domain to the oxyferrous center is very slow at this temperature. The ability to prepare stable homogeneous oxyferrous derivatives of both BMP and the CYP BM3 holoenzyme will enable these species to be used as starting materials for mechanistic investigation of dioxygen activation.     

Kinetic analysis of oxidation of coumarins by human cytochrome P450 2A6

Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b5 (b5). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (k(on) approximately 10(6) m(-1) s(-1)), and the k(off) rates range from 5.7 to 36 s(-1) (at 23 degrees C). Reduction of ferric P450 2A6 is rapid (7.5 s(-1)) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O2 is rapid (k > or = 10(6) m(-1) s(-1)), and the putative Fe2+.O2 complex decayed at a rate of approximately 0.3 s(-1) at 23 degrees C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by b5. Kinetic analyses showed that approximately 1/3 of the reduced b5 was rapidly oxidized in the presence of the Fe2+.O2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6-10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s.     

H93G myoglobin cavity mutant as versatile template for modeling heme proteins: magnetic circular dichroism studies of thiolate- and imidazole-ligated complexes

Recent ligand binding and spectroscopic investigations of the myoglobin H93G cavity mutant are reviewed, revealing it to be a versatile template for the preparation of model heme complexes of defined structure. The H93G myoglobin cavity mutant is shown to be capable of forming mixed ligand adducts because of the difference in accessibility of the two sides of the ferric heme iron. With imidazole bound in the proximal cavity, H93G myoglobin also forms reasonably stable oxyferrous and oxoferryl derivatives, thereby providing a potential system to use for the study of such complexes with proximal ligands other than imidazole. In addition, thiolate-ligated ferric H93G derivatives are described that serve as spectroscopic models for the high-spin ferric state of cytochrome P450. All of the complexes described are characterized with magnetic circular dichroism spectroscopy, and they are compared to the appropriate derivatives of native myoglobin and P450.     

Topology of catalytic portion of prostaglandin I(2) synthase: identification by molecular modeling-guided site-specific antibodies

Prostaglandin I(2) synthase (PGIS) is an eicosanoid-synthesizing cytochrome P450, located in the endoplasmic reticulum (ER) membrane. The membrane topology of the catalytic portion of PGIS is still unknown. General models of the membrane topology of microsomal P450s have been proposed in two forms: (a) large part of the polypeptide exposed on the cytoplasmic side with an NH(2)-terminal membrane anchor to the ER membrane and (b) deep immersion of the polypeptide in the membrane, as described by J. P. Miller et al. (1996, Biochemistry 35, 1466-1474). We have characterized the membrane topology of catalytic portion of PGIS using molecular modeling-guided site-specific antibodies. A 3D working model of PGIS was constructed by homology modeling using P450(BM-3) crystal structure as a template (S. K. Shyue et al., 1997, J. Biol. Chem. 272, 3657-3662). Three hydrophilic peptides corresponding to different regions of the surface portion of PGIS with residues 109-127 (P109-127), 353-368 (P353-368), and 411-431 (P411-431) predicted from the model and an NH(2)-terminal hydrophobic peptide (residues 1-28, P1-28) were synthesized and used to prepare site-specific antibodies. All three of the hydrophilic peptide antibodies have high titer and are specifically recognized human PGIS, as shown by binding assays and Western blot analysis. In contrast, the hydrophobic NH(2)-terminal peptide has a much lower titer binding to the PGIS protein. The overall arrangement of the PGIS polypeptide with respect to the endoplasmic reticulum (ER) membrane was examined by immunocytochemistry techniques in transiently transfected COS-1 cells with recombinant human PGIS cDNA and in ECV cells expressing endogenous PGIS. The immunofluorescence staining for the cells with selective permeabilization of the plasma membrane using streptolysin O indicated that all three of the hydrophilic peptide antibodies bound to the cytoplasmic surface of the ER membrane. These results provide direct experimental evidence supporting the predicted 3D protein topological model in which the segments are located on the protein surface and the membrane topological model in which PGIS is largely exposed on the cytoplasmic side of the ER membrane. It also led us to conclude that the PGIS substrate, prostaglandin H(2) (PGH(2)), produced by prostaglandin H(2) synthase (PGHS) in the ER lumenal side must pass through the ER membrane barrier to the catalytic site of the PGIS in the cytoplasmic side of the ER membrane.     

