Three-dimensional structure model of azoreductase AZR of Rhodobacter sphaeroides was constructed using homology modeling method. It is a flavodoxin adopting a/b structure. Structure alignment of two different types of flavin-dependent azoreductases revealed that they possessed high similarity. Based on sequence and structure analysis, site-directed mutagenesis of K109H and K109A were performed. The optimal pH values are pH 6 and pH 9 for K109H and K109A mutant protein, respectively. The optimal temperature (30℃) is not affected by mutagenesis. Positively charged residues at position 109 is necessary for the binding of methyl red, while K109H is not a conserved mutagenesis for the binding of NADPH. K109 may only be involved in the binding of the 2’-phosphate group of NADPH and have no effect on the binding of NADH.