Human heme oxygenase (hHO-1) is an enzyme that degrades heme through a multi-step reaction into biliverdin with the release of CO molecule and ferrous iron. Oxidation of the heme by hHO-1 is specific to the α - meso carbon and is facilitated by a hydrogen bond network cluster that comprises the aspartate 140 residue and two water molecules present in the distal heme active site. The mechanism of heme degradation is complex and has been actively debated. It has been proposed that the reactive intermediates that catalyze the first step, i.e. the α – meso hydroxylation, are the following species:  ferric hydroperoxo intermediate, hydroxide / ferryl π cation radical (Compound I) or hydroxyl radical / ferryl heme (Compound II). All these are fast decaying intermediates; hence they present a challenge in regard to their structural characterization. To overcome this, we utilized a combination of cryoreduction and resonance Raman spectroscopy to cryogenically generate and characterize these intermediates. In this work we present spectroscopic studies of reactive intermediates of the wild type (WT) hHO-1 and its D140 and G139 mutants.  The modes associated with the Fe-O-O fragments, such as ν(Fe−O2) and ν(O-O) stretching modes were identified based on oxygen isotopic substitution and H2O/D2O exchange. This structural characterization of reactive intermediates provides unique insight into oxygen activation chemistry which is a critical function of a wide variety of dioxygen utilizing enzymes.

Resonance Raman studies of the oxygenated species involved in heme hydroxylation

ACS Spring 2022

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