Modulation of heme peroxo nucleophilicities with axial ligands reveal key insights into the mechanistic landscape of nitric oxide synthase.

Mid-valent heme-oxygen intermediates are central to a medley of pivotal physiological transformations in humans, and such systems are increasingly becoming more relevant therapeutic targets for challenging disease conditions. Nonetheless, precise mechanistic details pertaining to mid-valent heme intermediates as well as key structure-activity relationships remain enigmatic. To this end, this study strives to describe the influence of heme proximal ligation on the nucleophilic reactivity patterns of heme peroxo intermediates. A functional model system in which organic oxime substrates are used as -hydroxy-l-arginine mimics reproduces the second mechanistic step of nitric oxide synthase. Our findings reveal that axial ligation of heme peroxo adducts escalates the rates of nucleophilic reactivity, wherein the anionic ligands exhibited the most pronounced "push effect". Coordination of these axial ligands are accompanied by distinct geometric and electronic perturbations, which are supported by complementary theoretical studies. Kinetic interrogations reveal that the axially ligated heme peroxo adducts presumably mediate oxime oxidation the same mechanism as the parent (, with only solvent ligation) heme peroxo adduct, where the initial nucleophilic attack from the peroxo moiety on the oxime substrate is rate-limiting. All reaction products, including the final ketone as well as NO, have been characterized in detail.