Investigating the active oxidants involved in cytochrome P450 catalyzed sulfoxidation reactions

Published 27 December 2022Cytochrome P450 (CYP) heme-thiolate monooxygenases catalyze the hydroxylation of the C H bonds of organic molecules. This reaction is initiated by a ferryl-oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric-hydroperoxy complex (Cpd 0) and the Fe(III)-H2O2 complex have been proposed as alternative oxidants for this transformation. To investigate this, the oxidation of 4-alkylthiobenzoic acids and 4-methoxybenzoic acid by the CYP199A4 enzyme from Rhodopseudomonas palustris HaA2 was compared using both monooxygenase and peroxygenase pathways. By examining mutants at the mechanistically important, conserved acid alcohol-pair (D251N, T252A and T252E) the relative amounts of the reactive intermediates that would form in these reactions were disturbed. Substrate binding and X-ray crystal structures helped to understand changes in the activity and enabled a attempt to evaluate whether multiple oxidants can participate in these reactions. In peroxygenase reactions the T252E mutant had higher activity towards sulfoxidation than Odemethylation but in the monooxygenase reactions with the WT enzyme the activity of both reactions was similar. The peroxygenase activity of the T252A mutant was greater for sulfoxidation reactions than the WT enzyme, which is the reverse of the activity changes observed for O-demethylation. The monooxygenase activity and coupling efficiency of sulfoxidation and oxidative demethylation were reduced by similar degrees with the T252A mutant. These observations infer that while Cpd I is required for O-dealkylation, another oxidant may contribute to sulfoxidation. Based on the activity of the CYP199A4 mutants it is proposed that this is the Fe(III)- H2O2 complex which would be more abundant in the peroxide-driven reactions.Matthew N. Podgorski, Tom Coleman, Luke R. Churchman, John B. Bruning, James J. De Voss, Stephen G. Bel

Structural insights into the role of the acid-alcohol pair of residues required for dioxygen activation in cytochrome P450 enzymes

The cytochrome P450 heme monooxygenases commonly use an acid-alcohol pair of residues, within the I-helix, to activate iron-bound dioxygen. This work aims to clarify conflicting reports on the importance of the alcohol functionality in this process. Mutants of the P450, CYP199A4 (CYP199A4D251N and CYP199A4T252A), were prepared, characterised and their crystal structures were solved. The acid residue of CYP199A4 is not part of a salt bridge network, a key feature of paradigmatic model system P450cam. Instead, there is a direct proton delivery network, via a chain of water molecules, extending to the surface. Nevertheless, CYP199A4D251N dramatically reduced the activity of the enzyme consistent with a role in proton delivery. CYP199A4T252A decreased the coupling efficiency of the enzyme with a concomitant increase in the hydrogen peroxide uncoupling pathway. However, the effect of this mutation was much less pronounced than reported with P450cam. Its crystal structures revealed fewer changes at the I-helix, compared to the P450cam system. The structural changes observed within the I-helix of P450cam during oxygen activation do not seem to be required in this P450. These differences are due to the presence of a second threonine residue at position 253, which is absent in P450cam. This threonine forms part of the hydrogen bonding network, resulting in subtle structural changes and is also present across the majority of the P450 superfamily. Overall, the results suggest that while the acid-alcohol pair is important for dioxygen activation this process and the method of proton delivery can differ across P450s.Tom Coleman, Jeanette E. Stok, Matthew N. Podgorski, John B. Bruning, James J. De Voss, Stephen G. Bel

An altered heme environment in an engineered cytochrome P450 enzyme enables the switch from monooxygenase to peroxygenase activity

Cytochrome P450 heme-thiolate monooxygenases are exceptionally versatile enzymes which insert an oxygen atom into the unreactive C–H bonds of organic molecules. They source O2 from the atmosphere and usually derive electrons from nicotinamide cofactors via electron transfer proteins. The requirement for an expensive nicotinamide adenine dinucleotide (phosphate) cofactor and the redox protein partners can be bypassed by driving the catalysis using hydrogen peroxide (H₂O₂). We demonstrate that the mutation of a highly conserved threonine residue, involved in dioxygen activation, to a glutamate shuts down monooxygenase activity in a P450 enzyme and converts it into a peroxygenase. The reason for this switch in the threonine to glutamate (T252E) mutant of CYP199A4 from Rhodopseudomonas palustris HaA2 was linked to the lack of a spin state change upon the addition of the substrate. The crystal structure of the substrate-bound form of this mutant highlighted a modified oxygen-binding groove in the I-helix and the retention of the iron-bound aqua ligand. This ligand interacts with the glutamate residue, which favors its retention. Electron paramagnetic resonance confirmed that the ferric heme aqua ligand of the mutant substrate-bound complex had altered characteristics compared to a standard ferric heme aqua complex. Significant improvements in peroxygenase activity were demonstrated for the oxidative demethylation of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and veratric acid to vanillic acid (up to 6-fold). The detailed characterization of this engineered heme peroxygenase will facilitate the development of new methods for driving the biocatalytic generation of oxygenated organic molecules via selective C–H bond activation using heme enzymes.Matthew N. Podgorski, Joshua S. Harbort, Joel H.Z. Lee, Giang T.H. Nguyen, John B. Bruning, William A. Donald, Paul V. Bernhardt, Jeffrey R. Harmer, and Stephen G. Bel

Cytochrome P450-catalyzed oxidation of halogen-containing substrates.

