Abstract
Molecular oxygen (O(2))-utilizing enzymes are among the most important in biology. The abundance of O(2), its thermodynamic power, and the benign nature of its end products have raised interest in oxidases and oxygenases for biotechnological applications. Although most O(2)-dependent enzymes have an absolute requirement for an O(2)-activating cofactor, several classes of oxidases and oxygenases accelerate direct reactions between substrate and O(2) 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 O(2) Here, using enzyme-kinetic, cyclic voltammetry, and mutagenesis methods, we demonstrate that NMO initially activates the substrate, lowering its pK(a) 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 ΔΔG(0)' 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 O(2)-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.