Cytochrome P-450cam and putidaredoxin interaction during electron transfer.

Cytochrome P-450cam, the bacterial hemeprotein which catalyzes the 5-exo-hydroxylation of d-camphor, requires two electrons to activate molecular oxygen for this monooxygenase reaction. These two electrons are transferred to cytochrome P-450cam in two one-electron steps by the physiological reductant, putidaredoxin. The present study of the kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin has shown that the reaction obeys first order kinetics with a rate constant of 33 s-1 at 25 degrees C with respect to: 1) the appearance of the carbon monoxide complex of Fe(II) cytochrome P-450cam; 2) the disappearance of the 645 nm absorbance band of high-spin Fe(III) cytochrome P-450cam; and 3) the disappearance of the g = 1.94 EPR signal of reduced putidaredoxin. This data was interpreted as indicative of the rapid formation of a bimolecular complex between reduced putidaredoxin Fe(III) cytochrome P-450cam. The existence of the complex was first shown indirectly by kinetic analysis and secondly directly by electron paramagnetic resonance spectroscopic analysis of samples which were freeze-quenched approximately 16 ms after mixing. The direct evidence for complex formation was the loss of the EPR signal of Fe(III) cytochrome P-450cam upon formation of the complex while the EPR signal of reduced putidaredoxin decays with the same kinetics as the appearance of Fe(II) cytochrome P-450. The mechanism of the loss of the EPR signal of cytochrome P-450 upon formation of the complex is not apparent at this time but may involve a conformational change of cytochrome P-450cam following complex formation.     

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.     

Single turnover studies with oxy-cytochrome P-450cam

The catalytic step of bacterial cytochrome P-450cam, i.e., the step of the reaction cycle in which the product 5-exo-hydroxycamphor is formed and released by the enzyme, has been studied by stopped-flow spectrophotometry. Our approach has been to observe a single-turnover reaction between reduced putidaredoxin and oxygenated camphor-bound cytochrome P-450cam. Multiple turnovers are prevented by using the inhibitor metyrapone to trap the cytochrome after product release, which prevents binding of another camphor molecule. The time course of the reaction has been measured at several wavelengths and has been found to be biphasic. The relatively slow second phase of the reaction is the reduction of ferric, metyrapone-bound cytochrome P-450cam. The first phase coincides with the formation of product stoichiometrically with cytochrome P-450cam, as measured by gas chromatography. A detailed kinetic study of the first phase reveals a hyperbolic dependence of initial rate upon putidaredoxin concentration at a fixed, limiting concentration of cytochrome P-450cam. The Vmax is 53 microM per second per microM cytochrome, and the Km for putidaredoxin is 33 microM. The hyperbolic relationship between initial rate and putidaredoxin concentration supports a model in which the cytochrome rapidly binds putidaredoxin, then undergoes one or more slower intracomplex steps.     

Nonalcoholic steatosis and steatohepatitis. II. Cytochrome P-450 enzymes and oxidative stress

Oxidative stress is present in the liver of humans with steatosis and nonalcoholic steatohepatitis (NASH) and is a plausible mediator of cellular injury, inflammatory recruitment, and fibrogenesis. CYPs 2E1 and 4A are the microsomal oxidases involved with fatty acid oxidation. Both enzymes can reduce molecular oxygen to produce prooxidant species, which, if not countered efficiently by antioxidants, create oxidative stress. In this theme article, we present the evidence that, in the context of hepatic steatosis, CYPs 2E1 and 4A could generate the "second hit" of cellular injury, particularly when antioxidant reserves are depleted, and propose ways in which this could contribute to the pathogenesis of NASH.     

The interaction of aliphatic analogs of methylene-dioxyphenyl compounds with cytochromes P-450 and P-420.

The spectra resulting from the interaction of a series of substituted dioxolanes with microsomal cytochromes P-450 or P-420, as well as purified cytochrome P-450, were measured. With the exception of dioxolane, 4-methyldioxolane and 4-ethyldioxolane, these compounds interacted with ferric cytochrome P-450 to give complexes exhibiting type I optical difference spectra, and, after incubation with NADPH, spectra with peaks at about 430 nm. These complexes, as well as those formed from dioxolanes in the presence of cumene hydroperoxide, inhibit the binding of CO to the cytochrome. Consideration of the known chemistry of dioxolanes, together with recent advances in the understanding of double Soret spectra, lead to a possible explanation for the differences between the spectra of dioxolanes and their aromatic analogs, the methylenedioxyphenyl compounds.     

Hydroxylation of taurolithocholate by isolated human liver microsomes II. Cytochrome P-450 dependency

The conversion of taurolithocholate to taurohyodeoxycholate by 6α-hydroxylation by isolated human liver microsomes and NADPH is inhibited by CO and the inhibition is maximally reversedby monochromatic light at 450 nm indicating that the reaction is cytochrome P-450 dependent.     

Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes.     

Cytochrome P-450 reduction exhibits burst kinetics

Cytochrome P-450 reduction kinetics can be described by sequential reactions involving a rapid reduction of cytochrome P-450 in the high spin state, followed by a slower reduction controlled by formation of high spin P-450 from the low spin configuration. The burst kinetics observed would be the result of the equilibrium between low and high spin states prior to addition of reducing equivalents. The initial reduction velocity (burst) can therefore be described as v_i=k₃m_hs⁰ and the slower velocity observed at longer times is controlled by the net rate of formation of the high spin conformation.     

Cytochrome P-450 and halothane metabolism. Decrease in rat liver microsomal P-450 in vitro

Anaerobic in vitro incubation of microsomes from phenobarbital(PB)-induced rats with halothane results in an irreversible decrease of measurable cytochrome P-450. There is a parallel decrease in heme content under the same incubation conditions. However, microsomes from 3-methylcholanthrene(3-MC)-induced or untreated animals do not show a reduction in cytochrome P-450 content. Aerobic incubation with halothane results in a decrease of cytochrome P-450 which can be completely reversed by dialysis or the addition of potassium ferricyanide. These latter treatments only partially restore the cytochrome P-450 levels following anaerobic incubations. The decrease in cytochrome caused by halothane is not associated with measureable heme N-alkyl adduct formation; lipid peroxidation does not play a role as indicated by the lack of effect of 1 mM EDTA or a decrease in glucose-6-phosphatase activity. Halothane metabolites are bound irreversibly to microsomal protein as determined by gel electrophoresis only when the oxygen concentration is very low. The mechanism of cytochrome P-450 decrease is consistent with the formation of a reactive metabolite which binds to the protein portion and also destroys heme.     

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.