Effect of phospholipids on the activity of highly-purified liver microsome cytochrome P-450 in a reaction of aniline and naphthalene hydroxylation by hydroperoxides

The effect of different phospholipids on the functional activity of highly purified cytochrome P-450 used as a co-substrate of cumene hydroperoxide was examined. At the molar ratio of phospholipids to cytochrome P-450 that was equal to 30, phosphatidyl serine, phosphatidyl inositol, the total fraction of microsomal phospholipids, and lysophosphatidyl choline increased the hydroxylation rate of aniline and naphthalene. The effect of the above phospholipids on the rate of naphthalene oxidation was much more pronounced. Phosphatidyl choline and sphingomyelin in similar large quantities did not exert a stimulating effect on the reactions studied. The kinetic parameters of aniline oxidation in the systems containing phospholipids that produced an activating effect were investigated.     

Possible higher valence states of cytochrome P-450 during oxidative reactions

The addition of the organic hydroperoxide, cumene hydroperoxide, to liver microsomes results in the appearance of a transient spectral change associated with cytochrome P-450. In addition, unique electron paramagnetic resonance signals are observed with liver microsomal cytochrome P-450 comparable to signals obtained when peroxides interact with metmyoglobin. It is suggested that higher valence states of cytochrome P-450 may function during the activation of oxygen for the hydroxylation of a variety of xenobiotics.     

Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450.

Highly purified liver microsomal cytochrome P-450 catalyzes the hydroperoxide-dependent hydroxylation of a variety of substrates in the absence of NADPH, NADPH-cytochrome P-450 reductase, and molecular oxygen. The addition of phosphatidylcholine is necessary for maximal activity. The absence of flavoproteins and cytochrome b₅ from the cytochrome P-450 preparations rules out the involvement of other known microsomal electron carriers. The ferrous form of cytochrome P-450 is not involved in peroxide-dependent hydroxylation reactions, as indicated by the lack of inhibition by carbon monoxide. With cumene hydroperoxide present, a variety of substrates is attacked, including N-methylaniline, N,N-dimethylaniline, cyclohexane, benzphetamine, and aminopyrine. With benzphetamine as the substrate, cumene hydroperoxide may be replaced by other peroxides, including hydrogen peroxide, or by peracids or sodium chlorite. A study of the stoichiometry indicated that equimolar amounts of N-methylaniline, formaldehyde, and cumyl alcohol (α,α-dimethylbenzyl alcohol) are formed in the reaction of N,N-dimethylaniline with cumene hydroperoxide. Since H₂¹⁸O is incorporated only slightly into cyclohexanol in the reaction of cyclohexane with cumene hydroperoxide, it appears that the oxygen atom in cyclohexanol is derived primarily from the peroxide. The data obtained are in accord with a peroxidase-like mechanism for the action of cytochrome P-450.     

The mechanism of cumene hydroperoxide-dependent lipid peroxidation: the significance of oxygen uptake

The addition of limiting amounts of cumene hydroperoxide to rat liver microsomes prepared from phenobarbital-treated rats resulted in the rapid uptake of molecular oxygen, the formation of thiobarbituric acid reactive products, and the loss of hydroperoxide over a similar time course. Maximal activity was observed at pH 7-8. The addition of cumene hydroperoxide to boiled microsomes did not initiate oxygen uptake or produce thiobarbituric acid reactive products. Oxygen uptake was required for the formation of thiobarbituric acid reactive products, but not for the loss of hydroperoxide. The extent of oxygen uptake and thiobarbituric acid reactive product formation was linearly dependent on the concentration of cumene hydroperoxide and independent of the amount of microsomes. For each nanomole of cumene hydroperoxide utilized, 1.5 nmol of oxygen was consumed and 0.11 nmol of thiobarbituric acid reactive products was formed. In addition, a saturable reaction having a high affinity for cumene hydroperoxide was observed that was associated with little or no oxygen uptake and thiobarbituric acid reactive product formation. Butylated hydroxytoluene at substoichiometric concentrations inhibited the extents and initial rates of oxygen uptake and thiobarbituric acid reactive product formation, indicating that cumene hydroperoxide-dependent lipid peroxidation may be an autocatalytic free radical process.     

Benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae. Substrate binding, spectral and kinetic data

Saccharomyces cerevisiae, brewer's yeast, produces a microsomal benzo(a)pyrene hydroxylase when grown at high glucose concentrations of which the haemoprotein, cytochrome P-450 (RH, reduced-flavoprotein:oxygen oxidoreductase (RH-hydroxylating) EC 1.14.14.1) is a component. We report here kinetic data derived from Lineweaver-Burk plots of benzo(a)pyrene hydroxylation. The Michaelis constant was decreased by growth of the yeast in the presence of benzo(a)pyrene showing the induction of a form of the enzyme more specific for this compound. NADPH or cumene hydroperoxide could be used as cofactors by this enzyme, although with different Km and V values for benzo(a)pyrene. A solubilised and a solubilised, immobilised enzyme preparation were capable of benzo(a)pyrene hydroxylation, using cumene hydroperoxide but not NADPH as the cofactor. Benzo(a)pyrene was found to produce a modified type I spectral change with yeast and rat liver microsomes. The interaction of benzo(a)pyrene with cytochrome P-450 was investigated further by means of an equilibrium gel filtration technique. There appeared to be 20 binding sites per mol ofcytochrome P-450 for benz(a)pyrene, in both yeast and rat liver microsomes.     

Role of THR501 residue in substrate binding and catalytic activity of cytochrome P4501A1

A putative binding region for cumene hydroperoxide in the active site of cytochrome P4501A1 was identified using photoaffinity labeling. Thr501 was determined as the most likely site of modification by azidocumene used as the photoaffinity label (T. Cvrk and H. W. Strobel, (1998) Arch. Biochem. Biophys. 349, 95-104). To evaluate further the role of this amino acid residue a site-directed mutagenesis approach was employed. P4501A1 wild type and two mutants, P4501A1Glu501 and P4501A1Phe501, were expressed in and purified from Escherichia coli and used for kinetic analysis to confirm the role of Thr501 residue in cumene hydroperoxide binding. The mutation resulted in a two- to fourfold decrease in the rate of heme degradation in the presence of 0.5 mM cumene hydroperoxide. The mutations do not prevent or significantly alter binding of the tested substrates; however, binding of 2-phenyl-2-propanol (product generated from cumene hydroperoxide) to P4501A1Glu501 and P4501A1Phe501 exhibited four- and eightfold decreases, respectively, suggesting that the mutations strongly affected the affinity of cumene hydroperoxide for the P4501A1 active site. The kinetic analysis of cumene hydroperoxide-supported reactions showed that both mutants exhibit increased Km and decreased VMax values for all tested substrates. Furthermore, the mutations affected product distribution in testosterone hydroxylation. On the basis of P4501A1Glu501 and P4501A1Phe501 characterization, it can be concluded that Thr501 plays an important role in cumene hydroperoxide/P4501A1 interaction.     

Effects of cumene hydroperoxide on cellular cation composition in frog kidney proximal tubular cells

Effects of cumene hydroperoxide were studied on the peritubular membrane potential and cellular cation composition in frog kidney proximal tubular cells. After perfusion of isolated frog kidneys for 30 min with 1.3x10(-4) mol l(-1) cumene hydroperoxide Ringer solution, the peritubular membrane potential gradually declined. The ouabain-like effects were demonstrated on cell Na and K activities after 1 h of perfusion with cumene hydroperoxide. The peritubular apparent transference number for potassium was decreased. Intracellular pH was not altered in the presence of cumene hydroperoxide. Intracellular free Ca(2+) concentration increased slowly and moderately. The concentration of the malondialdehyde in the kidney homogenates, measured as an index of lipid peroxidation, was increased. A previously observable effect of cumene hydroperoxide on the peritubular membrane potential was prevented by oxygen radical scavengers.     

Further characterization of the 2-deoxyecdysone C-2 hydroxylase from Locusta migratoria

Additional data are provided on the enzyme 2-deoxyecdysone C-2 hydroxylase which has been shown in a previous study (Kappler et al., 1986) to be a mitochondrial hydroxylase with some classical characteristics of a cytochrome P-450 monooxygenase but which appeared to be insensitive to CO. Using ¹⁸O₂, we have now demonstrated that molecular oxygen is directly incorporated into ecdysone during the process of C-2 hydroxylation. Neither cumene hydroperoxide nor linoleyl hydroperoxide could support C-2 hydroxylation. When the reaction was sustained by α-ketoglutarate, addition of cofactors like Fe²⁺, ascorbate and catalase caused only a slight increase of the enzymatic activity whereas the α-ketoglutarate-dependent hydroxylation was largely decreased in the presence of malonate; these data eliminate the possible existence of a dioxygenase mechanism for C-2 hydroxylation.The paper also provides inhibition kinetics which indicate that 2-deoxy-20-hydroxyecdysone, 2,22-bisdeoxyecdysone and 2,22,25-trideoxyecdysone are competitive inhibitors of the C-2 hydroxylase whereas the 3-epi isomer of 2-deoxyecdysone is a non-competitive inhibitor.     

