The catalytic mechanism of peptidylglycine α-hydroxylating monooxygenase investigated by computer simulation

The molecular basis of the hydroxylation reaction of the C alpha of a C-terminal glycine catalyzed by peptidylglycine alpha-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 O-2 binds to Cu-B to generate Cu-B(II)-O-2(center dot)-followed by electron transfer (ET) from Cu-A to form Cu-B(I)-O-2(center dot-). The computed potential energy profiles for the H-abstraction reaction for Cu-B(II)-O-2(center dot-), Cu-B(I)-O-2(center dot-), and [Cu-B(II)-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.