A Radical Addition–Diradical Coupling Switch Unifies Cytochrome P450-Catalyzed Diketopiperazine Dimerization

Abstract Cytochrome P450 dimerases generate the remarkable structural diversity of dimeric diketopiperazine (DKP) natural products through highly selective intermolecular radical-mediated reactions. Although radical addition mechanisms have been established for most bacterial DKP dimerization modes, the molecular basis underlying the formation of the unusual C3–N1′ linkage has remained unresolved, preventing a unified mechanistic understanding of P450-catalyzed DKP dimerization. Here, we combine structure-guided protein engineering, X-ray crystallography, biochemical characterization, molecular dynamics simulations, and QM/MM calculations to elucidate the mechanism of the C3–N1′ dimerase using an engineered soluble surrogate derived from its C3–C3′ homolog TtpB1. We show that C3–N1′ bond formation proceeds through a previously unrecognized diradical coupling mechanism, in which sequential hydrogen atom abstraction generates discrete N10- and N1′-centered radicals prior to intramolecular cyclization and intermolecular radical coupling. This pathway is fundamentally distinct from the radical addition mechanism employed by all previously characterized bacterial DKP dimerases. Guided by these mechanistic insights, we further reprogram a single P450 scaffold to catalyze four distinct intermolecular coupling modes, including native C3–C3′ and engineered C3–N1′, N1–C7′, and C3–C7′ linkages. Together, these findings reveal that divergent DKP dimerization is governed by a tunable radical addition–diradical coupling switch operating on a conserved substrate-binding architecture, establishing a unified mechanistic framework for cytochrome P450-catalyzed DKP dimer biosynthesis and providing a blueprint for engineering new radical-mediated oxidative coupling reactions.