Mechanisms and Control of O2 and C–H Activation within "Privileged" Enzyme Architectures

/Abstract Enzymes that activate dioxygen or (su)peroxide at transition metal cofactors to cleave strong X– H bonds (X = C/N/O) are prevalent in central and specialized metabolic pathways in organisms from all three domains of life. Their abilities to generate highly reactive carbon and heteroatom radicals and direct them down specific reaction pathways bestow a chemical versatility that inspires admiration and mimicry among synthetic chemists. Within several highly represented (privileged) protein architectures, Nature has evolved diverse reactivities from nearly identical cofactors and common reactivities from structurally distinct cofactors, including cofactors made up of different metal ions. The three classes of metalloenzymes under study in the proposed program exemplify these phenomena. Iron(II) and 2-oxoglutarate-dependent (Fe/2OG) oxygenases (Project 1) use a common iron(II) cofactor and oxoiron(IV) intermediate to promote hydroxylation, halogenation, desaturation, chain/ring-expansion, and cyclization reactions. Heme- oxygenase like diiron enzymes (HDOs) (Project 2) use unstable diiron(II) clusters that are (dis)assembled in concert with profound protein conformational changes to form µ- (hydro)peroxodiiron(III) intermediates that cleave strong X–H bonds to initiate N-oxygenation and complex fragmentation reactions. Class I ribonucleotide reductases (RNRs) (Project 3) use diverse diiron, manganese/iron, dimanganese, or flavin cofactors to form protein radicals that initiate – again, by cleaving a C–H bond – the common reduction reaction that provides all organisms with DNA precursors. The proposed program aims to use biochemical, biophysical and computational methods to understand (1) how largely conserved protein scaffolds can initiate different reactivities from similar cofactors or a common outcome from diverse cofactors and (2) how these diverse outcomes and cofactor usage have evolved. The emerging understanding will be tested in experiments aiming either to rationally redirect reactions to different outcomes (Projects 1 and 2) or to alter cofactor usage (Project 3) and will ultimately be useful in formulating directed evolution of new-to-nature reactivities. Project Number: 1R35GM161700-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: JOSEPH BOLLINGER | Institution: PENNSYLVANIA STATE UNIVERSITY, THE, UNIVERSITY PARK, PA | Award Amount: $750,554 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - E Study Section[MRAE] View on NIH RePORTER: https://reporter.nih.gov/project-details/11260901