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J. Biol. Chem., Vol. 280, Issue 7, 5195-5204, February 18, 2005

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Involvement of a Flavosemiquinone in the Enzymatic Oxidation of Nitroalkanes Catalyzed by 2-Nitropropane Dioxygenase^*

Kevin Francis, Bethany Russell, and Giovanni Gadda¶||

From the Departments of Chemistry and Biology and ^¶The Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia 30302-4098

2-Nitropropane dioxygenase (EC 1.13.11.32) catalyzes the oxidation of nitroalkanes into their corresponding carbonyl compounds and nitrite. In this study, the ncd-2 gene encoding for the enzyme in Neurospora crassa was cloned, expressed in Escherichia coli, and the resulting enzyme was purified. Size exclusion chromatography, heat denaturation, and mass spectroscopic analyses showed that 2-nitropropane dioxygenase is a homodimer of 80 kDa, containing a mole of non-covalently bound FMN per mole of subunit, and is devoid of iron. With neutral nitroalkanes and anionic nitronates other than propyl-1- and propyl-2-nitronate, for which a non-enzymatic free radical reaction involving superoxide was established using superoxide dismutase, substrate oxidation occurs within the enzyme active site. The enzyme was more specific for nitronates than nitroalkanes, as suggested by the second order rate constant k_cat/K_m determined with 2-nitropropane and primary nitroalkanes with alkyl chain lengths between 2 and 6 carbons. The steady state kinetic mechanism with 2-nitropropane, nitroethane, nitrobutane, and nitrohexane, in either the neutral or anionic form, was determined to be sequential, consistent with oxygen reacting with a reduced form of enzyme before release of the carbonyl product. Enzyme-monitored turnover with ethyl nitronate as substrate indicated that the catalytically relevant reduced form of enzyme is an anionic flavin semiquinone, whose formation requires the substrate, but not molecular oxygen, as suggested by anaerobic substrate reduction with nitroethane or ethyl nitronate. Substrate deuterium kinetic isotope effects with 1,2-[²H₄]nitroethane and 1,1,2-[²H₃ ethyl nitronate at pH 8 yielded normal and inverse effects on the k_cat/K_m value, respectively, and were negligible on the k_cat value. The k_cat/K_m and k_cat pH profiles with anionic nitronates showed the requirement of an acid, whereas those for neutral nitroalkanes were consistent with the involvement of both an acid and a base in catalysis. The kinetic data reported herein are consistent with an oxidasestyle catalytic mechanism for 2-nitropropane dioxygenase, in which the flavin-mediated oxidation of the anionic nitronates or neutral nitroalkanes and the subsequent oxidation of the enzyme-bound flavin occur in two independent steps.

Received for publication, October 1, 2004 , and in revised form, November 5, 2004.

^* This work was supported in part by Grant PRF 37351-G4 from the American Chemical Society (to G. G.), a Research Initiation Grant and a Quality Improvement Fund from Georgia State University (to G. G.), and a McNair Research Fellowship (to K. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. of Chemistry, GA State University, P.O. Box 4098, Atlanta, GA 30302-4098. Tel.: 404-651-4737; Fax: 404-651-1416; E-mail: ggadda{at}gsu.edu.

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