Monooxygenation Reaction Research Articles

Binding of steroid substrates reveals the key to the productive transition of the cytochrome P450 OleP

OleP is a bacterial cytochrome P450 involved in oleandomycin biosynthesis as it catalyzes regioselective epoxidation on macrolide intermediates. OleP has recently been reported to convert lithocholic acid (LCA) into murideoxycholic acid through a highly regioselective reaction and to unspecifically hydroxylate testosterone (TES). Since LCA and TES mainly differ by the substituent group at the C17, here we used X-ray crystallography, equilibrium binding assays, and molecular dynamics simulations to investigate the molecular basis of the diverse reactivity observed with the two steroids. We found that the differences in the structure of TES and LCA affect the capability of these molecules to directly form hydrogen bonds with N-terminal residues of OleP internal helix I. The establishment of these contacts, by promoting the bending of helix I, fosters an efficient trigger of the open-to-closed structural transition that occurs upon substrate binding to OleP and contributes to the selectivity of the subsequent monooxygenation reaction.

Exploring the carbonic anhydrase-mimetic [(PMDTA)2ZnII2(OH-)2]2+ for nitric oxide monooxygenation.

In biology, nitrite (NO2-) serves as a storage pool of nitric oxide (NO); however, the formation of NO2- from NO is still under investigation. Here, we report the NO monooxygenation (NOM) reaction of a ZnII-hydroxide complex (1), producing a ZnII-nitrito complex {2, (ZnII-NO2-)} + H2.

Experimental Evidence and Mechanistic Description of the Phenolic H-Transfer to the Cu2O2 Active Site of oxy-Tyrosinase.

Tyrosinase is a ubiquitous coupled binuclear copper enzyme that activates O2 toward the regioselective monooxygenation of monophenols to catechols via a mechanism that remains only partially defined. Here, we present new mechanistic insights into the initial steps of this monooxygenation reaction by employing a pre-steady-state, stopped-flow kinetics approach that allows for the direct measurement of the monooxygenation rates for a series of para-substituted monophenols by oxy-tyrosinase. The obtained biphasic Hammett plot and the associated solvent kinetic isotope effect values provide direct evidence for an initial H-transfer from the protonated phenolic substrate to the Cu2O2 core of oxy-tyrosinase. The correlation of these experimental results to quantum mechanics/molecular mechanics calculations provides a detailed mechanistic description of this H-transfer step. These new mechanistic insights revise and expand our fundamental understanding of Cu2O2 active sites in biology.

HMDB: A curated database of genes involved in hydrocarbon monooxygenation reaction with homologous genes as background

The investigation of hydrocarbon degradation potential of environmental microorganisms is an important research topic, whether for the global carbon cycle or oil pollution remediation. Under aerobic conditions, the microorganisms employ a range of monooxygenases to use hydrocarbons substrates as a source of carbon and energy. With the explosion of sequencing data, mining genes in genomes or metagenomes has become computationally expensive and time-consuming. We proposed the HMDB, which is a professional gene database of hydrocarbon monooxygenases. HMDB contains 38 genes, which encode 11 monooxygenases responsible for the hydroxylation of 8 hydrocarbons. To reduce false positives, the strategy of using homologous genes as background noise was applied for HMDB. We added 10,095 gene sequences of homologous enzymes which took non-hydrocarbons as substrates to HMDB. The classic BLAST method and best-hit strategy were recommended for HMDB usage, but not limited. The performance of HMDB was validated using 264,402 prokaryote genomes from RefSeq and 51 metagenomes from SRA. The results showed that HMDB database had high sensitivity and low false positive rate. We release the HMDB database here, hoping to speed up the process for investigation of hydrocarbon monooxygenases in massive metagenomic data. HMDB is freely available at http://www.orgene.net/HMDB/.

Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis

Melanins are highly conjugated biopolymer pigments that provide photoprotection in a wide array of organisms, from bacteria to humans. The rate-limiting step in melanin biosynthesis, which is the ortho-hydroxylation of the amino acid L-tyrosine to L-DOPA, is catalyzed by the ubiquitous enzyme tyrosinase (Ty). Ty contains a coupled binuclear copper active site that binds O2 to form a μ:η2:η2-peroxide dicopper(II) intermediate (oxy-Ty), capable of performing the regioselective monooxygenation of para-substituted monophenols to catechols. The mechanism of this critical monooxygenation reaction remains poorly understood despite extensive efforts. In this study, we have employed a combination of spectroscopic, kinetic, and computational methods to trap and characterize the elusive catalytic ternary intermediate (Ty/O2/monophenol) under single-turnover conditions and obtain molecular-level mechanistic insights into its monooxygenation reactivity. Our experimental results, coupled with quantum-mechanics/molecular-mechanics calculations, reveal that the monophenol substrate docks in the active-site pocket of oxy-Ty fully protonated, without coordination to a copper or cleavage of the μ:η2:η2-peroxide O-O bond. Formation of this ternary intermediate involves the displacement of active-site water molecules by the substrate and replacement of their H bonds to the μ:η2:η2-peroxide by a single H bond from the substrate hydroxyl group. This H-bonding interaction in the ternary intermediate enables the unprecedented monooxygenation mechanism, where the μ-η2:η2-peroxide O-O bond is cleaved to accept the phenolic proton, followed by substrate phenolate coordination to a copper site concomitant with its aromatic ortho-hydroxylation by the nonprotonated μ-oxo. This study provides insights into O2 activation and reactivity by coupled binuclear copper active sites with fundamental implications in biocatalysis.

Following Nature's Footprint: Mimicking the High-Valent Heme-Oxo Mediated Indole Monooxygenation Reaction Landscape of Heme Enzymes.

Pathways for direct conversion of indoles to oxindoles have accumulated considerable interest in recent years due to their significance in the clear comprehension of various pathogenic processes in humans and the multipotent therapeutic value of oxindole pharmacophores. Heme enzymes are predominantly responsible for this conversion in biology and are thought to proceed with a compound-I active oxidant. These heme-enzyme-mediated indole monooxygenation pathways are rapidly emerging therapeutic targets; however, a clear mechanistic understanding is still lacking. Additionally, such knowledge holds promise in the rational design of highly specific indole monooxygenation synthetic protocols that are also cost-effective and environmentally benign. We herein report the first examples of synthetic compound-I and activated compound-II species that can effectively monooxygenate a diverse array of indoles with varied electronic and steric properties to exclusively produce the corresponding 2-oxindole products in good to excellent yields. Rigorous kinetic, thermodynamic, and mechanistic interrogations clearly illustrate an initial rate-limiting epoxidation step that takes place between the heme oxidant and indole substrate, and the resulting indole epoxide intermediate undergoes rearrangement driven by a 2,3-hydride shift on indole ring to ultimately produce 2-oxindole. The complete elucidation of the indole monooxygenation mechanism of these synthetic heme models will help reveal crucial insights into analogous biological systems, directly reinforcing drug design attempts targeting those heme enzymes. Moreover, these bioinspired model compounds are promising candidates for the future development of better synthetic protocols for the selective, efficient, and sustainable generation of 2-oxindole motifs, which are already known for a plethora of pharmacological benefits.

Co-Cross-Linked Aggregates of Baeyer–Villiger Monooxygenases and Formate Dehydrogenase for Repeated Use in Asymmetric Biooxidation

Baeyer–Villiger monooxygenases (BVMOs) are versatile biocatalysts, but their applications are hindered by their poor stability and cofactor dependence. In this study, cross-linked enzyme aggregate (CLEA) technology was adopted to coimmobilize BVMO and its accessory cofactor-regeneration enzyme. Combi-CLEAs of a pyrmetazole monooxygenase from Acinetobacter calcoaceticus (AcPSMO) and a formate dehydrogenase from Burkholderia stabili (BstFDH) were prepared for the synthesis of (S)-omeprazole. After optimization, AcPSMO and BstFDH were coprecipitated with an activity ratio of 1:6 using ammonium sulfate and then cross-linked with glutaraldehyde (0.12% w/v). The activity recoveries of AcPSMO and BstFDH in the prepared combi-CLEAs were 43% and 38%, respectively. Compared with the free enzymes AcPSMO and BstFDH, the thermostabilities of AcPSMO and BstFDH in combi-CLEAs were improved by 2.5- and 1.6-fold, respectively. Both enzymes were more stable against alkaline buffer after being immobilized. The combi-CLEAs could be reused for seven cycles in the biooxidative synthesis of (S)-omeprazole without significant activity loss, indicating the excellent operational stability and reusability in repeated reactions for the enzymatic synthesis of (S)-omeprazole. Another two combi-CLEAs prepared under the same conditions, TmCHMO–BstFDH and RpBVMO–BstFDH, can be reused for at least 15 consecutive batches for the cyclohexanone mono-oxygenation reaction, which indicates the promising potential for coimmobilization of BVMOs and FDH with CLEA methodology.

