The protein stability of the Met16Leu pseudoazurin (PAz) variant was examined by electrospray ionization mass spectrometry (ESI-MS), and the protein stability was similar to that of WT, which has an S-pi interaction in the second coordination sphere. The stability of Met16Leu PAz was remarkably higher than that of the aliphatic amino acid variants Met16Val and Met16Ile PAz. The CH-pi interactions between the Cu-coordinated His81 imidazole ring and the two methyl groups of the substituted leucine residue acts to stabilize the protein.