H-Bonding Networks Dictate the Molecular Mechanism of H₂O₂ Activation by P450

Abstract

Understanding the molecular basis for controlled H 2 O 2 activation is of fundamental importance for peroxide-driven catalysis by metalloenzymes. In addition to O 2 activation in the presence of stoichiometric reductants, an increasing number of metalloenzymes are found to activate the H 2 O 2 cosubstrate for oxidative transformations in the absence of stoichiometric reductants. Herein, we characterized the X-ray structure of the P450BM3 F87A mutant in complex with the dual-functional small molecule (DFSM) N-(ω-imidazolyl)-hexanoyl-Lphenylalanine (Im-C6-Phe), which enables an efficient peroxygenase activity for P450BM3. Our computational investigations show that the H 2 O 2 activations by P450BM3 are highly dependent on the substrate and the DFSM. In the absence of both the substrate and the DFSM, H 2 O 2 activation via the O−O homolysis mechanism is significantly inhibited by the H-bonding network from the proximal H of H 2 O 2 . However, the presence of the substrate expels the solvation waters and disrupts the H-bonding network from the proximal H of H 2 O 2 , thus remarkably favoring homolytic O−O cleavage toward Cpd I formation. However, the presence of the DFSM forms a proton channel between the imidazolyl group of the DFSM and the proximal H of H 2 O 2 , thus enabling a heterolytic O−O cleavage and Cpd I formation that is greatly favored over the homolysis mechanism. Meanwhile, our simulations demonstrate that the H-bonding network from the distal H of H 2 O 2 is the key to control of the H 2 O 2 activation in the homolytic route. These findings are in line with all available experimental data and highlight the key roles of H-bonding networks in dictating H 2 O 2 activations.

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

“…[80] Based the crystal structure of the P450BM3-F87A mutant in complex with Im-C6-Phe and NH 2 OH, multiple polar interactions and hydrophobic interactions were identified between the enzyme and the anchor group of DFSM (Figure 9B). [81] The computational study demonstrated that the H 2 O 2 maintained a strong H-bonding network with a bridging water and the imidazolyl group of the DFSM, which speeds up the Cpd I formation via a favored heterolytic OÀ O cleavage pathway, thus to affect the overall kinetics. [81] Among the P450 peroxygenases, OleT is the only one that can efficiently use H 2 O 2 to catalyze the oxidative decarboxylation of fatty acids to produce terminal olefins, which have potential applications as next generation of biofuels.…”

“…With the DFSM strategy, different P450BM3 variants displayed high O ‐demethylation on aromatic ethers [79] and peroxidase activity on guaiacol, 2,6‐dimethoxyphenol, o ‐phenylenediamine, and p ‐phenylenediamine [80] . Based the crystal structure of the P450BM3‐F87A mutant in complex with Im‐C6‐Phe and NH 2 OH, multiple polar interactions and hydrophobic interactions were identified between the enzyme and the anchor group of DFSM (Figure 9B) [81] . The computational study demonstrated that the H 2 O 2 maintained a strong H‐bonding network with a bridging water and the imidazolyl group of the DFSM, which speeds up the Cpd I formation via a favored heterolytic O−O cleavage pathway, thus to affect the overall kinetics [81] …”

A Unique P450 Peroxygenase System Facilitated by a Dual-Functional Small Molecule: Concept, Application, and Perspective

“…The mechanism for H 2 O 2 activation was further elucidated by QM-MM computational investigations. These computational chemistry results revealed the crucial role of DFSM in promoting a heterolytic O-O cleavage to favor Cpd I formation [74]. The DFSM facilitates the formation of a proton channel between the imidazolyl group of the DFSM and proximal H of H 2 O 2 , thus enabling a heterolytic O-O cleavage and Cpd I formation, which is similar to the proposed mechanism for H 2 O 2 activation in natural peroxygenases (e.g., UPO) or peroxidases (e.g., HRP).…”

“…This discovery provides a unique choice of protein engineering sites for developing catalytic promiscuity of the current peroxygenase system (will be discussed below by combination with other results). The catalytic role and mechanism of DFSMs have been further disclosed by combining structural biology and computational chemistry [74]. To mimic the pre-reaction state The catalytic role and mechanism of DFSMs have been further disclosed by combining structural biology and computational chemistry [74].…”

“…The catalytic role and mechanism of DFSMs have been further disclosed by combining structural biology and computational chemistry [74]. To mimic the pre-reaction state The catalytic role and mechanism of DFSMs have been further disclosed by combining structural biology and computational chemistry [74]. To mimic the pre-reaction state of P450-bounded H 2 O 2 and avoid the H 2 O 2 -initiated reaction, Jiang et al skillfully adopted the NH 2 OH molecule as the analog of H 2 O 2 to prepare the co-crystal (Figure 4A,B).…”