How a cofactor-free protein environment lowers the barrier to O₂ reactivity

Molecular oxygen (O₂)-utilizing enzymes are among the most important in biology. The abundance of O₂, its thermodynamic power, and the benign nature of its end products have raised interest in oxidases and oxygenases for biotechnological applications. Although most O₂-dependent enzymes have an absolute requirement for an O₂-activating cofactor, several classes of oxidases and oxygenases accelerate direct reactions between substrate and O₂ using only the protein environment. Nogalamycin monooxygenase (NMO) from Streptomyces nogalater is a cofactor-independent enzyme that catalyzes rate-limiting electron transfer between its substrate and O₂ Here, using enzyme-kinetic, cyclic voltammetry, and mutagenesis methods, we demonstrate that NMO initially activates the substrate, lowering its pK by 1.0 unit (ΔG* = 1.4 kcal mol^-1). 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 ΔΔG⁰' of 2.0 kcal mol^-1 (0.087 eV) and that the activation barrier, ΔG^‡, is lowered by 4.8 kcal mol^-1 (0.21 eV). Applying the Marcus model, we observed that this suggests a sizable decrease of 28 kcal mol^-1 (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 O₂-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.