The flavoenzyme fructosyl amino acid oxidase (FAOD) catalyzes the oxidative deglycation of fructosyl amino acids, model compounds of glycated proteins. The high oxygen reactivity of FAODs limits their potential utility in amperometric enzyme sensors employing artificial electron mediators. To alter their electron acceptor availability, site-directed mutagenesis was carried out on conserved residues predicted to be involved in the proton relay system (PRS) of two eukaryotic FAODs, the FAOD from the marine yeast Pichia sp. N1-1 and amadoriase II from the fungus Aspergillus fumigatus. The substitution of a single conserved Asn residue in the putative PRS, Asn47Ala of N1-1 FAOD and Asn52Ala of amadoriase II, resulted in significant loss in the catalytic ability to employ O-2 as the electron acceptor, while having little effect on the dye-mediated dehydrogenase activity employing artificial electron acceptors instead of O-2.