Selective acceleration of 2-hydroxyl pyridine mono-oxygenation using specially acclimated biomass

Biodegradation of pyridine starts with two mono-oxygenation reactions, and 2-hydroxyl pyridine (2-HP) accumulates as pyridine is mono-oxygenated in the first reaction. The accumulation of 2-HP inhibits both initial reactions. Therefore, selective acceleration of the second mono-oxygenation reaction should significantly enhance pyridine transformation and mineralization. Activated-sludge biomass was separately acclimated with pyridine or 2-HP to produce pyridine- and 2-HP-acclimated biomasses. The pyridine-acclimated biomass was superior for pyridine biodegradation, but the 2-HP-acclimated biomass was superior for 2-HP biodegradation. As a consequence, the pyridine-acclimated biomass by itself achieved faster mono-oxygenation of pyridine to 2-HP, but 2-HP accumulated, which limited mineralization to 60%. In contrast, mineralization reached 90% when one-third of the pyridine-acclimated was replaced with 2-HP-acclimated biomass, because 2-HP did not accumulate during pyridine biodegradation. The lack of 2-HP accumulation relieved its inhibition: e.g., the pyridine removal rates, normalized to the mass of pyridine-acclimated biomass, increased from 0.52 to 0.57 mM0.5⋅h−1 when one-third of the pyridine-acclimated biomass was replaced by 2-HP-acclimated biomass. Phylogenetic analysis showed that microbiological communities of pyridine-acclimated biomass and 2-HP-acclimated biomass differed in important ways. On the one hand, the 2-HP-acclimated biomass was richer and dominated by a rare biosphere, or genera having <0.1% of total reads. On the other hand, the most-enriched genus in the pyridine-acclimated community (Methylibium) is associated with the first mono-oxygenation of pyridine, while enriched genera in the 2-HP-acclimated community (Sediminibacterium and Dokdonella) are associated with the second mono-oxygenation of pyridine.

How bioaugmentation with Comamonas testosteroni accelerates pyridine mono-oxygenation and mineralization

Pyridine is a common heterocycle found in industrial wastewaters. Its biodegradation begins with a mono-oxygenation reaction, and bioaugmentation with bacteria able to carry out this mono-oxygenation is one strategy to improve pyridine removal and mineralization. Although bioaugmentation has been used to enhance the biodegradation of recalcitrant organic compounds, the specific role played by the bioaugmented bacteria usually has not been addressed. We acclimated activated-sludge biomass for pyridine biodegradation and then isolated a strain -- Comamonas testosteroni -- based on its ability to biodegrade and grow on pyridine alone. Pyridine was removed faster by C. testosteroni, compared to pyridine-acclimated biomass, but pyridine mineralization was slower. Pyridine biodegradation and mineralization rates were accelerated when C. testosteroni was bioaugmented into the acclimated biomass, which increased the amount of C. testosteroni, but otherwise had minimal effects on the microbial community. The key role of C. testosteroni was to accelerate the first step of pyridine biodegradation, mono-oxygenation to 2-hydroxylpyridine (2HP), and the acclimated biomass was better able to complete downstream reactions leading to mineralization. Thus, bioaugmentation increased the rates of pyridine mono-oxygenation and subsequent mineralization through the synergistic roles of C. testosteroni and the main community in the acclimated biomass.

Nitric oxide monooxygenation (NOM) reaction of cobalt-nitrosyl {Co(NO)}8 to CoII-nitrito {CoII(NO2 -)}: base induced hydrogen gas (H2) evolution.

Here, we report the nitric oxide monooxygenation (NOM) reactions of a CoIII-nitrosyl complex (1, {Co-NO}8) in the presence of mono-oxygen reactive species, i.e., a base (OH−, tetrabutylammonium hydroxide (TBAOH) or NaOH/15-crown-5), an oxide (O2− or Na2O/15-crown-5) and water (H2O). The reaction of 1 with OH− produces a CoII-nitrito complex {3, (CoII-NO2−)} and hydrogen gas (H2), via the formation of a putative N-bound Co-nitrous acid intermediate (2, {Co-NOOH}+). The homolytic cleavage of the O–H bond of proposed [Co-NOOH]+ releases H2via a presumed CoIII-H intermediate. In another reaction, 1 generates CoII-NO2− when reacted with O2−via an expected CoI-nitro (4) intermediate. However, complex 1 is found to be unreactive towards H2O. Mechanistic investigations using 15N-labeled-15NO and 2H-labeled-NaO2H (NaOD) evidently revealed that the N-atom in CoII-NO2− and the H-atom in H2 gas are derived from the nitrosyl ligand and OH− moiety, respectively.

