Peroxidase-like activity of uncoupled cytochrome P450: studies with bilirubin and toxicological implications of uncoupling.

The NADPH-dependent consumption of O(2) by cytochrome P450 BM3 was stimulated by either laurate or perfluorolaurate, but the NADPH/O(2) molar consumption ratios were approximately 1 and 2, respectively, indicating that perfluorolaurate does not become oxygenated by BM3 and oxygen undergoes full reduction to water. The nature of this catalytic cycle uncoupled to hydroxylation was explored using bilirubin as a molecular probe. During uncoupling with perfluorolaurate bilirubin was degraded and stimulated O(2) uptake by an approximately equimolar amount. No stimulation of oxygen uptake was caused by bilirubin in presence of NADPH alone or in presence of laurate together with NADPH; under these conditions little degradation of bilirubin was observed. Mesobilirubin was also degraded during uncoupling with perfluorolaurate, whereas biliverdin (which lacks the central methene bridge present in rubins) was unaffected. It is suggested that the CYP ferryl oxygen species abstracts a hydrogen atom from the central methene bridge of bilirubin to generate a radical, which is further dehydrogenated to biliverdin or else binds O(2) and undergoes fragmentation. We conclude that the uncoupled catalytic cycle of cytochrome P450 has properties resembling those of a peroxidase and that bilirubin is rapidly oxidized as a peroxidase substrate. The potential toxicological significance of cytochrome P450 uncoupling is considered.

Transglutaminase 1-deficient recessive lamellar ichthyosis associated with a LINE-1 insertion in Jack Russell terrier dogs.

BACKGROUND: Congenital, nonepidermolytic cornification disorders phenotypically resembling human autosomal recessive ichthyosis have been described in purebred dog breeds, including Jack Russell terrier (JRT) dogs. One cause of gene mutation important to humans and dogs is transposon insertions. OBJECTIVES: To describe an autosomal recessive, severe nonepidermolytic ichthyosis resembling lamellar ichthyosis (LI) in JRT dogs due to insertion of a long interspersed nucleotide element (LINE-1) in the transglutaminase 1 (TGM1) gene. METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Phenotypic information and genotyping with a canine microsatellite marker suggested TGM1 to be a candidate gene. Genomic DNA samples and cDNA generated from epidermal RNA were examined. Consequences of the mutation were evaluated by Western blotting, quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme activity from cultured keratinocytes. RESULTS: Affected dogs had generalized severe hyperkeratosis. Histological examination defined laminated to compact hyperkeratosis without epidermolysis; ultrastructurally, cornified envelopes were thin. Affected dogs were homozygous for a 1980-bp insertion within intron 9 of TGM1. The sequence of the insertion was that of a canine LINE-1 element. Quantitative RT-PCR indicated a significant decrease in TGM1 mRNA in affected dogs compared with wild-type. TGM1 protein was markedly decreased on immunoblotting, and membrane-associated enzyme activity was diminished in affected dogs. CONCLUSIONS: Based on morphological and molecular features, this disease is homologous with TGM1-deficient LI in humans, clinically models LI better than the genetically modified mouse and represents its first spontaneous animal model. This is the first reported form of LI due to transposon insertion.

Desaturase reactions complicate the use of norcarane as a mechanistic probe. Unraveling the mixture of twenty-plus products formed in enzyme-catalyzed oxidations of norcarane.

Norcarane, bicyclo[4.1.0]heptane, has been widely used as a mechanistic probe in studies of oxidations catalyzed by several iron-containing enzymes. We report here that, in addition to oxygenated products, norcarane is also oxidized by iron-containing enzymes in desaturase reactions that give 2-norcarene and 3-norcarene. Furthermore, secondary products from further oxidation reactions of the norcarenes are produced in yields that are comparable to those of the minor products from oxidation of the norcarane. We studied oxidations catalyzed by a representative spectrum of iron-containing enzymes including four cytochrome P450 enzymes, CYP2B1, CYPDelta2B4, CYPDelta2E1, and CYPDelta2E1 T303A, and three diiron enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. 2-Norcarene and 3-norcarene and their oxidation products were found in all reaction mixtures, accounting for up to half of the oxidation products in some cases. In total, more than 20 oxidation products were identified from the enzyme-catalyzed reactions of norcarane. The putative radical-derived product from the oxidation of norcarane, 3-hydroxymethylcyclohexene (21), and the putative cation-derived product from the oxidation of norcarane, cyclohept-3-enol (22), coelute with other oxidation products on low-polarity GC columns. The yields of product 21 found in this study are smaller than those previously reported for the same or similar enzymes in studies where the products from norcarene oxidations were ignored, and therefore, the limiting values for lifetimes of radical intermediates produced in the enzyme-catalyzed oxidation reactions are shorter than those previously reported.

