Alternative modes of O2 activation in P450 and NOS enzymes are clarified by DFT modeling and resonance Raman spectroscopy.

The functions of heme proteins are modulated by hydrogen bonds (H-bonds) directed at the heme-bound ligands by protein residues. When the gaseous ligands CO, NO, or O2 are bound, their activity is strongly influenced by H-bonds to their atoms. These H-bonds produce characteristic changes in the vibrational frequencies of the heme adduct, which can be monitored by resonance Raman spectroscopy and interpreted with density functional theory (DFT) computations. When the protein employs a cysteinate proximal ligand, bound O2 becomes particularly reactive, the course of the reaction being controlled by H-bonding and proton delivery. In this work, DFT modeling is used to examine the effects of H-bonding to either the terminal (Ot) or proximate (Op) atom of methylthiolate-Fe(II)porphine-O2, as well as to the thiolate S atom. H-bonds to Op produce a positive linear correlation between ν(Fe - O) and ν(O - O), because they increase the sp2 character of Op, weakening both the Fe - O and O - O bonds. H-bonds to Ot produce a negative correlation, because they increase Fe backbonding, strengthening the Fe - O but weakening the O - O bond. Available experimental data accommodate well to the computed pattern. In particular, this correspondence supports the interpretation of cytochrome P450 data by Kincaid and Sligar [M. Gregory, P.J. Mak, S.G. Sligar, J.R. Kincaid, Angew. Chem. Int. Ed. 125 (2013) 5450-5453], involving steering between hydroxylation and lyase reaction channels by differential H-bonds. Similar channeling between the first and second steps of the nitric oxide synthase reaction is likely.