Insights Into How Heme Reduction Potentials Modulate Enzymatic Activities of a Myoglobin-based Functional Oxidase

Heme-copper oxidase (HCO) is a class of respiratory enzymes that use a heme-copper center to catalyze O₂ reduction to H₂O. While heme reduction potential (E°′) of different HCO types has been found to vary >500 mV, its impact on HCO activity remains poorly understood. Here, we use a set of myoglobin-based functional HCO models to investigate the mechanism by which heme E°′ modulates oxidase activity. Rapid stopped-flow kinetic measurements show that increasing heme E°′ by ca. 210 mV results in increases in electron transfer (ET) rates by 30-fold, rate of O₂ binding by 12-fold, O₂ dissociation by 35-fold, while decreasing O₂ affinity by 3-fold. Theoretical calculations reveal that E°′ modulation has significant implications on electronic charge of both heme iron and O₂, resulting in increased O₂ dissociation and reduced O₂ affinity at high E°′ values. Overall, this work suggests that fine-tuning E°′ in HCOs and other heme enzymes can modulate their substrate affinity, ET rate and enzymatic activity.