Products from enzyme-catalyzed oxidations of norcarenes.

Recent studies revealed that norcarane (bicyclo[4.1.0]heptane) is oxidized to 2-norcarene (bicyclo[4.1.0]-hept-2-ene) and 3-norcarene (bicyclo[4.1.0]hept-3-ene) by iron-containing enzymes and that secondary oxidation products from the norcarenes complicate mechanistic probe studies employing norcarane as the substrate (Newcomb, M.; Chandrasena, R. E. P.; Lansakara-P., D. S. P.; Kim, H.-Y.; Lippard, S. J.; Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg, P. F.; Coon, M. J. J. Org. Chem. 2007, 72, 1121-1127). In the present work, the product profiles from the oxidations of 2-norcarene and 3-norcarene by several enzymes were determined. Most of the products were identified by GC and GC-mass spectral comparison to authentic samples produced independently; in some cases, stereochemical assignments were made or confirmed by 2D NMR analysis of the products. The enzymes studied in this work were four cytochrome P450 enzymes, CYP2B1, CYPDelta2E1, CYPDelta2E1 T303A, and CYPDelta2B4, and three diiron-containing enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. The oxidation products from the norcarenes identified in this work are 2-norcaranone, 3-norcaranone, syn- and anti-2-norcarene oxide, syn- and anti-3-norcarene oxide, syn- and anti-4-hydroxy-2-norcarene, syn- and anti-2-hydroxy-3-norcarene, 2-oxo-3-norcarene, 4-oxo-2-norcarene, and cyclohepta-3,5-dienol. Two additional, unidentified oxidation products were observed in low yields in the oxidations. In matched oxidations, 3-norcarene was a better substrate than 2-norcarene in terms of turnover by factors of 1.5-15 for the enzymes studied here. The oxidation products found in enzyme-catalyzed oxidations of the norcarenes are useful for understanding the complex product mixtures obtained in norcarane oxidations.

The Ulcerated Leg Severity Assessment score for prediction of venous leg ulcer healing.

BACKGROUND: The aim of the study was to create a reliable scoring system for the prediction of venous ulcer healing in patients treated with compression. METHODS: A prospective baseline study to identify risk factors for venous ulcer healing was undertaken between March 1999 and August 2001. All patients were treated with multilayer compression. A number of variables were related to 24-week healing rates. A Cox regression model was used to identify risk factors that predicted ulcer healing, from which a scoring system was developed and validated prospectively between February 2004 and March 2005. RESULTS: In the baseline study of 229 patients, patient age, ulcer chronicity and venous refill time (VRT) of 20 s or less were identified as risk factors. Using these factors and hazard ratios from the Cox regression analysis, the following formula was devised: Ulcerated Leg Severity Assessment (ULSA) score=age+chronicity-50 (when VRT is greater than 20 s). Patients with an ULSA score of 50 or less had higher 24-week ulcer healing rates than those with higher scores in both the baseline study (P<0.001, log rank test) and the validation study performed in 86 patients (P=0.007, log rank test). CONCLUSION: The ULSA score may help to identify patients with venous ulcers unlikely to respond to conventional treatment who could be offered alternative therapy.

Accurate model predicting sustained response at week 4 of therapy with pegylated interferon with ribavirin in patients with chronic hepatitis C.