Cytochrome P450 (CYP) enzymes are heme-thiolate monooxygenases which catalyze the oxidation of aliphatic and aromatic C-H bonds and other reactions. The oxidation of halogens by cytochrome P450 enzymes has also been reported. Here we use CYP199A4, from the bacterium Rhodopseudomonas palustris strain HaA2, with a range of para-substituted benzoic acid ligands, which contain halogens, to assess if this enzyme can oxidize these species or if the presence of these electronegative atoms can alter the outcome of P450-catalyzed reactions. Despite binding to the enzyme, there was no detectable oxidation of any of the 4-halobenzoic acids. CYP199A4 was, however, able to efficiently catalyze the oxidation of both 4-chloromethyl- and 4-bromomethyl-benzoic acid to 4-formylbenzoic acid via hydroxylation of the α‑carbon. The 4-chloromethyl substrate bound in the enzyme active site in a similar manner to 4-ethylbenzoic acid. This places the benzylic α‑carbon hydrogens in an unfavorable position for abstraction indicating a degree of substrate mobility must be possible within the active site. CYP199A4 catalyzed oxidations of 4-(2'-haloethyl)benzoic acids yielding α-hydroxylation and desaturation metabolites. The α-hydroxylation product was the major metabolite. The desaturation pathway is significantly disfavored compared to 4-ethylbenzoic acid. This may be due to the electron-withdrawing halogen atom or a different positioning of the substrate within the active site. The latter was demonstrated by the X-ray crystal structures of CYP199A4 with these substrates. Overall, the presence of a halogen atom positioned close to the heme iron can alter the binding orientation and outcomes of enzyme-catalyzed oxidation.Tom Coleman, Matthew N. Podgorski, Maya L. Doyle, Jarred M. Scaffidi-Muta, Eleanor C. Campbell, John B. Bruning, James J. De Voss, Stephen G. Bel

Enabling Aromatic Hydroxylation in a Cytochrome P450 Monooxygenase Enzyme through Protein Engineering

Published 1 December 2022The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4-cyclohexylbenzoic acid but not the aromatic oxidation of 4-phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4-benzyl-, 4-phenoxy- and 4-benzoyl-benzoic acid and methoxy-substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4-benzylbenzoic acid. The methoxysubstituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4-(3’-methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4- phenylbenzoic acid. 4-Phenoxy- and 4-benzoyl-benzoic acid bound with the ether or ketone oxygen atom hydrogenbonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4-phenylbenzoic acid; however the F182L mutant was able to catalyse 4-phenylbenzoic acid oxidation generating 2’- hydroxy-, 3’-hydroxy- and 4’-hydroxy metabolites in a 83:9:8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild-type enzyme, the phenyl ring of 4-phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.Tom Coleman, Joel Z. H. Lee, Alicia M. Kirk, Daniel Z. Doherty, Matthew N. Podgorski, Dilshi K. Pinidiya, John B. Bruning, James J. De Voss, Elizabeth H. Krenske, and Stephen G. Bel

Biophysical techniques for distinguishing ligand binding modes in cytochrome P450 monooxygenases

Published: February 14, 2020The cytochrome P450 superfamily of heme monooxygenases catalyzes important chemical reactions across nature. The changes in the optical spectra of these enzymes, induced by the addition of substrates or inhibitors, are critical for assessing how these molecules bind to the P450, enhancing or inhibiting the catalytic cycle. Here we use the bacterial CYP199A4 enzyme (Uniprot entry Q2IUO2 ), from Rhodopseudomonas palustris HaA2, and a range of substituted benzoic acids to investigate different binding modes. 4-Methoxybenzoic acid elicits an archetypal type I spectral response due to a ≥95% switch from the low- to high-spin state with concomitant dissociation of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)benzoic acid induced different type II ultraviolet-visible (UV-vis) spectral responses in CYP199A4. The former induced a greater red shift in the Soret wavelength (424 nm vs 422 nm) along with a larger overall absorbance change and other differences in the α-, β-, and δ-bands. There were also variations in the ferrous UV-vis spectra of these two substrate-bound forms with a spectrum indicative of Fe-N bond formation with 4-(pyridin-3-yl)benzoic acid. The crystal structures of CYP199A4, with the pyridinyl compounds bound, revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua ligand remains. Continuous wave and pulse electron paramagnetic resonance data in frozen solution revealed that the substrates are bound in the active site in a form consistent with the crystal structures. The redox potential of each CYP199A4-substrate combination was measured, allowing correlation among binding modes, spectroscopic properties, and the observed biochemical activity.Matthew N. Podgorski, Joshua S. Harbort, Tom Coleman, Jeanette E. Stok, Jake A. Yorke, Luet-Lok Wong, John B. Bruning, Paul V. Bernhardt, James J. De Voss, Jeffrey R. Harmer, and Stephen G. Bel