Mutational dissection of a hole hopping route that can be tuned to boost peroxygenase activity in LPMOs

Abstract Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial lytic polysaccharide monooxygenase (LPMO), member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a Tyr-His pair in the protein’s surface. Real-time monitoring of radical formation revealed a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a mutant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.