Current models used to predict response to peginterferon plus ribavirin treatment, based on viral decline during the first 12 weeks of therapy, have focused on creating an early stopping rule to avoid unnecessary prolongation of therapy. We developed a multivariate model that predicted sustained virological response and nonresponse at baseline and during the first 12 weeks of therapy using collected data from 186 unselected patients with chronic hepatitis C treated with peginterferon plus ribavirin. This model employed ordinal regression with similarity least squares technology to assign the probability of a given outcome. Model variables include sex, age, prior treatment status, genotype, baseline serum alanine aminotransferase levels, histologic necroinflammation and fibrosis scores and serum hepatitis C virus RNA concentration at baseline and weeks, 4, 8, and 12. A multivariate model demonstrated high performance values at all time points. At baseline, the model demonstrated a negative predictive value (NPV) and a positive predictive value (PPV) of 91% and 95%, respectively. At week 4, these values improved to 97% and 100%, respectively, with 95% sensitivity, 89% specificity and 93% accuracy. At week 4, the model was equally efficient for naïve or previously treated patients. Internal validation demonstrated 90% PPV, 94% NPV, 95% sensitivity, 88% specificity and 92% accuracy. A week 4 stopping rule for patients with chronic hepatitis C treated with peginterferon with ribavirin might be proposed by using the model developed in our study.

Oxidative aldehyde deformylation catalyzed by NADPH-cytochrome P450 reductase and the flavoprotein domain of neuronal nitric oxide synthase.

We report here the unexpected finding that recombinant or hepatic microsomal NADPH-cytochrome P450 reductase catalyzes the oxidative deformylation of a model xenobiotic aldehyde, 2-phenylpropionaldehyde, to the n-1 alcohol, 1-phenylethanol, in the absence of cytochrome P450. The flavoprotein and NADPH are absolute requirements, and the reaction displays a dependence on time and on NADPH and reductase concentration. Not surprisingly, the hydrophobic tail of the flavoprotein is not required for catalytic competence. The reductase domain of neuronal nitric oxide synthase is about 30% more active than P450 reductase, and neither flavoprotein catalyzes conversion of the aldehyde to the carboxylic acid, by far the predominant metabolite with P450s in a reconstituted system. Reductase-catalyzed deformylation is unaffected by metal ion chelators and oxygen radical scavengers, but is strongly inhibited by catalase, and the catalase-mediated inhibition is prevented by azide. These results, together with observed parallel increases in 1-phenylethanol and H(2)O(2) formation as a function of NADPH concentration, are evidence that free H(2)O(2) is rate-limiting in aldehyde deformylation by the flavoprotein reductases. This contrasts sharply with the P450-catalyzed reaction, which is brought about by iron-bound peroxide that is inaccessible to catalase.

Omega oxygenases: nonheme-iron enzymes and P450 cytochromes.

Enzymes that effect with ease one of the most difficult chemical reactions, hydroxylation of an unfunctionalized alkyl group, are of particular interest because highly reactive intermediates must be produced. A typical example, the hydroxylation of fatty acids in the omega position, is now known to occur widely in nature. The catalysts, which can be called "omega-oxygenases," also insert molecular oxygen into a variety of other substrates at positions removed from activating functional groups, as in steroids, eicosanoids, and numerous drugs and other xenobiotics. Progress in the characterization of bacterial nonheme-iron enzymes, and plant, bacterial, and mammalian P450 cytochromes that catalyze fatty acid omega-oxidation, and evidence for multiple functional oxidants are summarized.

Comparative sequence analysis and radiation hybrid mapping of the canine keratin 10 gene.

The type I keratin, K10, is expressed in epidermal keratinocytes undergoing terminal differentiation to form the stratum corneum, a barrier essential for life. In order to facilitate the study of keratinization disorders in the dog, the sequence and mapping of KRT10 is reported. The coding region of KRT10 is 1707 bp and is comprised of eight exons. Although the length of KRT10 has been reported to be polymorphic in humans, this was not observed in the eight domestic dog breeds studied, although one wild canid displayed a size difference. The structure and sequence of this gene is highly conserved across mammalian species. Canine K10 had an 86% amino acid identity with the human gene. KRT10 was localized to the on-going canine radiation hybrid map to chromosome 9 in the type I keratin gene cluster.

Mild recessive epidermolytic hyperkeratosis associated with a novel keratin 10 donor splice-site mutation in a family of Norfolk terrier dogs.

