Cobalt-substituted cytochrome P-450cam

Reconstitution of the apo-cytochrome with cobalt protoporphyrin provides a faithful P-450cam analogue as characterized by optical, ligand-binding, and enzymatic parameters. The thiol and cyanide complexes exhibit Soret "hyper" spectra, not previously observed in cobalt porphyrins. Substrate-induced spectral changes and limited stereospecific hydroxylation activity are retained in the cobalt P-450cam. The EPR (electron paramagnetic resonance) spectra of the reduced cobaltous protein indicate clearly an endogenous axial ligand other than a nitrogenous base and support an assignment of thiolate coordination. A thiolate ligand is also indicated by EPR measurements in the oxygenated cobaltous analogue. By analogy, these studies suggest that the native ferrous and oxygenated P-450cam states retain a thiolate axial ligand.     

Ethylbenzene hydroxylation by cytochrome P450cam.

The metabolism of ethylbenzene by cytochrome P450cam was analyzed by experimental and theoretical methods. The present experiments indicate that ethylbenzene is hydroxylated almost exclusively at the secondary ethyl carbon with about a 2:1 ratio of R:S product. Several molecular dynamics trajectories were performed with different starting conformations of ethylbenzene in the active site of P450cam. The stereochemistry of hydroxylation predicted from the molecular dynamics simulations was found to be in good agreement with the observed products.     

Hydroperoxide catalyzed liver microsomal aromatic hydroxylation reactions involving cytochrome P-450

Cumene hydroperoxide is capable of supporting the aromatic hydroxylation of a variety of compounds in the presence of hepatic microsomes. NADPH and molecular oxygen are not required. Cytochrome P-450 acts as the catalyst and could not be replaced by other hemoproteins. One mole of hydroperoxide is consumed for every mole of substrate hydroxylated. It is suggested that the oxenoid species of cytochrome P-450 involved in microsomal aromatic hydroxylation is present in a form equivalent to the ferryl from.     

Dynamics underlying hydroxylation selectivity of cytochrome P450cam

Structural heterogeneity and the dynamics of the complexes of enzymes with substrates can determine the selectivity of catalysis; however, fully characterizing how remains challenging as heterogeneity and dynamics can vary at the spatial level of an amino acid residue and involve rapid timescales. We demonstrate the nascent approach of site-specific two-dimensional infrared (IR) spectroscopy to investigate the archetypical cytochrome P450, P450cam, to better delineate the mechanism of the lower regioselectivity of hydroxylation of the substrate norcamphor in comparison to the native substrate camphor. Specific locations are targeted throughout the enzyme by selectively introducing cyano groups that have frequencies in a spectrally isolated region of the protein IR spectrum as local vibrational probes. Linear and two-dimensional IR spectroscopy were applied to measure the heterogeneity and dynamics at each probe and investigate how they differentiate camphor and norcamphor recognition. The IR data indicate that the norcamphor complex does not fully induce a large-scale conformational change to a closed state of the enzyme adopted in the camphor complex. Additionally, a probe directed at the bound substrate experiences rapidly interconverting states in the norcamphor complex that explain the hydroxylation product distribution. Altogether, the study reveals large- and small-scale structural heterogeneity and dynamics that could contribute to selectivity of a cytochrome P450 and illustrates the approach of site-selective IR spectroscopy to elucidate protein dynamics.     

Proton activities for three states of cytochrome P-450cam.

Changes in proton concentration during the binding of dioxygen, carbon monoxide, and for the exchange of dioxygen by carbon monoxide, at ferrous-cytochrome P-450cam were measured by direct titration. Insufficient proton release was observed to support protonation-deprotonation of an axial cysteinyl sulfur donor as a mechanism for generation of hyper spectra in only the carbonylated ferrous state. Measurement of the $\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ value for CO binding as a function of pH (the carbon monoxide Bohr effect) confirms the direct titration data.     

Binding sites of pyridine in cytochrome P-450cam

Careful titration of oxidized cytochrome P-450cam from Pseudomonas putida with pyridine revealed deviations of the Eadie plot from linearity in the substrate-bound as well as in the substrate-free protein. A binding model which assumes two binding sites for pyridine--the iron and the camphor binding site--is able to describe completely the nonlinear Eadie plot.     

Apoprotein formation and heme reconstitution of cytochrome P-450cam

Apoprotein suitable for heme reconstitution has been prepared by an acid/butanone extraction of cytochrome P-450cam at pH 2.5. Absorption spectra of apo-P-450cam indicate less than 2% residual holoenzyme. Four tryptophan residues per molecule were estimated from the aromatic absorbance region of denatured apoprotein. Heme-reconstituted holoprotein was purified in 30% yield to a specific activity equivalent to the native enzyme. Absorption and EPR spectra of 57Fe- and 54Fe-heme-enriched P-450cam reveal complete restoration of the native active site.     

NADPH: cytochrome P-450 reductase in olfactory epithelium. Relevance to cytochrome P-450-dependent reactions.

The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue.     

