Investigation of the Mechanism of Multiple Cytochrome P450-catalysed Reactions

Abstract

The cytochrome P450 heme-thiolate enzymes catalyse a multitude of oxidation reactions and, in humans, carry out drug metabolism. P450s perform hydroxylation, epoxidation, N-, O- and Sdealkylation, sulfoxidation, alkyne oxidation and aldehyde oxidation of organic molecules (and many other reactions). These reactions are predominantly performed by the reactive intermediate Compound I, but other intermediates in the catalytic cycle may mediate some types of reactions. It would be appealing to exploit these enzymes as environmentally benign catalysts in the synthesis of fine chemicals. Their widespread use in industrial synthesis is, however, impractical given the high cost of the required cofactor NAD(P)H, but engineering P450s to instead use cheap H₂O₂ would overcome this problem. CYP199A4 is a soluble bacterial P450 enzyme from Rhodopseudomonas palustris HaA2 that favours 4-methoxybenzoic acid and other para-substituted benzoic acids as substrates. It tightly binds these substrates and the para-substituent is rapidly oxidised. This enzyme has been used as a model system to study the mechanism of P450-catalysed reactions. While 4-methoxybenzoic acid is oxidatively demethylated at a rate of 1220 μM (μM-P450)⁻¹ min⁻¹, CYP199A4 displays no detectable activity towards the meta isomer, 3-methoxybenzoic acid. In vitro reactions were performed with a range of other meta-substituted benzoic acids (3-methylthio-, 3-methylamino-, 3- formyl-, 3-methyl-, 3-isopropyl-, 3-tert-butyl- and 3-ethoxy-benzoic acid) to assess whether CYP199A4 had activity towards these substrates. These meta-substituted substrates, except for 3- tert-butylbenzoic acid, were all metabolised by CYP199A4, but with low activity compared to the corresponding para isomers. Compared to the para isomers, the meta isomers had lower binding affinity and induced smaller type I spin-state shifts to high-spin. To rationalise CYP199A4’s preference for para- over meta-substituted benzoic acid substrates and to investigate the requirements for efficient monooxygenase activity, crystal structures were solved of CYP199A4 in complex with 3-methoxy-, 3-methylamino-, 3-methylthio-, and 3- and 4-methyl-benzoic acid. These structures revealed that the heme-bound water ligand to the heme is retained when these substrates bind (water occupancy 21-90%) and is hydrogen-bonded to the heteroatom (N, S, O) of the substrate. The corresponding para isomers displace the iron-bound water. 3-Ethoxybenzoic acid, which has a bulkier meta-substituent, shifted the spin-state to 85% high-spin and in the crystal structure the iron-bound water was removed. The meta-substituent of each substrate is held in close proximity to the iron. 3-Methoxybenzoic acid is positioned near the iron but is not oxidised. This was attributed to the fact that the C-H bonds are oriented away from the heme, whereas those of 4-methoxybenzoic acid are ideally oriented for H-atom abstraction by Cpd I. These results emphasise that close proximity of the methyl carbon to the heme iron does not guarantee that hydroxylation will occur if the C-H bonds are not oriented appropriately for abstraction, and subtle modification of the substrate’s position relative to the heme can abolish catalytic activity. X-ray crystallography, CW and HYSCORE EPR and other experiments were performed to elucidate the binding modes of 4-pyridin-2-yl-, 4-pyridin-3-yl- and 4-imidazol-1-yl-benzoic acid in the CYP199A4 active site. These heterocyclic aromatic compounds are not metabolised and induce substantially different type II UV-Vis spectra. 4-Pyridin-3-yl- and 4-imidazol-1-yl-benzoic acid redshifted the Soret band from 419 to 424 nm. They induced ‘normal’ type II spectra, characterised by a less intense α-band than β-band and an increase in δ-band intensity. The UV-Vis spectra of ferrous CYP199A4 in complex with these ligands indicated that 4-pyridin-3-ylbenzoic acid was directly ligated to the heme iron via the pyridine nitrogen, but the Fe-N bond between the iron and 4-imidazol-1-ylbenzoic acid was ruptured upon heme reduction. 