The Catalytic Mechanism of Peptidylglycine α-Hydroxylating Monooxygenase Investigated by Computer Simulation

The molecular basis of the hydroxylation reaction of the Cα of a C-terminal glycine catalyzed by
peptidylglycine α-hydroxylating monooxygenase (PHM) was investigated using hybrid quantum-classical
(QM-MM) computational techniques. We have identified the most reactive oxygenated species and presented
new insights into the hydrogen abstraction (H-abstraction) mechanism operative in PHM. Our results suggest
that O2 binds to CuB to generate CuBII−O2•- followed by electron transfer (ET) from CuA to form CuBI−O2•-.
The computed potential energy profiles for the H-abstraction reaction for CuBII−O2•-, CuBI−O2•-, and [CuBII−OOH]+ species indicate that none of these species can be responsible for abstraction. However, the latter
species can spontaneously form [CuBO]+2 (which consists of a two-unpaired-electrons [CuBO]+ moiety
ferromagneticaly coupled with a radical cation located over the three CuB ligands, in the quartet spin ground
state) by abstracting a proton from the surrounding solvent. Both this monooxygenated species and the
one obtained by reduction with ascorbate, [CuBO]+, were found to be capable of carrying out the
H-abstraction; however, whereas the former abstracts the hydrogen atom concertedly with almost no
activation energy, the later forms an intermediate that continues the reaction by a rebinding step. We propose
that the active species in H-abstraction in PHM is probably [CuBO]+2 because it is formed exothermically
and can concertedly abstract the substrate HA atom with the lower overall activation energy. Interestingly,
this species resembles the active oxidant in cytochrome P450 enzymes, Compound I, suggesting that
both PHM and cytochrome P450 enzymes may carry out substrate hydroxylation by using a similar
mechanism.