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The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrification of a broad range of primary and secondary nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. With nitroethane as substrate the D2O(k(cat)/K(M)) value is 0.6 and the D2Ok(cat) value is 2.4. The k(cat) proton inventory is consistent with a single exchangeable proton in flight, while the k(cat)/K(M) is consistent with either a single proton in flight in the transition state or a medium effect. Increasing the solvent viscosity did not affect the k(cat) or k(cat)/K(M) value significantly, establishing that nitroethane binding is at equilibrium and that product release does not limit k(cat)., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)