4-Pyridin-2-ylbenzoic acid induced a smaller Soret band red-shift (to 422 nm) when added to the ferric enzyme. It produced an ‘abnormal’ type II spectrum, with no decrease in the α-band intensity. 4-Pyridin-2-ylbenzoic acid also induced a smaller Soret band trough in the difference spectrum than 4-pyridin-3-ylbenzoic acid. HYSCORE EPR and X-ray crystallography revealed that 4-pyridin-3-yl- and 4-imidazol-1-ylbenzoic acid were directly ligated to the ferric heme iron, but 4-pyridin-2-ylbenzoic acid was hydrogen-bonded to the heme-bound water. This study revealed that optical spectroscopy can distinguish between water-bridged and directly bound nitrogen donor ligands. 4-Pyridin-3-yl- and 4-imidazol-1-yl-benzoic acid-bound CYP199A4 were both reduced to the ferrous form by ferredoxin, ferredoxin reductase and NADH. On the other hand, binding of 4-pyridin-2-ylbenzoic acid to CYP199A4 lowered the reduction potential and prevented heme reduction by even the powerful reductant dithionite. This implies that water-bridged nitrogen ligands may in some instances be more effective P450 inhibitors than those that bind directly to the iron. The T252E mutant of CYP199A4 was produced and characterised. This variant was no longer able to operate using NADH but was a more efficient peroxygenase (H₂O₂-utilising enzyme) than the wild-type (WT) enzyme. EPR indicated that the sixth axial ligand to the heme was a mixture of hydroxide and water. Crystal structures showed that this aqua/hydroxo ligand was tightly bound due to strong interactions with the carboxylate of E252. The axial aqua/hydroxo ligand was not displaced by substrates, even sterically bulky substrates, explaining the lack of substrate-induced spin-state shifts and the exceedingly slow rate of electron transfer from the ferredoxin to the P450. Because this ligand is not displaced by substrates, the active species could potentially be generated via light-driven oxidation of the water-bound ferric resting state to Compound I. Type II nitrogen ligands were also unable to displace the aqua/hydroxo ligand. 4-Pyridin-3-ylbenzoic acid, when added to the T252E mutant, induced an ‘abnormal’ type II spectrum, confirming that optical spectroscopy can distinguish between water-bridged and directly bound type II ligands. X-ray crystallography revealed that the T252 → E mutation only subtly altered the orientation of substrates in the CYP199A4 active site. In the absence of substrate, the heme signal of the T252E mutant was rapidly bleached by 50 mM H₂O₂. When substrate was present, the T252E variant remained catalytically active for several hours. The T252E variant was able to perform a range of reactions using H₂O₂ (O-dealkylation, hydroxylation/desaturation, epoxidation, sulfoxidation, alkyne oxidation and aldehyde oxidation). When the surrogate oxygen donor tert-butyl hydroperoxide was substituted for H₂O₂, the T252E mutant had negligible activity. t-BuOOH is presumably too bulky to access the heme iron in this P450. WT CYP199A4 and the T252A and D251N mutants also catalysed these reactions using H₂O₂ but afforded less product over a 4-hour period than the T252E mutant. H₂O₂- and NADH-driven epoxidation of 4-vinylbenzoic acid catalysed by WT and mutant CYP199A4 proceeded with high enantioselectivity, yielding almost exclusively the (S)-enantiomer. In NADH-supported reactions, WT CYP199A4 catalysed O-demethylation of 4- methoxybenzoic acid more efficiently than sulfoxidation of 4-methylthiobenzoic acid. In H₂O₂- driven reactions, the T252E variant had higher activity towards sulfoxidation compared to Odemethylation, hydroxylation, epoxidation, aldehyde oxidation and alkyne oxidation. This may indicate the involvement of a second oxidant in sulfoxidation (e.g. the FeIII–H₂O₂ species), allowing sulfoxidation to occur in the absence of Compound I as proposed by Shaik.