Nitroalkane oxidase (NAO) catalyzes the oxidation of neutral nitroalkanes to aldehydes and ketones with oxygen consumption and the production of hydrogen peroxide and nitrite. The enzyme is a flavoprotein from the fungus Fusarium oxysporum. The active site base, Asp402, abstracts one proton from the substrate to give a carbanion which then attacks the flavin adenine dinucleotide (FAD). The three dimensional crystal structure of NAO shows that Arg409 is 3.6 Å from Asp402. When Arg409 is mutated to Lys, the rate constant for proton abstraction decreases 100-fold. The three-dimensional structure of NAO also reveals the existence of a tunnel which extends from the protein exterior and terminates at the FAD N5 atom and the residues Asp402 and Phe401. We mutated amino acids in the tunnel into tryptophan, phenylalanine and leucine. The L99W, S276W and S276A enzymes showed the biggest decreases in both kcat and kcat/Km; these amino acids are closest to the FAD molecule and the active site. Mutation of amino acids farther away from the active site showed very small changes in the kinetic parameters. Ser276 is hydrogen bonded to Asp402 in the wild-type enzyme. When this amino acid is mutated to alanine or tryptophan, k3, the rate constant for proton abstraction, decreases around 35 fold. Asp402, Arg409 and Ser276 constitute a catalytic triad in the active site of nitroalkane oxidase, and both Arg409 and Ser276 are important for positioning Asp402 and catalysis.