Mechanistic Study of the Stereoselective Hydroxylation of [2‐2H1,3‐2H1]Butanes Catalyzed by Cytochrome P450 BM3 Variants

Engineered bacterial cytochrome P450s are noted for their ability in the oxidation of inert small alkanes. Cytochrome P450 BM3 L188P A328F (BM3 PF) and A74E L188P A328F (BM3 EPF) variants are able to efficiently oxidize n‐butane to 2‐butanol. Esterification of the 2‐butanol derived from this reaction mediated by the aforementioned two mutants gives diastereomeric excesses (de) of −56±1 and −52±1 %, respectively, with the preference for the oxidation occurring at the C−HS bond. When tailored (2R,3R)‐ and (2S,3S)‐[2‐2H1,3‐2H1]butane probes are employed as substrates for both variants, the obtained de values from (2R,3R)‐[2‐2H1,3‐2H1]butane are −93 and −92 % for BM3 PF and EPF, respectively; whereas the obtained de values from (2S,3S)‐[2‐2H1,3‐2H1]butane are 52 and 56 % in the BM3 PF and EPF systems, respectively. The kinetic isotope effects (KIEs) for the oxidation of (2R,3R)‐[2‐2H1,3‐2H1]butane are 7.3 and 7.8 in BM3 PF and EPF, respectively; whereas KIEs for (2S,3S)‐[2‐2H1,3‐2H1]butanes are 18 and 25 in BM3 PF and EPF, respectively. The discrepancy in KIEs obtained from the two substrates supports the two‐state reactivity (TSR) that is proposed for alkane oxidation in cytochrome P450 systems. Moreover, for the first time, experimental evidence for tunneling in the oxidation mediated by P450 is given through the oxidation of the C−HR bond in (2S,3S)‐[2‐2H1,3‐2H1]butane. Majorly distinct: Enzymatic oxy‐functionalization of tailored (2R,3R)‐ and (2S,3S)‐[2‐2H1,3‐2H1]butanes mediated by cytochrome P450 BM3 variants display two distinct major products, (S)‐ and (R)‐2‐butanols, and kinetic isotope effects (KIEs) of 7.3–7.8 and 18–25, respectively. The findings here experimentally support the two‐state reactivity (TSR) proposed for alkane hydroxylation in cytochrome P450 systems.