Enzymatically Produced Trimethylamine N-Oxide: Conserving It or Eliminating It

Trimethylamine N-Oxide (TMAO) is the product of the monooxygenation reaction catalyzed by a drug-metabolizing enzyme, human flavin-containing monooxygenase 3 (hFMO3), and its animal orthologues. For several years, researchers have looked at TMAO and hFMO3 as two distinct molecules playing specific but separate roles, the former to defend saltwater animals from osmotic or hydrostatic stress and the latter to process xenobiotics in men. The presence of high levels of plasmatic TMAO in elasmobranchs and other animals was demonstrated a long time ago, whereas the actual physiological role of hFMO3 is still unknown because the enzyme has been mainly characterized for its ability to oxidize drugs. Recently TMAO was found to be related to several human health conditions such as atherosclerosis, cardiovascular, and renal diseases. This correlation poses a striking question of how other vertebrates (and invertebrates) can survive in the presence of very high TMAO concentrations (micromolar in humans, millimolar in marine mammals and several hundred millimolar in elasmobranchs). Therefore, it is important to address how TMAO, its precursors, and FMO catalytic activity are interconnected.

Investigation on drug-binding in heme pocket of CYP2C19 with UV-visible and resonance Raman spectroscopies.

Cytochrome P450 (CYP) is a class of heme-containing enzymes which mainly catalyze a monooxygenation reaction of various chemicals, and hence CYP plays a key role in the drug metabolism. Although CYP2C19 isoform is a minor hepatic CYP, it metabolizes clinically important drugs such as omeprazole and S‑mephenytoin. In this work, the interaction of purified CYP2C19 WT (CYP2C19) with seven drugs (phenytoin, S‑mephenytoin, omeprazole, lansoprazole, cimetidine, propranolol, and warfarin) was investigated using spectroscopic methods. The binding of each drug and the induced structural change in the heme distal environment were evaluated. Ferric form of CYP2C19 was revealed to contain a six-coordinate low-spin heme with a water molecule as a sixth ligand in a distal site, and the addition of each drug caused varied minor fraction of five-coordinate heme. It was suggested that the ligated water molecule was partly moved away from the heme distal environment and that the degree of water removal was dependent on the type of drugs. The effect on the coordination was varied with the studied drugs with wide variation in the dissociation constants from 2.6 μM for lansoprazole to 5400 μM for warfarin. Phenytoin and S‑mephenytoin showed that binding to CYP2C19 occurred in a stepwise manner and that the coordination of a water molecule was facilitated in the second binding step. In the ferrous CO-bound state, ν(FeCO) stretching mode was clearly observed at 471 cm-1 in the absence of drugs. The Raman line was greatly up-shifted by omeprazole (487 cm-1) and lansoprazole (477 cm-1) but was minimally affected by propranolol, phenytoin, and S‑mephenytoin. These results indicate that slight chemical modification of a drug greatly affects the heme distal environments upon binding.

Competition for electrons between mono-oxygenations of pyridine and 2-hydroxypyridine.

Pyridine and its heterocyclic derivatives are widely encountered in industrial wastewaters, and they are relatively recalcitrant to biodegradation. Pyridine biodegradation is initiated by two mono-oxygenation reactions that compete for intracellular electron donor (2H). In our experiments, UV photolysis of pyridine generated succinate, whose oxidation augmented the intracellular electron donor and accelerated pyridine biodegradation and mineralization. The first mono-oxygenation reaction always was faster than the second one, because electrons provided by intracellular electron donors were preferentially utilized by the first mono-oxygenase; this was true even when the concentration of 2HP was greater than the concentration of pyridine. In addition, the first mono-oxygenation had faster kinetics because it had higher affinity for its substrate (pyridine), along with less substrate self-inhibition.

Heme degradation in Mycobacterium tuberculosis

Bacterial pathogens must acquire host iron for their survival and proliferation. Because of limited availability of free iron in the host, they have evolved to overcome this restriction by employing a family of heme oxygenase enzymes that mediate the oxidative cleavage of heme into biliverdin, carbon monoxide, and iron by three step monooxygenation reaction. However, MhuD, a heme degradation enzyme of Mycobacterium tuberculosis, the etiological agent of tuberculosis, accomplishes this task without generating carbon monoxide, which induces the M. tuberculosis latent state. This unusual heme degradation reaction proceeds through sequential mono- and di-oxygenation events within the single active center of MhuD, a mechanism unprecedented in oxygenase enzyme catalysis. Like canonical heme oxygenase, the initial MhuD monooxygenation converts heme to meso-hydroxyheme, which subsequently reacts with O2 at an unusual position to completely suppress monooxygenation but to allow ring cleavage through dioxygenation. This mechanistic change, due to heavy deformation of hydroxyheme, rationally explains the unique heme catabolites of MhuD. Coexistence of mechanistically distinct functions is a newly identified strategy to expand the physiological outcome of enzymes.

