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HPLC analysis of the NADH/O2‐driven oxidation of 4‐methylthiobenzoic acid by T252E (black) and WT CYP199A4 (blue). In red is a control reaction omitting the P450. 4‐Methylsulfinylbenzoic acid elutes at 4.7 min and 4‐mercaptobenzoic acid at 12.5 min. The substrate appears at 13.6 min. Negligible levels of 4‐mercaptobenzoic acid were detected. No sulfone was detected. Gradient: 20–95 % acetonitrile in H2O with 0.1 % TFA. Detection wavelength: 254 nm.
