From Ferrous HRP to Compound I and Back: The Role of Protein and the Mediators in Reversible Redox Transitions

Abstract

Highly oxidized iron species play key role in the catalysis of many heme and non-heme enzymes due to their ability to activate substrates via electron or hydrogen atom transfer. In Nature, such highly reactivate and unstable states are typically generated by activation and reduction of molecular oxygen by ferrous porphyrin with the ensuing transfer of redox equivalents onto the cofactor and/or protein itself. Oxygen activation is also associated with a variety of side-reactions, including generation of ROS and self-inactivation. This study investigates alternative approaches to sustaining catalytic activity of highly-oxidized heme species via direct oxidation of cofactor-bound solvent ligand under externally applied electric potential. We developed experimental framework that allows to quickly and reversibly traverse a range of oxidation states of heme cofactor between ferrous iron and Compound I while monitoring redox changes spectroscopically in the UV-Vis or IR domains. We investigate the role of redox mediators, their structures and redox potentials on the ability to catalyze electron transfer to and from the active site of horseradish peroxidase. We show that self-catalyzed modification of the protein moiety enhances the rate the electron transfer, eventually enabling sustained redox transitions in the absence of a mediator. Our results also demonstrate the role on intra-protein solvent molecule as an intrinsic reductant, rationalizing spontaneous conversion of Compounds I and II to the ferric state.