Oxygen activation by homoprotocatechuate 2,3-dioxygenase: a QM/MM study reveals the key intermediates in the activation cycle

Oxygen activation by homoprotocatechuate 2,3-dioxygenase (HPCD) was investigated by quantum mechanical/molecular mechanical (QM/MM) calculations. Our results demonstrated that the semiquinone substrate radical-Fe-II-superoxo (SQ(center dot)-Fe-II-O-2(center dot)) and the corresponding Fe-III-superoxo species are both present within the protein environment. Moreover, we also located a species, which possesses a hybrid SQ(center dot)-Fe-II-O-2(center dot-)/Fe-III-O-2(center dot-) character (so-called hybrid state) with a hydrogen bond between His200 and the proximal oxygen. His200 was found to play an important role in controlling the electronic configuration of the superoxide species. A mere reorientation of the hydrogen bonding donated by His200, from its interaction with the substrate's oxygen to interaction with the proximal oxygen of the dioxygen moiety, causes a fast rearrangement from Fe-III-superoxo to the hybrid state with partial electron transfer from the substrate to the Fe center. Since the hybrid state reacts further with a low barrier, then during the oxidation process all the Fe-III-superoxo species are converted to the hybrid state, which is consumed rapidly by the substrate oxidation process. This theoretical result agrees quite well with the mechanism proposed in previous experimental investigation by Lipscomb et al., Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 16788-16793, where the Fe-III-O-2(center dot-) was suggested to be able to convert to the true reactive species, the SQ(center dot)-Fe-II-O-2(center dot) species, rapidly with one-electron transfer from the substrate to iron.