BACKGROUND: Epidermolytic hyperkeratosis in humans is caused by dominant-negative mutations in suprabasal epidermal keratins 1 and 10. However, spontaneous keratin mutations have not been confirmed in a species other than human. OBJECTIVES: To describe an autosomal recessive, mild, nonpalmar/plantar epidermolytic ichthyosis segregating in an extended pedigree of Norfolk terrier dogs due to a splice-site mutation in the gene encoding keratin 10 (KRT10). METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Genomic DNA samples and cDNA from skin RNA were sequenced and defined a mutation in KRT10. Consequences of the mutation were evaluated by assessing protein expression with immunohistochemistry and Western blotting and gene expression with real-time RT-PCR (reverse transcriptase-polymerase chain reaction). RESULTS: Adult dogs with the disease had generalized, pigmented hyperkeratosis with epidermal fragility. Light microscopic examination defined epidermolysis with hyperkeratosis; ultrastructural changes included a decrease in tonofilaments and abnormal filament aggregation in upper spinous and granular layer keratinocytes. Affected dogs were homozygous for a single base GT-->TT change in the consensus donor splice site of intron 5 in KRT10. Keratin 10 protein was not detected with immunoblotting in affected dogs. Heterozygous dogs were normal based on clinical and histological appearance and keratin 10 protein expression. The mutation caused activation of at least three cryptic or alternative splice sites. Use of the cryptic sites resulted in transcripts containing premature termination codons. One transcript could result in shortening of the proximal portion of the 2B domain before the stutter region. Quantitative real-time PCR indicated a significant decrease in KRT10 mRNA levels in affected dogs compared with wild-type dogs. CONCLUSIONS: This disease is the first confirmed spontaneous keratin mutation in a nonhuman species and is the first reported recessive form of epidermolytic hyperkeratosis.

Cytochrome P450: nature's most versatile biological catalyst.

The author describes studies that led to the resolution and reconstitution of the cytochrome P450 enzyme system in microsomal membranes. The review indicates how purification and characterization of the cytochromes led to rigorous evidence for multiple isoforms of the oxygenases with distinct chemical and physical properties and different but somewhat overlapping substrate specificities. Present knowledge of the individual steps in the P450 and reductase reaction cycles is summarized, including evidence for the generation of multiple functional oxidants that may contribute to the exceptional diversity of the reactions catalyzed.

Abolition of oxygenase function, retention of NADPH oxidase activity, and emergence of peroxidase activity upon replacement of the axial cysteine-436 ligand by histidine in cytochrome P450 2B4.

A fundamental aspect of cytochrome P450 function is the role of the strictly conserved axial cysteine ligand, replacement of which by histidine has invariably resulted in mammalian and bacterial preparations devoid of heme. Isolation of the His-436 variant of NH2-truncated P450 2B4 partly as the holoenzyme was achieved in the present study by mutagenesis of the I-helix Ala-298 residue to Glu and subsequent conversion of the axial Cys-436 to His. The expressed A298E/C436H double mutant, cloned with a hexahistidine tag, had a molecular mass equivalent to that of the primary structure of His-tagged truncated 2B4 and the sum of the two mutated residues, and contained a heme group which, when released on HPLC, showed a retention time and spectrum identical to those of iron protoporphyrin IX. The absolute spectra of A298E/C436H indicate a change in heme coordination structure from low- to high-spin, and, as expected for a His-ligated hemeprotein, the Soret maximum of the ferrous CO complex is at 422 nm. The double mutant has no oxygenase activity with representative substrates known to undergo transformation by the oxene [(FeO)3+] or peroxo activated oxygen species, but catalyzes significant H2O2 formation that is NADPH- and time-dependent, and directly proportional to the concentration of A298E/C436H in the presence of saturating reductase. Moreover, the catalytic efficiency of A298E/C436H in the H2O2-supported peroxidation of pyrogallol is more than two orders of magnitude greater than that of wild-type 2B4 or the A298E variant. The results unambiguously demonstrate that the proximal thiolate ligand is essential for substrate oxygenation by P450.

The influence of superficial venous surgery and compression on incompetent calf perforators in chronic venous leg ulceration.

