NADPH-cytochrome c oxidoreductase from the mosquito Anopheles minimus lacking the first 55 amino acid residues was expressed in Escherichia coli. The purified enzyme loses FMN, leading to an unstable protein and subsequent aggregation. To understand the basis for the instability, we constructed single and triple mutants of L86F, L219F, and P456A, with the first two residues in the FMN domain and the third in the FAD domain. The triple mutant was purified in high yield with stoichiometries of 0.97 FMN and 0.55 FAD. Deficiency in FAD content was overcome by addition of exogenous FAD to the enzyme. Both wild-type and the triple mutant follow a two-site Ping-Pong mechanism with similar kinetic constants arguing against any global structural changes. Analysis of the single mutants indicates that the proline to alanine substitution has no impact, but that both leucine to phenylalanine substitutions are essential for FMN binding and maximum stability of the enzyme.