Dynamic Metal--Metal Distance Modulation Controls Oxygen Activation in Non-heme Diiron Enzymes

Abstract

Multinuclear metal centers catalyze some of the most challenging oxidative transformations in biological and chemical catalysis, yet the molecular principles controlling oxygen activation in non-heme diiron enzymes remain unclear. Structural studies of desaturase-like diiron enzymes frequently reveal elongated Fe-Fe separations lacking canonical bridging motifs, whereas spectroscopic measurements indicate substantial metal-metal coupling during catalysis, creating a longstanding mechanistic paradox between structural and spectroscopic observations. Here we show that oxygen activation can be dynamically regulated by fluctuations in metal-metal distance through extensive multiscale simulations of the membrane-bound fatty-acid decarboxylase UndB. UndB catalyses the conversion of naturally abundant free fatty acids into terminal 1-alkenes-valuable hydrocarbon precursors for sustainable biofuels, and exhibits unusual hydrogen peroxide formation that has led to conflicting mechanistic proposals. Combining quantum mechanics/molecular mechanics (QM/MM) simulations with free energy analysis and quantum chemical calculations, we identify a catalytically competent ensemble in which transient Fe-Fe contraction establishes effective metal-metal coupling and enables formation of a peroxodiiron(III/III) intermediate despite elongated resting-state structures and the absence of canonical bridging motifs. The computed free-energy landscape quantitatively reproduces experimentally inferred catalytic barriers and accounts for the substantial H2O2 formation observed during catalysis. The reactive state resembles a P-like intermediate rather than the canonical diamond-core Q species commonly invoked for related diiron enzymes. Comparison with magnetic coupling and mechanistic features reported for related non-heme diiron enzymes, including Alkane monooxygenase AlkB, Stearoyl-CoA Desaturase 1 (SCD1) and the soluble decarboxylase UndA, suggests that dynamic metal-metal distance modulation represents a general mechanism governing oxygen activation across diverse diiron enzyme families. These findings establish metal-metal distance modulation as a previously unrecognized control parameter for oxygen activation in diiron and related multinuclear metalloenzymes, reconciling structural and spectroscopic observations and revealing how protein conformational dynamics regulate electronic coupling to control oxidative catalysis, thereby suggesting a general principle for tuning reactivity in multinuclear catalysts.