Molecular Oxygen Activation and Proton Transfer Mechanisms in Lanosterol 14α-Demethylase Catalysis

The CYP51 lanosterol 14 alpha-demethylases are evolutionarily ancient enzymes ubiquitously distributed throughout the biological domains. The experimental X-ray crystal structure of Mycobacterium tuberculosis (Mtb) CYP51 is the first of an enzyme capable of catalyzing inert C-C bond cleavage. Amino acid sequence comparisons of CYP51 family members with other members of the CYP superfamily reveal the almost universally conserved acid-alcohol pair, putatively involved in proton transport and O-2 activation, is replaced with a His-Thr dyad. In this study, extended molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics calculations (QM/MM) are applied to characterize reactive oxygen intermediates and to unravel mechanisms of O-2 activation vis-a-vis proton transport for this important enzyme. MD confirms stable His259 delta H+-Thr260OH-O-2 (Mtb numbering) hydrogen bonding early in the simulations, suggesting these amino acids could function similarly to the Asp251-Thr252 pair in CYP101. QM/MM calculations support this dyad competently catalyzes the peroxo to Compound 0 (Cmpd 0) reaction, albeit an endothermic homolytic. O-O scission mechanism affording Compound I (Cmpd 1) was identified. Disruption of the His259H(+)-Thr260OH hydrogen bond in MD simulation divulges a second previously unidentified hydrogen-bond network, including three water molecules linking Glu 173 in the CYP51 F-helix to the distal O-2 atom. Expansion of the QM region to contain these atoms unveils an unprecedented triradicaloid electronic structure of the peroxo intermediate characterized by spin polarization to the Glu173 side chain, attributable to the protein electrostatic environment. This amino acid, in concert with an active-site water network, catalyzes a facile protonation of the peroxo intermediate and offers a series of redundant heterolytic and homolytic mechanisms, affording exothermic formation of the ultimate oxidant Cmpd I. In summary, this study highlights the importance of the protein electrostatic environment to tune the electronic structure of CYP catalytic intermediates in addition to Cmpd I and illustrates the diversity of proton transport pathways available to these enzymes to drive catalysis.