Studies on the cytochrome P-450 product complexes formed during the metabolism of N,N-dimethylaniline

The oxidative metabolism of N,N-dimethylaniline by partially solubilized cytochrome P-450 from rabbit liver was found to be associated with the formation of a 424- and 448-nm product adduct of the hemoprotein. From the effects of temperature, hydrogen ion concentration, n-octylamine, extraction of the enzyme preparations with organic solvents and pretreatment of the animals with inducers of drug metabolism on both the formation of the spectral species and the enzymic C- and N-oxidation of N,N-dimethylaniline it is concluded that the 424-nm spectral change is generated from an intermediate in the C-oxidation reaction, whereas formation of the 448-nm spectral perturbation is the result of binding to cytochrome P-450 of a metabolite arising from N-oxidation of the arylamine; N-dealkylation of the parent amine is not a obligatory intermediary step in 448-nm complex formation.The 448-nm ferrohemochrome is supposed to be formed through coordination of the N-oxidized intermediate via the oxygen atom. This type of interaction appears to require considerably stronger thermal activation as compared with the 424-nm complex. The 448-nm product adduct of cytochrome P-450 is unstable in the ferric state or in the presence of sodium dithionite.     

Studies on the equilibria and kinetics of the reactions of ferrous catalase with ligands

The optical absorption spectrum of bovine liver catalase was found to change on light irradiation in the presence of proflavin and EDTA in a deaerated solution. Upon addition of CO to the photolyzed product, the spectrum changed to an another form, suggesting that the photolyzed product is the ferrous form of the enzyme and CO is bound to the ferrous enzyme. When O2 was introduced into the ferrous enzyme, the absorption spectrum returned to its original ferric state. An intermediate spectrum was obtained in this reaction at -20 degrees C in 33% v/v ethylene glycol. Judged from the spectral characteristics of this compound, it is probably an oxyferrous enzyme. It was converted into ferric enzyme gradually when the sample was left at room temperature. The ferrous enzyme, which was generated by flash photolysis of the CO complex of the enzyme in an air-saturated buffer, reacted with O2 to form the oxyferrous enzyme with a second order rate constant of 9.2 x 10(3) M-1.s-1 at pH 8.6 and 20 degrees C. The oxyferrous enzyme thus obtained autodecomposed into the ferric form with a rate constant of 0.1 s-1.     

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.     

Resonance Raman investigations of Escherichia coli-expressed Pseudomonas putida cytochrome P450 and P420.

High-resolution resonance Raman spectra of the ferric, ferrous, and carbonmonoxy (CO)-bound forms of wild-type Escherichia coli-expressed Pseudomonas putida cytochrome P450cam and its P420 form are reported. The ferric and ferrous species of P450 and P420 have been studied in both the presence and absence of excess camphor substrate. In ferric, camphor-bound, P450 (mos), the E. coli-expressed P450 is found to be spectroscopically indistinguishable from the native material. Although substrate binding to P450 is known to displace water molecules from the heme pocket, altering the coordination and spin state of the heme iron, the presence of camphor substrate in P420 samples is found to have essentially no effect on the Raman spectra of the heme in either the oxidized or reduced state. A detailed study of the Raman and absorption spectra of P450 and P420 reveals that the P420 heme is in equilibrium between a high-spin, five-coordinate (HS,5C) form and low-spin six-coordinate (LS,6C) form in both the ferric and ferrous oxidation states. In the ferric P420 state, H2O evidently remains as a heme ligand, while alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys(357) thiolate binding to the heme iron. Ferrous P420 also consists of an equilibrium between HS,5C and LS,6C states, with the spectroscopic evidence indicating that H2O and histidine are the most likely axial ligands. The spectral characteristics of the CO complex of P420 are found to be almost identical to those of a low pH of Mb. Moreover, we find that the 10-ns transient Raman spectrum of the photolyzed P420 CO complex possesses a band at 220 cm-1, which is strong evidence in favor of histidine ligation in the CO-bound state. The equilibrium structure of ferrous P420 does not show this band, indicating that Fe-His bond formation is favored when the iron becomes more acidic upon CO binding. Raman spectra of stationary samples of the CO complex of P450 reveal VFe-CO peaks corresponding to both substrate-bound and substrate-free species and demonstrate that substrate dissociation is coupled to CO photolysis. Analysis of the relative band intensities as a function of photolysis indicates that the CO photolysis and rebinding rates are faster than camphor rebinding and that CO binds to the heme faster when camphor is not in the distal pocket.