Mnemiopsin 2 from Mnemiopsis leidy has three Ca2+-binding motifs and has luminescence properties in the presence of calcium and coelenterazine. It has been reported that the pattern of calcium binding among EF-hand loops of various photoproteins is different. Here, we designed and constructed two variants of mnemiopsin 2 (E50G/D47N and E50G/E53T mutants) with modified EF-hand I in which the negative charge in the first loop was reduced. According to the activity measurements, the initial intensity of mutants decreases; while the decay rate increases in E50G/D47N. We concluded that the presence of negative charge at positions 47 and 53 of mnemiopsin 2 is critical for both calcium coordination and the interaction of the substrate with the core structure of mnemiopsin 2. Structural studies accompanied by equilibrium denaturation experiments were also performed and it was found that negative charges at the aforementioned positions also have structural consequences which can affect the conformational stability of photoproteins.