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.

中文翻译：

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P450-BM3 催化的磺化氧化与羟基化：一种常见的还是两种不同的催化活性物质？

虽然 P450 催化的有机化合物氧化羟基化的机制已被详细研究多年，但对磺化氧化的了解却很少。根据各自底物的结构，血红素-Fe=O（Cpd I）、血红素-Fe（III）-OOH（Cpd 0）和血红素-Fe（III）-H2O2（质子化的Cpd 0）已被提议为反应中间体。在本研究中，我们分别考虑了通过定向进化构建的 P450-BM3 区域选择性和对映选择性突变体催化的等排底物转化。1-硫代色满酮和 1-四氢萘酮被用作等排底物，因为与之前的研究分别涉及硫代脂肪酸和脂肪酸等完全柔性化合物的研究不同，这些化合物是刚性的，不能在大 P450-BM3 结合口袋中以多种不同的构象和结合​​模式出现。包括活性和区域选择性和对映选择性在内的实验结果，辅以 300 ns 时间尺度内的分子动力学计算和过渡态能量的 QM/MM 计算，明确表明血红素-Fe = O（Cpd I）是常见的催化磺化和氧化羟基化的活性中间体。