We report a unique strategy for the development of a H O -dependent cytochrome P450BM3 system, which catalyzes the monooxygenation of non-native substrates with the assistance of dual-functional small molecules (DFSMs), such as N-(ω-imidazolyl fatty acyl)-l-amino acids. The acyl amino acid group of DFSM is responsible for bounding to enzyme as an anchoring group, while the imidazolyl group plays the role of general acid-base catalyst in the activation of H O . This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450-H O system previously reported. This work provides the first example of the activation of the normally H O -inert P450s through the introduction of an exogenous small molecule. This approach improves the potential use of P450s in organic synthesis as it avoids the expensive consumption of the reduced nicotinamide cofactor NAD(P)H and its dependent electron transport system. This introduces a promising approach for exploiting enzyme activity and function based on direct chemical intervention in the catalytic process.

The direct hydroxylation of alkanes under mild conditions is a key issue in catalytic chemistry that addresses an increasing number of industrial and economic requirements. Cytochrome P450s are monooxygenases that are capable of oxidizing less reactive C−H bonds; however, wild-type P450s are unavailable for many important nonnative substrates such as gaseous alkanes. Here, we report the enhanced hydroxylation activities and crystallographic evidence for the role of decoy molecules in wild-type P450BM3-catalyzed hydroxylation of gaseous ethane and propane by using the next generation of decoy molecule. A cocrystal structure of P450BM3 and a decoy molecule reveals that an N-perfluoroacyl amino acid (decoy molecule) partially occupies the substrate-binding site of P450BM3. This binding of the decoy re-forms the active site pocket to allow the accommodation of small substrates and simultaneously influences the formation of compound I species by expelling water molecules from the active site.

The selective hydroxylation of benzene to phenol, without the formation of side products resulting from overoxidation, is catalyzed by cytochrome P450BM3 with the assistance of amino acid derivatives as decoy molecules. The catalytic turnover rate and the total turnover number reached 259 min P450BM3 and 40 200 P450BM3 when N-heptyl-l-proline modified with l-phenylalanine (C7-l-Pro-l-Phe) was used as the decoy molecule. This work shows that amino acid derivatives with a totally different structure from fatty acids can be used as decoy molecules for aromatic hydroxylation by wild-type P450BM3. This method for non-native substrate hydroxylation by wild-type P450BM3 has the potential to expand the utility of P450BM3 for biotransformations.

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.