Abstract
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 pKa 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.
Highlights
Molecular oxygen (O2)-utilizing enzymes are among the most important in biology
We found that the oneelectron reduction potential, measured for the deprotonated substrate both inside and outside the protein environment, increases by 85 mV inside Nogalamycin monooxygenase (NMO), corresponding to a ⌬⌬G0 of 2.0 kcal mol؊1 (0.087 eV) and that the activation barrier, ⌬G‡, is lowered by 4.8 kcal mol؊1 (0.21 eV)
Pure WT and mutant NMO were prepared in high yields
Summary
His6-NMO was purified as a homodimer in average yields of 50 mg/liter of culture for WT, 20 mg/liter for the N18A, and 30 mg/liter for the N63A variant. Using the measured values for ⌬G0, ⌬G‡, and Equation 2, the reorganization energy is 40 kcal molϪ1 for the reaction between O2 and dithranolϪ, and 12 kcal molϪ1 for the NMO– dithranolϪ complex (Table 1). NMO acts to lower the activation barrier of rate-limiting electron transfer via a 28 kcal molϪ1 shift in the reorganization energy. low represents the counterintuitive case where the chemical environment lowers ⌬G‡ for electron transfer by increasing the reorganization energy and/or ⌬G0 This inversion is physically possible; the range of ⌬E0 over which a normal (i.e. proportional) relationship between ⌬G‡ and is predicted by Marcus theory is given by the square root of the product of the two solutions for the reorganization energy, i.e. Ϯ(low high).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
