Radical Clock Substrates Measure Nonstatistical Dynamical Effects in Cytochrome P450-Mediated C–H Functionalization Reactions

Cytochrome P450 enzymes functionalize strong C–H bonds via a two-step hydrogen atom abstraction-radical rebound mechanism that involves a transient radical pair intermediate. Specially designed strained hydrocarbon substrates with predetermined rearrangement rate constants (radical clocks) are typically used to probe the apparent lifetime of the intermediate and measure the rate of rebound. A long-standing mystery of P450 clock reactions is that the experimentally measured unrearranged (U) versus rearranged (R) clock product selectivity (U/R) has no correlation with the rate of radical rearrangements. Using ab initio direct dynamics trajectories, this work demonstrates that dynamic (momentum) nonstatistical effects control the fate of the radical pair intermediate in reactions of P450 Fe-oxo with clock substrates, and this explains the lack of correlation between U/R selectivity and radical rearrangement rates. It also means that the results of clock reactions cannot always be interpreted within the context of transition state theory. Trajectories also revealed that some clocks react through a carbocation intermediate rather than a radical intermediate, which also impacts the formation of U or R products. This discovery resolves the discrepancy in the experimentally observed U/R selectivity data by incorporating the clock ionization energy and C–C bond elongation during cation formation. Overall, this work reveals that understanding the outcome of radical clock experiments, especially for P450 reactions, requires the integration of two-state reactivity concepts with dynamic effects and nonstatistical pathway branching.