Cumene hydroperoxide effected hydroperoxidation by cytochrome P-450

9-Methylfluorene was found to be oxygenated to 9-hydroperoxy-9-methylfluorene and 9-hydroxy-9-methylfluorene by cytochrome P-450 in the presence of cumene hydroperoxide. Molecular oxygen is required and carbon monoxide is inhibitory. The reaction is inhibited by SKF-525A and metyrapone. Metyrapone and cumene hydroperoxide also retard the conversion of 9-hydroperoxy-9-methylfluorene to 9-hydroxy-9-methylfluorene. The reaction is different from hydroperoxide-supported oxygenation, since the cumene hydroperoxide appears to act as an effector of the enzyme rather than oxygen donor. It is suggested that substrates with stable radicals can be dioxygenated in this manner.     

Indole hydroxylation by bacterial cytochrome P450 BM-3 and modulation of activity by cumene hydroperoxide

Cytochrome P450 BM-3 from Bacillus megaterium catalyzed NADPH-supported indole hydroxylation under alkaline conditions with homotropic cooperativity toward indole. The activity was also found with the support of H2O2, tert-butyl hydroperoxide (tBuOOH), or cumene hydroperoxide (CuOOH). Enhanced activity and heterotropic cooperativity were observed in CuOOH-supported hydroxylation, and both the Hill coefficient and substrate concentration required for half-maximal activity in the CuOOH-supported reaction were much lower than those in the H2O2-, tBuOOH-, or NADPH-supported reactions. CuOOH greatly enhanced NADPH consumption and indole hydroxylation in the NADPH-supported reaction. However, when CuOOH was replaced by tBuOOH or H2O2, heterotropic cooperativity was not observed. Spectral studies also confirmed that CuOOH stimulated indole binding to P450 BM-3. Interestingly, a mutant enzyme with enhanced indole-hydroxylation activity, F87V (Phe87 was replaced by Val), lost homotropic cooperativity towards indole and heterotropic cooperativity towards CuOOH, indicating that the active-site structure affects the cooperativities.     

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts

The objectives of this study were to determine 1) whether reactive oxygen species generated upon postischemic reperfusion lead to oxidative stress in rat hearts, and 2) whether an exogenous prooxidant present in the early phase of reperfusion causes additional injury. Isolated buffer-perfused rat hearts were subjected to 30 min of hypothermic no-flow ischemia followed by 30 min of reperfusion. Increased myocardial content of glutathione disulfide (GSSG) and increased active transport of GSSG were used as indices of oxidative stress. To impose a prooxidant load, cumene hydroperoxide (20 M) was administered during the first 10 min of reperfusion to a separate group of postischemic hearts. Reperfusion after 30 min of hypothermic ischemia resulted in a recovery of myocardial ATP from 28% at end-ischemia to 50–60%, a release of 5% of total myocardial LDH, and an almost complete recovery of both coronary flow rate and left ventricular developed pressure. After 5 and 30 min of reperfusion, neither myocardial content of GSSG nor active transport of GSSG were increased. These indices were increased, however, if cumene hydroperoxide was administered during early reperfusion. After stopping the administration of cumene hydroperoxide, myocardial GSSG content returned to control values and GSH content increased, indicating an unimpaired glutathione reductase reaction. Despite the induction of oxidative stress, reperfusion with cumene hydroperoxide did not cause additional metabolic, structural, or functional injury when compared to reperfusion without cumene hydroperoxide. We conclude that reactive oxygen species generated upon postischemic reperfusion did not lead to oxidative stress in isolated rat hearts. Moreover, even a superimposed prooxidant load during early reperfusion did not cause additional injury.     

The involvement of cytochrome P-450 in hepatic microsomal steroid hydroxylation reactions supported by sodium periodate, sodium chlorite, and organic hydroperoxides.