OBJECTIVES: Previous studies have suggested that perforating vein incompetence is reduced by surgery to superficial veins. This study analysed the effect in a randomised clinical trial. DESIGN: Retrospective analysis of duplex data. METHODS: Patients in this study were part of the ESCHAR randomised controlled trial. All patients had chronic venous leg ulceration with superficial venous reflux. Patients were treated with compression bandaging alone or compression plus superficial venous surgery. Legs were assessed using colour venous duplex prior to treatment and at 3 and 12 months. RESULTS: Of 500 patients recruited to the ESCHAR trial, 261 were included in this study. One hundred and forty six of 261 legs were treated with compression alone and 115/261 underwent compression and superficial venous surgery. In the compression group, more legs had incompetent perforators at 12 months (77/131) compared to baseline (61/146, p =0.010, Wilcoxon Signed Ranks test for paired data in 131 legs). Following surgery, significantly fewer legs had incompetent calf perforators (59/115 vs 44/104 at 12 months, p =0.001, Wilcoxon Signed Ranks test for paired data in 104 legs). In addition, significantly fewer legs in the compression and surgery group developed new perforator incompetence in comparison to the group treated with compression alone (12/104 vs 36/131, p =0.003, Chi-Squared test). CONCLUSION: Surgical correction of superficial reflux may abolish incompetence in some calf perforators and offer protection against developing new perforator incompetence.

Hydroxylation by the hydroperoxy-iron species in cytochrome P450 enzymes.

Intramolecular and intermolecular kinetic isotope effects (KIEs) were determined for hydroxylation of the enantiomers of trans-2-(p-trifluoromethylphenyl)cyclopropylmethane (1) by hepatic cytochrome P450 enzymes, P450s 2B1, Delta2B4, Delta2B4 T302A, Delta2E1, and Delta2E1 T303A. Two products from oxidation of the methyl group were obtained, unrearranged trans-2-(p-trifluoromethylphenyl)cyclopropylmethanol (2) and rearranged 1-(p-trifluoromethylphenyl)but-3-en-1-ol (3). In intramolecular KIE studies with dideuteriomethyl substrates (1-d(2)) and in intermolecular KIE studies with mixtures of undeuterated (1-d(0)) and trideuteriomethyl (1-d(3)) substrates, the apparent KIE for product 2 was consistently larger than the apparent KIE for product 3 by a factor of ca. 1.2. Large intramolecular KIEs found with 1-d(2) (k(H)/k(D) = 9-11 at 10 degrees C) were shown not to be complicated by tunneling effects by variable temperature studies with two P450 enzymes. The results require two independent isotope-sensitive processes in the overall hydroxylation reactions that are either competitive or sequential. Intermolecular KIEs were partially masked in all cases and largely masked for some P450s. The intra- and intermolecular KIE results were combined to determine the relative rate constants for the unmasking and hydroxylation reactions, and a qualitative correlation was found for the unmasking reaction and release of hydrogen peroxide from four of the P450 enzymes in the absence of substrate. The results are consistent with the two-oxidants model for P450 (Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3555), which postulates that a hydroperoxy-iron species (or a protonated analogue of this species) is a viable electrophilic oxidant in addition to the consensus oxidant, iron-oxo.

Intrinsic proportions of the human tarsus: an original approach to tarsal biometry.

Few precise biometrical data on human tarsal bones are available, and those published consist principally of linear and angular measurements made on dried bones or on radiographs. The material consisted of 86 complete adult human tarsi (dried bones). The tarsal proportions were determined using the relative tarsal weights (Weight of each of the seven tarsal bones/Weight of the total tarsusx100). The calcaneus was the greatest tarsal bone (41.95%), and the talus the second-largest (28.45%). The medial cuneiform, the cuboid, and the navicular had very similar proportions (7.00-8.38%), as did the intermediate and lateral cuneiforms (2.98% and 3.81%). The study of the ranks of each bone by decreasing proportion allowed the determination of individual variability. The posterior tarsal row was considerably larger (70.40%) than the anterior row (29.60%), resulting in an antero-posterior ratio of 0.42. The medial and lateral tarsal columns had quite similar proportions (50.63% and 49.37%), resulting in a latero-medial ratio of 0.98. The intrinsic tarsal proportions are fundamental biometrical data which seem to be of interest for a better characterization of the human foot in clinical practice and for quantitative approaches in functional and comparative morphology and in paleontology.

