43763-AC4
Probing the Mechanism of 2-Nitropropane Dioxygenase: A Model for Flavin Semiquinone Intermediates in Catalysis
2-Nitropropane dioxygenase (2NPD; E.C. 1.13.11.32) catalyzes the oxidative denitrification of nitroalkanes to their corresponding carbonyl compounds and nitrite. Both Neurospora crassa and Hansenula mrakii 2NPD have been demonstrated to stabilize an anionic flavin semiquinone during turnover. The formation of a transient flavin semiquinone during catalysis is an unusual feature of 2NPD that distinguishes these enzymes from other flavin-dependent enzymes. Thus, the enzyme serves as suitable model system to understand the reactivity of flavin semiquinone intermediates in enzymatic catalysis.
The biochemical properties of 2NPD from N. crassa and H. mrakii have been characterized. The enzymes are similar in their biochemical properties in that they both contain a non-covalently bound flavin mononucleotide cofactor in a 1:1 stoichiometry and are devoid of iron. Anaerobic substrate reductions and direct measurements of the reductive half reactions of each enzyme have revealed that substrate oxidation occurs through a single electron transfer reaction between an enzyme bound nitronate and the flavin cofactor. N. crassa 2NPD can effectively utilize both the neutral (nitroalkane) and anionic (nitronate) forms of substrate, whereas the H. mrakii enzyme is active only with the nitronate form.
Extensive investigation of the pH and kinetic isotope effects on the reaction catalyzed by N. crassa 2NPD have been carried out to establish that the enzyme utilizes a branched catalytic mechanism. Branching occurs at an enzyme-nitronate complex and involves the partitioning of this intermediate between oxidative and nonoxidative pathways.
Measurements of the secondary deuterium kinetic isotope effects with [1-2H]ethylnitronate as substrate for the enzyme have demonstrated that at low pH a fraction of the nitronate is protonated to form nitroethane as a product of nonoxidative turnover, rather than undergoing oxidative catalysis. A quantitative assay for ethylnitronate formation has also been developed and applied to the reaction of N. crassa 2NPD with nitroethane as substrate. This has demonstrated that branching also occurs with neutral substrates after an initial proton abstraction reaction between an enzymatic base and the nitroalkane to generate an enzyme-nitronate intermediate. This intermediate partitions between nitronate release and flavin reduction to create the branch point in the catalytic mechanism of the enzyme.
Mutagenesis studies have revealed that the catalytic base of N. crassa 2NPD with neutral substrates is histidine 196. This was demonstrated through a lack of either oxygen consumption or nitronate formation when nitroethane was used as substrate for a H196N form of the enzyme. The H196N form of the enzyme is a better catalyst for oxidative denitrification of ethylnitronate as shown through a comparison of the kinetic parameters of the mutant and wild-type 2NPD obtained by measuring rates of oxygen consumption. The increase in activity is likely due to the abolishment of the nonoxidative pathway upon mutation as suggested by a lack of secondary kinetic isotope effects with [1-2H]ethylnitronate as substrate for H196N 2NPD. An anionic flavosemiquinone was observed upon anaerobic mixing of the H196N variant with ethylnitronate suggesting that histidine 196 is not required for the reductive half reaction of the enzyme.
Current studies are aimed at investigating the role of branching on the observed kinetic isotope effect of N. crassa 2NPD with nitroethane as substrate. The primary kinetic isotope effects with [1,1-2H2]nitroethane as substrate measured by following nitronate formation are larger than those determined previously by monitoring oxygen consumption. Analytical expressions for the kinetic parameters of the enzyme differ according to which method is used to assay activity and can be used to determine how the enzyme partitions between oxidative and nonoxidative turnover. An analysis of the kinetic isotope effects obtained in the pH independent regions reveal that an isotope effect on branching modulates the observed value of the overall kinetic isotope effects measured by each assay. The insights gained from the study of the kinetic isotope effects of the 2NPD catalyzed reaction with nitroethane as substrate are generally applicable to any enzymatic reaction occurring with branching of intermediates during turnover.
Investigations of the reductive half reactions of both the N. crassa and H. mrakii enzymes are also currently being carried out using anaerobic stopped-flow spectrophotometery. The pH dependence of the reductive half reaction of N. crassa 2NPD has suggested that a positive charge in the active site of the enzyme acts as an electrostatic catalyst for anionic flavin semiquinone formation. In addition, measurements of both the reductive half reactions and the steady-state kinetic parameters of the H. mrakii 2NPD suggest that the single electron transfer reaction is rate-limiting for overall turnover of the enzyme. Future experiments are planned to probe the reactivity of the anionic flavosemiquinone formed during catalysis with molecular oxygen. These results will be used in conjunction with that obtained for the reductive half reaction to provide a comprehensive understanding of the role of the anionic flavin semiquinone in catalysis by 2NPD.