The mechanism of steroid hydroxylation in rat liver microsomes has been investigated by employing NaIO4, NaClO2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. Androstenedione, testosterone, progesterone, and 17beta-estradiol were found to act as good substrates. NaIO4 was by far the most effective hydroxylating agent followed by cumene hydroperoxide, NADPH, NaClO2, pregnenolone 17alpha-hydroperoxide, tert-butyl hydroperoxide, and linoleic acid hydroperoxide. Androstenedione was chosen as the model substrate for inducer and inhibitor studies. The steroid was converted to its respective 6beta-, 7alpha, 15-, and 16alpha-hydroxy derivatives when incubated with microsomal fractions fortified with hydroxylating agent. Evidence for cytochrome P-450 involvement in androstenedione hydroxylation included a marked inhibition by substrates and modifiers of cytochrome P-450 and by reagents which convert cytochrome P-450 to cytochrome P-420. The ratios of the steroid products varied according to the type of hydroxylating agent used and were also modified by in vivo phenobarbital pretreatment. It was suggested that multiple forms of cytochrome P-450 exhibiting different affinities for hydroxylating agent are responsible for these different ratios. Horse-radish peroxidase, catalase, and metmyoglobin could not catalyze androstenedione hydroxylation. Addition of NaIO4, NaClO2, cumene hydroperoxide and other organic hydroperoxides to microsomal suspensions resulted in the appearance of a transient spectral change in the difference spectrum characterized by a peak at about 440 nm and a trough at 420 nm. The efficiency of these oxidizing agents in promoting steroid hydroxylation in microsomes appeared to be related to their effectiveness in eliciting the spectral complex. Electron donors, substrates, and modifiers of cytochrome P-450 greatly diminished the magnitude of the spectral change. It is proposed that NaIO4, NaClO2, and organic hydroperoxides promote steroid hydroxylation by forming a transient ferryl ion (compound I) of cytochrome P-450 which may be the common intermediate hydroxylating species involved in hydroxylations catalyzed by cytochrome P-450.     

The role of lipid peroxidation products in cumene hydroperoxide-induced Ca2+ efflux from mitochondria

Cumene hydroperoxide-induced calcium release from mitochondria has been studied. Activation of lipid peroxidation by increasing concentrations of cumene hydroperoxide does not enhance calcium efflux induced by low (up to 50 microM) concentration of cumene hydroperoxide. It is concluded that cumene hydroperoxide-induced calcium release depends mainly on processes coupled to hydroperoxide reduction by an endogenous enzyme system.     

The mechanism of hydroperoxide-dependent reactions with participation of cytochrome P-450

Cytochrome P-450 destruction kinetics by cumene hydroperoxide (CHP) has been studied at 25 degrees C in phosphate buffer, pH 7.25-7.50, in various systems: intact and induced rat or rabbit microsomes, highly purified LM2- and LM2- and LM4-forms of cytochrome P-450 from rabbit liver microsomes. The destruction kinetics is characterized by three phases in all systems. The CHP-influenced cytochrome P-450 destruction is a radical chain process with linear termination of the chains. The acidic phospholipids, phosphatidylserine and phosphatidylinositol and total microsomal phospholipids containing the acidic lipid components activate cytochrome P-450 in the hydroxylation of aniline and naphthalene by CHP. Phosphatidylcholine and sphingomyelin have no effect upon the cytochrome P-450 activity in the type I and II substrates oxidation by CHP. The phase transitions of the microsomal phospholipids influence the interaction of cytochrome P-450 with its reductase, altering the activation energy of type I substrates oxidation. The type II substrate oxidation is not affected by phase transitions in the full microsomal hydroxylating system.     

Cytochrome P-450 from mitochondria of bovine adrenal cortex: comparison of cholesterol side-chain cleavage P-450 with steroid 11beta-hydroxylation P-450 and immunochemical cross-reactivity between adrenal mitochondrial and liver microsomal cytochromes P-450.

Adrenocortical mitochondrial cytochrome P-450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11beta-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0 degrees C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11beta-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H2O2, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents. Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.     

