1. How a cofactor-free protein environment lowers the barrier to O 2 reactivity.
- Author
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Machovina MM, Ellis ES, Carney TJ, Brushett FR, and DuBois JL
- Subjects
- Catalytic Domain, Electron Transport, Mixed Function Oxygenases chemistry, Mixed Function Oxygenases genetics, Models, Molecular, Mutagenesis, Streptomyces enzymology, Temperature, Thermodynamics, Mixed Function Oxygenases metabolism, Nogalamycin metabolism, Oxygen metabolism
- Abstract
Molecular oxygen (O
2 )-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 p Ka by 1.0 unit (Δ G * = 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 ΔΔ G0 ' of 2.0 kcal mol-1 (0.087 eV) and that the activation barrier, Δ G‡ , 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., (© 2019 Machovina et al.)- Published
- 2019
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