Project Summary/Abstract Enzymatic redox catalysis, imperative to all organisms, is a showcase of nature's mastery in tuning the activity and selectivity at the electronic level. The reduction potential, E0, of the metal performing the redox transformation is precisely controlled beyond the primary coordination sphere through the microenvironment of the metal in the protein. Interactions of consequence for the physiologically relevant modulations of E0 are often weak; they include hydrogen bonds, hydrophobic contacts, and long- to intermediate-range electrostatics. It is a challenge to study the impact of these individual factors on metalloenzymatic redox processes, and even more so to design metalloproteins that would perform selective redox catalysis. We propose an approach that allows elaboration of the individual factors that govern redox properties of metalloproteins en route to the design artificial Co and Mn metalloenzymes with selective oxygen reduction or oxidative reactivity. We propose metalloprotein constructs that combine synthetic redox active complexes of Co and Mn with salen ligands, and the protein streptavidin (Sav) to which biotinylated organometallic complexes will be attached. In these systems, the intermediate-range electrostatics will be controlled largely within the organometallic complex that has a unique feature, a secondary metal binding site containing a redox innocent metal ion exerting an electric field on the active cation. Electrochemistry and reactivity of these complexes will be measured and computed. The protein matrix of Sav will be used for incorporating other weak interactions in the microenvironment of the metal, such as H-bonds and hydrophobic contacts, through mutagenesis. Mixed quantum mechanical and quantum-classical simulations will guide the choice for mutations to consider for the desired redox activity. Importantly, while in natural metalloenzymes all the weak factors influencing E0 also influence the protein structure and couple to each other, in the proposed systems, they are decoupled to the extent possible, and thus more amendable to studying and strategic modifications. Broadly, this research will allow learning how redox chemistry is controlled in nature, and how to approach the design of redox enzymes. Our target catalytic reactions of oxygen reduction and aerobic substrate oxidation are of interest to both natural enzymology and the broader field of catalysis, and understanding these reactions is essential for realizing how aerobic metabolism efficiently utilizes oxygen while avoiding the formation deleterious concentrations of ROS.

Project Narrative Redox activity of metalloenzymes is critical for survival of all organisms, including humans, while the ways by which nature tunes their functionality so precisely remain elusive. For example, the target catalytic reactions in this proposal are oxygen reduction, related to aerobic metabolism and efficient oxygen utilization while avoiding the formation of toxic reactive oxygen species. We plan to elucidate the various factors in proteins that impact the redox behavior of the metal sites, through the use of artificial organometallic-protein constructs, and a combination of tools of synthetic chemistry, analytical physical measurements, multiscale simulations, and protein mutagenesis.