The mechanism of cumene hydroperoxide-dependent lipid peroxidation: the function of cytochrome P-450

The addition of limiting amounts of cumene hydroperoxide to rat liver microsomes resulted in the rapid uptake of molecular oxygen, the formation of thiobarbituric acid reactive products, and the loss of hydroperoxide. The stoichiometry of lipid peroxidation and the yields of 2-phenyl-2-propanol (a major product of the reaction) and acetophenone (a minor product) observed with liver microsomes prepared from untreated rats is greater than that seen with liver microsomes from ciprofibrate-treated rats which, in turn, is greater than that observed with liver microsomes from phenobarbital-treated rats. The Km's and Vmax's of oxygen uptake varied with the type of rat liver microsomes used. Cytochrome P-450 substrates and inhibitors decreased the extents and initial rates of oxygen uptake and thiobarbituric acid reactive product formation. A mechanism is proposed involving the cytochrome P-450-catalyzed homolytic cleavage of the cumene hydroperoxide O-O bond to give the cumyloxyl radical. It is proposed that this oxygen-centered radical abstracts a hydrogen atom from an unsaturated fatty acid associated with a lipid (initiating lipid peroxidation) to give 2-phenyl-2-propanol or that the radical undergoes beta-scission to produce acetophenone and a methyl radical.     

Hemin-mediated para-hydroxylation of aniline: A potential model for oxygen activation and insertion reactions of mixed function oxidases

The activation of molecular oxygen by alkaline hemin (ferriprotoporphyrin IX) has been studied. In the presence of reductant nicotineamide adenine dinucleotide (NADH) or nicotineamide adenine dinucleotide phosphate (NADPH) and organic substrate, aniline, hemin activates oxygen to the hydroperoxide anion (HO₂^?) and subsequently mediates insertion of active oxygen into the benzene ring of the substrate to form p-aminophenol, with a high degree of regiospecificity. Oxygen activation does not occur in the absence of aniline. Stoichiometry of the reaction indicates that two electrons are required per molecule of oxygen activated or atom of oxygen inserted into the substrate aromatic ring system. Direct measurements of H₂O₂ and of the pK_a for maximum rate of p-aminophenol formation (11.7 ± 0.1) indicate participation of the hydroperoxide anion as the active oxygen species in the rate-determining step of the insertion reaction. Powerful scavengers of the hydroxyl radical (OH′) have little effect on the formation of H₂O₂ or p-aminophenol by the system. Superoxide dismutase (10^?7 mol dm^?3) inhibited both p-aminophenol and H₂O₂ formation, when added to the system immediately prior to initiation of the reaction. Studies involving N-phenylhydroxylamine indicate that aromatic ring hydroxylation is occurring directly and not by rearrangement of an N-hydroxylated intermediate. Implications of hemin-mediated hydroxylation reactions for those of enzymatic mixed function oxidase activity are discussed.     

Roles of the axial push effect in cytochrome P450cam studied with the site-directed mutagenesis at the heme proximal site

To examine the roles of the axial thiolate in cytochrome P450-catalyzed reactions, a mutant of cytochrome P450cam, L358P, was prepared to remove one of the conserved amide protons that are proposed to neutralize the negative charge of the thiolate sulfur. The increased push effect of the thiolate in L358P was evidenced by the reduced reduction potential of the heme. The 15N-NMR and resonance Raman spectra of the mutant in the ferric-CN and in the ferrous-CO forms, respectively, also supported the increased push effect. The maintenance of stereo- and regioselectivities for d-camphor hydroxylation by the mutant suggests the minimum structural change at the distal site. The heterolysis/homolysis ratios of cumene hydroperoxide were the same for wild-type and L358P. However, we observed the enhanced monooxygenations of the unnatural substrates using dioxygen and electrons supplied from the reconstituted system, which indicate the significant role of the push effect in dioxygen activation. We interpret that the enhanced push effect inhibits the protonation of the inner oxygen atom and/or promotes the protonation of the outer oxygen atom in the putative iron-hydroperoxo intermediate (Fe3+ -O-OH) of P450cam. This work is the first experimental indication of the significance of the axial cysteine for the P450 reactivity.     

Generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of oligopeptides.

The generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of several oligopeptides was investigated by electron spin resonance (ESR) utilizing 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Incubation of Ni2+ with cumene hydroperoxide or t-butyl hydroperoxide did not generate any detectable free radical. In the presence of glycylglycylhistidine (GlyGlyHis), however, Ni2+ generated cumene peroxyl (ROO.) radical from cumene hydroperoxide, with the free radical generation reaching its saturation level within about 3 min. The reaction was first order with respect to both cumene hydroperoxide and Ni2+. Similar results were obtained using t-butyl hydroperoxide, but the yield of t-butyl peroxyl radical generation was about 7-fold lower. Other histidine-containing oligopeptides such as beta-alanyl-L-histidine (carnosine), gamma-aminobutyryl-L-histidine (homocarnosine), and beta-alanyl-3-methyl-L-histidine (anserine) caused the generation of both cumene alkyl (R.) and cumene alkoxyl (RO.) radicals in the reaction of Ni2+ with cumene hydroperoxide. Similar results were obtained using t-butyl hydroperoxide. Glutathione also caused generation of R. and RO. radicals in the reaction of Ni2+ with cumene hydroperoxide but the yield was approximately 25-fold greater than that produced by the histidine-containing peptides, except GlyGlyHis. The ratio of DMPO/R. and DMPO/RO. produced with glutathione and cumene hydroperoxide was approximately 3:1. Essentially the same results were obtained using t-butyl hydroperoxide except that the ratio of DMPO/R. to DMPO/RO. was approximately 1:1. The free radical generation from cumene hydroperoxide reached its saturation level almost instantaneously while in the case of t-butyl hydroperoxide, the saturation level was reached in about 3 min. In the presence of oxidized glutathione, the Ni2+/cumene hydroperoxide system caused DMPO/.OH generation from DMPO without forming free hydroxyl radical. Since glutathione, carnosine, homocarnosine, and anserine are considered to be cellular antioxidants, the present work suggests that instead of protecting against oxidative damage, these oligopeptides may facilitate the Ni(2+)-mediated free radical generation and thus may participate in the mechanism(s) of Ni2+ toxicity and carcinogenicity.     

Sodium periodate, sodium chlorite, and organic hydroperoxides as hydroxylating agents in steroid hydroxylation reactions catalyzed by adrenocortical microsomal and mitochondrial cytochrome P450.

This study has investigated the mechanism of steroid hydroxylation in bovine adrenocortical microsomes and mitochondria by employing NaIO₄, NaClO₂, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. In the microsomal hydroxylating system, progesterone, 17α-hydroxyprogesterone, and androstenedione were found to act as substrates. Progesterone was chosen as the model substrate and was converted mainly to the 21-hydroxylated derivative in the presence of microsomal fractions fortified with hydroxylating agent. Using saturating levels of hydroxylating agent, NaIO₄ was found to be the most effective in promoting progesterone hydroxylation followed by cumene hydroperoxide, t-butyl hydroperoxide, NADPH, NaClO₂, and pregnenolone 17α-hydroperoxide. Evidence for cytochrome P₄₅₀ involvement included a marked inhibition of the activity by substrates and modifiers of cytochrome P₄₅₀ and by reagents that convert cytochrome P₄₅₀ to cytochrome P₄₂₀. Steroid hydroxylation was studied in adrenocortical mitochondria that had been previously depleted of endogenous pyridine nucleotides by aging for 1 h at 30 dgC in a phosphate-supplemented medium. Androstenedione was converted to its respective 6β-, 11β-, 16β-, and 19-hydroxylated derivatives when incubated with aged mitochondrial fractions fortified with hydroxylating agent whereas progesterone was hydroxylated in the 1β-, 6β-, and 15β- positions. These hydroxylations were completely abolished by preheating the mitochondria for 5 min at 95 dgC prior to assay, indicating the enzymic nature of the reactions. Deoxycorticosterone and deoxycortisol were effective substrates for NADPH-dependent enzymic 11β-hydroxylation but were extensively degraded nonenzymically to unidentified products in the presence of NaIO₄ and hydroxylating agents other than NADPH and consequently could not be utilized as substrates in these reactions. Using androstenedione as substrate, NaIO₄ was the most effective hydroxylating agent, followed by cumene hydroperoxide, NaClO₂, t-butyl hydroperoxide, and NADPH. These hydroxylations were inhibited by substrates and modifiers of cytochrome P₄₅₀ and by reagents that convert cytochrome P₄₅₀ to cytochrome P₄₂₀. A mechanism for steroid hydroxylation in adrenocortical microsomes and mitochondria is proposed in which the ferryl ion (compound I) of cytochrome P₄₅₀ functions as the common “activated oxygen” species.