How a cofactor-free protein environment lowers the barrier to O 2 reactivity

Molecular oxygen (O )-utilizing enzymes are among the most important in biology. The abundance of O , its thermodynamic power, and the benign nature of its end products have raised interest in oxidases and oxygenases for biotechnological applications. Although most O -dependent enzymes have an absolute requirement for an O -activating cofactor, several classes of oxidases and oxygenases accelerate direct reactions between substrate and O using only the protein environment. Nogalamycin monooxygenase (NMO) from is a cofactor-independent enzyme that catalyzes rate-limiting electron transfer between its substrate and O Here, using enzyme-kinetic, cyclic voltammetry, and mutagenesis methods, we demonstrate that NMO initially activates the substrate, lowering its p by 1.0 unit (Δ * = 1.4 kcal mol ). We found that the one-electron reduction potential, measured for the deprotonated substrate both inside and outside the protein environment, increases by 85 mV inside NMO, corresponding to a ΔΔ ' of 2.0 kcal mol (0.087 eV) and that the activation barrier, Δ , is lowered by 4.8 kcal mol (0.21 eV). Applying the Marcus model, we observed that this suggests a sizable decrease of 28 kcal mol (1.4 eV) in the reorganization energy (λ), which constitutes the major portion of the protein environment's effect in lowering the reaction barrier. A similar role for the protein has been proposed in several cofactor-dependent systems and may reflect a broader trend in O -utilizing proteins. In summary, NMO's protein environment facilitates direct electron transfer, and NMO accelerates rate-limiting electron transfer by strongly lowering the reorganization energy.