Electron paramagnetic resonance detectable states of cytochrome P-450cam

Cytochrome P-450cam is a low-spin Fe3+hemoprotein (g = 2.45, 2.26, and 1.91) which is made 60% high spin (g = 7.85, 3.97, and 1.78) at 12 K by the addition of 1 mol of substrate per mol of enzyme. Low-temperature EPR spectra show that the low-spin fraction of substrate-bound P-450cam contains two magnetic species. The majority species has an unusual EPR spectrum (g = 2.42, 2.24, and 1.97) which connot be simulated by using the range of crystal field parameters known for other heme proteins. The minority species has the same g values as substrate-free enzyme. Both low-spin species show Curie law temperature dependence below 50 K and have similar saturation behavior. Above 50 K the g = 2.42, 2.24, and 1.97 species rapidly loses signal intensity. The distribution of low-spin species is pH dependent (apparent pKa = 6.2) with the g = 2.42, 2.24, and 1.97 magnetic species favored at high pH. The substrate binding stoichiometry and the equilibria observed in the low-spin fraction suggest that there are not multiple protein forms of cytochrome P-450cam. Putidaredoxin and other effector molecules which specifically catalyze hydroxylation convert either the high-spin or the g = 2.42, 2.24, and 1.97 low-spin species to another new magnetic species (g = 2.47, 2.26, and 1.91). This species is only seen in the presence of substrate, and its stability reflects the catalytic potency of the effector molecule. The EPR and UV-visible spectra of cytochrome P-420 depend upon the manner in which the P-420 is generated. Incubation with acetone or reaction with N-ethylmaleimide or diethyl pyrocarbonate generates P-420 with different spectral characteristics. Through identification of active-site amino acids by chemical modification and comparison with porphyrin model complexes, the range of ligands likely to participate in each of the EPR detectable species is assigned. Mechanisms of interconversion of these species and their bearing on catalysis are discussed.     

Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450.

The stoichiometry of hydroxylation reactions catalyzed by cytochrome P-450 was studied in a reconstituted enzyme system containing the highly purified cytochrome from phenobarbital-induced rabbit liver microsomes. Hydrogen peroxide was shown to be formed in the reconstituted system in the presence of NADPH and oxygen; the amount of peroxide produced varied with the substrated added. NADPH oxidation, oxygen consumption, and total product formation (sum of hydroxylated compound and hydrogen peroxide) were shown to be equimolar when cyclohexane, benzphetamine, or dimethylaniline served as the substrate. The stoichiometry observed represents the sum of two activities associated with cytochrome P-450. These are (1) hydroxylase activity: NADPH + H⁺ + O₂ + RH → NADP⁺ + H₂O + ROH; and (2) oxidase activity: NADPH + H⁺ + O₂ → NADP⁺ + H₂O₂. Benzylamphetamine (desmethylbenzphetamine) acts as a pseudosubstrate in that it stimulates peroxide formation to the same extent as the parent compound (benzphetamine), but does not undergo hydroxylation. Accordingly, when benzylamphetamine alone is added in control experiments to correct for the NADPH and O₂ consumption not associated with benzphetamine hydroxylation, the expected 1:1:1 stoichiometry for NADPH oxidation, O₂ consumption, and formaldehyde formation in the hydroxylation reaction is observed.     

Determination of cytochrome b5 association reactions. Characterization of metmyoglobin and cytochrome P-450cam binding to genetically engineered cytochromeb5

Genetically engineered cytochrome b5 has been used to quantitative binding interactions of this protein with cytochrome P-450cam and sperm whale metmyoglobin by static fluorescence titration. Two cytochrome b5 mutants were constructed by cassette mutagenesis to replace a surface threonine residue with cysteine at two crystallographically defined positions, 65 and 8, located 11 and 21 A, respectively, from the nearest heme edge. The T65C and T8C mutant proteins were labeled with the sulfhydryl selective fluorescent reagent, acrylodan, which provided a spectral probe for monitoring protein-protein association. The fluorescence emission spectra of the acrylodan-labeled T65C mutant exhibited an ionic strength-dependent, blue-shifted fluorescence enhancement upon binding met-myoglobin, cytochrome c, and cytochrome P-450cam, whereas the acrylodan-labeled T8C mutant fluorescence emission remained unchanged during all titrations. Dissociation constants of 1.3, 0.6, and 0.5 microM, pH 7.15, were measured for metmyoglobin, cytochrome P-450cam, and cytochrome c, respectively. A similar averaged binding surface for cytochrome P-450cam and cytochrome c is suggested by their closely related degree of fluorescence enhancement, degree of emission blue shift, and binding free energies. Myoglobin binds less tightly, enhances fluorescence to a greater extent, and exhibits a larger blue shift in acrylodan emission spectra suggesting a different averaged binding orientation relative to the acrylodan probe.     

Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: a proposed molecular model for a cytochrome P-450cam electron-transfer complex

Cytochrome b5 has been genetically engineered to afford a fluorescent derivative capable of monitoring its association with cytochrome P-450cam from Pseudomonas putida [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. In the mutant cytochrome b5, threonine is replaced by a cysteine at position 65 (T65C) and has been labeled with the environmentally sensitive fluorophore acrylodan. In this paper, the physiological P-450cam reductant putidaredoxin, an Fe2S2.Cys4 iron-sulfur protein, is shown to competitively inhibit the cytochrome b5 association, suggesting that cytochrome b5 and putidaredoxin bind to a similar site on the cytochrome P-450cam surface. Since the crystal structures for both cytochrome b5 and cytochrome P-450cam have been solved to high resolution, the complex has been computer modeled, and a good fit was found on the proximal surface of nearest approach to the P-450cam heme prosthetic group. The proposed model includes electrostatic contacts between conserved cytochrome b5 carboxylates Glu-44, Glu-48, Asp-60, and the exposed heme propionate with cytochrome P-450cam basic residues Lys-344, Arg-72, Arg-112, and Arg-364, respectively. Putidaredoxin has similarly been shown to contain a carboxylate-based binding surface, and the current results suggest that if the model is correct, then it also interacts at the proposed site, probably utilizing similar P-450cam electrostatic contacts.