Integrating multi-omics analyses of Nonomuraea dietziae to reveal the role of soybean oil in [(4\u2032-OH)MeLeu]4-CsA overproduction

BackgroundNonomuraea dietziae is a promising microorganism to mediate the region-specific monooxygenation reaction of cyclosporine A (CsA). The main product [(4′-OH)MeLeu]4-CsA possesses high anti-HIV/HCV and hair growth-stimulating activities while avoiding the immunosuppressive effect of CsA. However, the low conversion efficiency restricts the clinical application. In this study, the production of [(4′-OH)MeLeu]4-CsA was greatly improved by 55.6% from 182.8 to 284.4 mg/L when supplementing soybean oil into the production medium, which represented the highest production of [(4′-OH)MeLeu]4-CsA so far.ResultsTo investigate the effect of soybean oil on CsA conversion, some other plant oils (corn oil and peanut oil) and the major hydrolysates of soybean oil were fed into the production medium, respectively. The results demonstrated that the plant oils, rather than the hydrolysates, could significantly improve the [(4′-OH)MeLeu]4-CsA production, suggesting that soybean oil might not play its role in the lipid metabolic pathway. To further unveil the mechanism of [(4′-OH)MeLeu]4-CsA overproduction under the soybean oil condition, a proteomic analysis based on the two-dimensional gel electrophoresis coupled with MALDI TOF/TOF mass spectrometry was implemented. The results showed that central carbon metabolism, genetic information processing and energy metabolism were significantly up-regulated under the soybean oil condition. Moreover, the gas chromatography-mass spectrometry-based metabolomic analysis indicated that soybean oil had a great effect on amino acid metabolism and tricarboxylic acid cycle. In addition, the transcription levels of cytochrome P450 hydroxylase (CYP) genes for CsA conversion were determined by RT-qPCR and the results showed that most of the CYP genes were up-regulated under the soybean oil condition.ConclusionsThese findings indicate that soybean oil could strengthen the primary metabolism and the CYP system to enhance the mycelium growth and the monooxygenation reaction, respectively, and it will be a guidance for the further metabolic engineering of this strain.

Characterization of an Isoeugenol Monooxygenase (Iem) from &lt;i&gt;Pseudomonas nitroreducens&lt;/i&gt; Jin1 That Transforms Isoeugenol to Vanillin

The isoeugenol monooxygenase (iem) gene from Pseudomonas nitroreducens Jin1, responsible for the conversion of isoeugenol to vanillin, was cloned and overexpressed in Escherichia coli. The purified Iem had a predicted molecular mass of 54 kDa. The V(max), K(M), and k(cat) values for it, using isoeugenol as substrate, were 4.2 µmol vanillin min(-1) mg(-1) of protein, 120 µM, and 3.8 s(-1), respectively. Maximum substrate turnover for Iem occurred at 30 °C and pH 9.0. An (18)Oxygen-labeling experiment revealed that oxidative cleavage of isoeugenol by Iem was catalyzed via a monooxygenation reaction, and that incorporation of the oxygen atom from O(2) into vanillin was preferable to incorporation from water. While the catalytic activity of Iem, as prepared, did not require the addition of any organic or metal cofactor, ICP-MS analysis showed 0.7 mol of iron per mol of Iem. Moreover site-directed mutagenesis of Iem of four conserved histidine residues individually, His(167), His(218), His(282), and His(471), which appear to be involved in ligand bonding with Fe(2+), resulted in a loss of activity. Enzyme activity was not appreciably influenced by preincubation of Iem with high concentrations of chelators, suggesting that iron is tightly bound. Iem has an iron-mediated mechanism that is widely spread among the three domains of life.

Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): Involvement of alternative redox partners

A fungal cytochrome P450 monooxygenase (CYP5150A2) from the white-rot basidiomycete Phanerochaete chrysosporium was heterologously expressed in Escherichia coli and purified as an active form. The purified CYP5150A2 was capable of hydroxylating 4-propylbenzoic acid (PBA) with NADPH-dependent cytochrome P450 oxidoreductase (CPR) as the single redox partner; the reaction efficiency was improved by the addition of electron transfer protein cytochrome b5 (Cyt-b5). Furthermore, CYP5150A2 exhibited substantial activity with redox partners Cyt-b5 and NADH-dependent Cyt-b5 reductase (CB5R) even in the absence of CPR. These results indicated that a combination of CB5R and Cyt-b5 may be capable of donating both the first and the second electrons required for the monooxygenation reaction. Under reaction conditions in which the redox system was associated with the CB5R-dependent Cyt-b5 reduction system, the exogenous addition of CPR and NADPH had no effect on the PBA hydroxylation rate or on coupling efficiency, indicating that the transfer of the second electron from Cyt-b5 was the rate-limiting step in the monooxygenase system. In addition, the rate of PBA hydroxylation was significantly dependent on Cyt-b5 concentration, exhibiting Michaelis–Menten kinetics. This study provides indubitable evidence that the combination of CB5R and Cyt-b5 is an alternative redox partner facilitating the monooxygenase reaction catalyzed by CYP5150A2.

Structural Basis of Drug Binding to CYP46A1, an Enzyme That Controls Cholesterol Turnover in the Brain

Cytochrome P450 46A1 (CYP46A1) initiates the major pathway of cholesterol elimination from the brain and thereby controls cholesterol turnover in this organ. We determined x-ray crystal structures of CYP46A1 in complex with four structurally distinct pharmaceuticals; antidepressant tranylcypromine (2.15 Å), anticonvulsant thioperamide (1.65 Å), antifungal voriconazole (2.35 Å), and antifungal clotrimazole (2.50 Å). All four drugs are nitrogen-containing compounds that have nanomolar affinity for CYP46A1 in vitro yet differ in size, shape, hydrophobicity, and type of the nitrogen ligand. Structures of the co-complexes demonstrate that each drug binds in a single orientation to the active site with tranylcypromine, thioperamide, and voriconazole coordinating the heme iron via their nitrogen atoms and clotrimazole being at a 4 Å distance from the heme iron. We show here that clotrimazole is also a substrate for CYP46A1. High affinity for CYP46A1 is determined by a set of specific interactions, some of which were further investigated by solution studies using structural analogs of the drugs and the T306A CYP46A1 mutant. Collectively, our results reveal how diverse inhibitors can be accommodated in the CYP46A1 active site and provide an explanation for the observed differences in the drug-induced spectral response. Co-complexes with tranylcypromine, thioperamide, and voriconazole represent the first structural characterization of the drug binding to a P450 enzyme.

Catalytic oxygenation of phenols by arthropod hemocyanin, an oxygen carrier protein, from Portunus trituberculatus

The hexamer (Pt-6Hc) of swimming crab Portunus trituberculatus hemocyanin (Pt-Hc) and one of its monomeric subunits (Pt-1Hc) have been purified and converted to an efficient phenol monooxygenase (phenolase) by treatment with urea. To explore the intrinsic chemical reactivity of the dicopper center of Pt-Hc, the spectroscopic features and phenol monooxygenase (phenolase) activity of the isolated proteins have been examined in detail. The oxy-forms involving a (mu-eta(2):eta(2)-peroxo)dicopper(II) species (oxy-Hc) of Pt-6Hc and Pt-1Hc are relatively stable in 0.5 M borate buffer (pH 9.0) even in the presence of a high concentration of urea (3 M) at 25 degrees C. The catalytic activity of monomeric Pt-1Hc in the oxygenation reaction (multi-turnover reaction) of 4-methylphenol to 4-methyl-1,2-dihydroxybenzene (4-methylcatechol) was higher than that of hexameric Pt-6Hc, and its catalytic activity was further accelerated by the addition of urea. Kinetic deuterium isotope effect analysis and Hammett analysis using a series of p-substituted phenol derivatives under anaerobic conditions (single-turnover reaction) have indicated that the monooxygenation reaction of phenols to catechols by the peroxo species of oxy-Hc proceeds via electrophilic aromatic substitution mechanism as in the case of tyrosinase (dinuclear copper monooxygenase). The effect of urea on the redox functions of oxy-Hc is discussed on the basis of spectroscopic analysis and reactivity studies.

The Role of Heme in Monooxygenation Reaction Mechanism by Cytochrome P-450

The Role of Heme in Monooxygenation Reaction Mechanism by Cytochrome P-450