P450-BM3-Catalyzed Sulfoxidation versus Hydroxylation: A Common or Two Different Catalytically Active Species?

Jian-Bo Wang,Bo Chen,Peng Wu,Da Yu,Manfred T Reetz,Binju Wang,Qun Huang,Wei Peng

doi:10.1021/jacs.9b13061

Abstract

While the mechanism of the P450-catalyzed oxidative hydroxylation of organic compounds has been studied in detail for many years, less is known about sulfoxidation. Depending upon the structure of the respective substrate, heme-Fe=O (Cpd I), heme–Fe(III)–OOH (Cpd 0), and heme–Fe(III)–H2O2 (protonated Cpd 0) have been proposed as reactive intermediates. In the present study, we consider the transformation of isosteric substrates via sulfoxidation and oxidative hydroxylation, respectively, catalyzed by regio- and enantioselective mutants of P450-BM3 which were constructed by directed evolution. 1-Thiochromanone and 1-tetralone were used as the isosteric substrates because, unlike previous studies involving fully flexible compounds such as thia-fatty acids and fatty acids, respectively, these compounds are rigid and cannot occur in a multitude of different conformations and binding modes in the large P450-BM3 binding pocket. The experimental results comprising activity and regio- and enantioselectivity, flanked by molecular dynamics computations within a time scale of 300 ns and QM/MM calculations of transition-state energies, unequivocally show that heme-Fe=O (Cpd I) is the common catalytically active intermediate in both sulfoxidation and oxidative hydroxylation.

Highlights

Cytochrome P450 monooxygenases catalyze a remarkable range of oxidative reaction types, including CH-activating hydroxylation, olefin epoxidation, sulfoxidation, and aminoxidation.[1−11] This raises the question of whether one and the same catalytically active species is involved or whether the enzyme utilizes a different active species for each type of transformation
All researchers in the field have agreed that it involves high-spin intermediate heme-Fe O (Cpd I) in a rate-determining radical abstraction process followed by rapid C−O bond formation.[1−11] In the most recent QM/MM study, Shaik and co-workers have studied the regio- and enantioselectivity of fatty acid hydroxylation catalyzed by wild type (WT) and mutants of P450-BM3.12 Again, the crucial role of Cpd I was documented, in addition to the dynamics of the flexible carbon chain of fatty acids
■ RESULTS AND DISCUSSION: EXPERIMENTAL RESULTS DERIVED FROM DIRECTED EVOLUTION We reasoned that isosteric compounds thiochromanone (1) and 1-tetralone (3) are ideal substrates for the present study (Scheme 2)

Summary

Introduction

Cytochrome P450 monooxygenases catalyze a remarkable range of oxidative reaction types, including CH-activating hydroxylation, olefin epoxidation, sulfoxidation, and aminoxidation.[1−11] This raises the question of whether one and the same catalytically active species is involved or whether the enzyme utilizes a different active species for each type of transformation. The experimental results comprising activity and regio- and enantioselectivity, flanked by molecular dynamics computations within a time scale of 300 ns and QM/MM calculations of transition-state energies, unequivocally show that heme-Fe O (Cpd I) is the common catalytically active intermediate in both sulfoxidation and oxidative hydroxylation.

Results

Conclusion