Nature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity. Understanding how different local metal-oxo coordination environments control intermediate reactivity and catalytic function is a long-standing objective. However, conducting structure-activity relationships directly in active sites has proven challenging due to the limited range of amino acid substitutions achievable within the constraints of the genetic code. Here, we use an expanded genetic code to examine the impact of hydro-gen bonding interactions on ferryl heme structure and reactivity, by replacing the N-H group of the active site Trp51 of cyto-chrome c peroxidase by an S atom. Removal of a single hydro-gen bond stabilizes the porphyrin π-cation radical state of CcP W191F compound I. In contrast, this modification leads to more basic and reactive neutral ferryl heme states, as found in CcP W191F compound II and the wildtype ferryl heme-Trp191 radical pair of compound I. This increased reactivity manifests in a >60-fold activity increase towards phenolic substrates but remarkably has negligible effects on oxidation of the biological redox partner cytc. Our data highlight how Trp51 tunes the life-times of key ferryl intermediates and works in synergy with the redox active Trp191 and a well-defined substrate binding site to regulate catalytic function. More broadly, this work shows how non-canonical substitutions can advance our understanding of active site features governing metal-oxo structure and reactivity.
|Publication status||Accepted/In press - 23 Apr 2021|
Research Beacons, Institutes and Platforms
- Manchester Institute of Biotechnology