AbstractTo elucidate the effect of various amino add residues on the heme‐binding pocket in cytochrome Tb5, several residues were chosen for replacement by means of site‐directed mutagenesis. Comparison of the mass spectrum between the F35Y mutant and the wild type shows that the relative abundance of holoprotein ion of F35Y is lower than that of the wild type in gas phase. It is concluded that mutation from Phe35 residue to tyrosine decreases the hydrophobic character of cytochrome Tb5 heme pocket, which decreases the stability of heme‐binding pocket. ESI‐MS spectra of the mutants V61E, V61K, V61H and V61Y show various contribution of amino acid to the stability of heme‐binding pocket. The small and non‐polar residue Val61 was replaced with large or polar residues, resulting in enhancing the trend of heme leaving from the pocket. In addition, comparison of the mass relative abundance of holo‐proteins among all the Vakil‐mutants, shows mat their stability in gas phase appropriately submit the following order: wild type > V61H > V61E > V61K ≈︁ V61Y. The extra great stability of quadruple sites mutant E44/48/56A/D60A shows that reduction of electrostatic or hydrogen bond interactions among the residues locating in the outside region of the heme edge remarkably affect the stability of heme. The results of analyzing the oxidation states of heme iron in Tb5 and its mutants by insource‐CAD experiment suggest that the charge states of heme iron Maintain inflexible in mutation process.