Kinetic isotope effects implicate two electrophilic oxidants in cytochrome p450-catalyzed hydroxylations.

Intramolecular kinetic isotope effects (KIEs) were determined for cytochrome P450-catalyzed hydroxylation reactions of methyl-dideuterated trans-2-phenylcyclopropylmethane-d2 (1-d2), which gives two products from oxidation of the methyl group, trans-2-phenylcyclopropylmethanol (2) and 1-phenyl-3-buten-1ol (3). In oxidations of each enantiomer of 1-d2 with three P450 enzymes (CYP2B1, CYPDelta2E1, and CYPDelta2E1 T303A), the apparent intramolecular KIEs were different for products 2 and 3 in all cases and different for each enzyme-substrate combination. In oxidations of each enantiomer of undeuterated 1-d0 and trideuteriomethyl 1-d3 by CYP2B1 and CYPDelta2E1, the ratio of products 2/3 decreased for 1-d3 in comparison to 1-d0 in all cases. The results require multiple pathways for P450-catalyzed hydroxylation and are consistent with the "two-oxidants" model, where hydroxylation is effected by both the hydroperoxy-iron species and the iron-oxo species. The results are not consistent with predictions of the "two-states" model for P450-catalyzed hydroxylations, where oxidations occur from a low-spin state and a high-spin state of iron-oxo.

Multiple mechanisms and multiple oxidants in P450-catalyzed hydroxylations.

Cytochrome P450 enzymes catalyze a number of oxidations in nature including the difficult hydroxylations of unactivated positions in an alkyl group. The consensus view of the hydroxylation reaction 10 years ago was that a high valent iron-oxo species abstracts a hydrogen atom from the alkyl group to give a radical that subsequently displaces the hydroxy group from iron in a homolytic substitution reaction (hydrogen abstraction-oxygen rebound). More recent mechanistic studies, as summarized in this review, indicated that the cytochrome P450-catalyzed "hydroxylation reaction" is complex, involving multiple mechanisms and multiple oxidants. In addition to the iron-oxo species, another electrophilic oxidant apparently exists, either the hydroperoxo-iron intermediate that precedes iron-oxo or iron-complexed hydrogen peroxide formed by protonation of the hydroperoxo-iron species on the proximal oxygen. The other electrophilic oxidant appears to react by insertion of OH(+) into a C-H bond to give a protonated alcohol. Computational work has suggested that iron-oxo can react through multiple spin states, a low-spin ensemble that reacts by insertion of oxygen, and a high-spin ensemble that reacts by hydrogen atom abstraction to give a radical.

Replacement of active-site cysteine-436 by serine converts cytochrome P450 2B4 into an NADPH oxidase with negligible monooxygenase activity.

The function of the unique axial thiolate ligand of cytochrome P450 has been investigated by mutagenesis of the active-site cysteine with other amino acids in NH(2)-truncated P450s 2B4 and 2E1. The expressed Ser-436 variant of P450 2B4 was highly purified but incurred considerable heme loss. The pyridine hemochrome spectrum of C436S is characteristic of protoporphyrin IX, and the absolute spectra display Soret maxima at 405 nm (ferric), 422 nm (ferrous), and 413 nm (ferrous CO). 2B4:C436S catalyzes the NADPH- and time-dependent formation of H(2)O(2) in the reconstituted enzyme system, with maximal rates at approximately equimolar amounts of P450 reductase and C436S hemeprotein. The 2-electron oxidase activity with saturating reductase is directly proportional to the concentration of 2B4:C436S, and the turnover is 60-70% of that of the wild-type enzyme. In contrast, the C436S variant is devoid of oxygenase activity with typical substrates such as d-benzphetamine, 1-phenylethanol, and 4-fluorophenol, and has only marginal 4-nitrophenol aromatic hydroxylation activity. H(2)O(2)-supported peroxidation of guaiacol and pyrogallol is comparable with 2B4 and mutant C436S and negligible relative to the turnover of peroxidases with these substrates. Neither 2B4 nor 2B4:C436S catalyzes H(2)O(2) decomposition. It is concluded that replacement of active-site Cys-436 by Ser converts P450 2B4 mainly into a 2-electron oxidase.