Atypical profiles and modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest diffusible radicals' obligatory involvement in such redox reactions.

Background: Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj et al., 2010; Andrew et al., 2011, Parashar et al., 2014 & Venkatachalam et al., 2016].

Methods: A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes.

Results: When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equivalent concentrations of diverse redox-active additives and this is owing to multiple redox equilibriums in the milieu. At low enzyme and peroxide concentrations, the enzyme is seen to recycle via a one-electron (oxidase) cycle, which does not require the substrate to access the heme centre. Schemes are provided that explain the complex mechanistic cycle, kinetics & stoichiometry.

Conclusion: It is not obligatory for an inhibitor or substrate to interact with the heme centre for influencing overall catalysis. Roles of diffusible reactive species explain catalytic outcomes at low enzyme and reactant concentrations.

Significance: The current work highlights the scope/importance of redox enzyme reactions that could occur "out of the active site" in biological or in situ systems.