Abstract
In this work, a proteolytic digest of cytochrome c (microperoxidase 11, MP-11) was used as a model to study the structural aspects of heme protein interactions and porphyrin networks. The MP-11 structural heterogeneity was studied as a function of the starting pH (e.g., pH 3.1-6.1) and concentration (e.g., 1-50 μM) conditions and adduct coordination. Trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) showed the MP-11 structural dependence of the charge state distribution and molecular ion forms with the starting pH conditions. The singly charged (e.g., [M]+, [M - 2H + NH4]+, [M - H + Na]+ and [M - H + K]+) and doubly charged (e.g., [M + H]2+, [M - H + NH4]2+, [M + Na]2+ and [M + K]2+) molecular ion forms were observed for all solvent conditions, although the structural heterogeneity (e.g., number of mobility bands) significantly varied with the pH value and ion form. The MP-11 dimer formation as a model for heme-protein protein interactions showed that dimer formation is favored toward more neutral pH and favored when assisted by salt bridges (e.g., NH4 +, Na+ and K+ vs. H+). Inspection of the dimer mobility profiles (2+ and 3+ charge states) showed a high degree of structural heterogeneity as a function of the solution pH and ion form; the observation of common mobility bands suggest that the different salt bridges can stabilize similar structural motifs. In addition, the salt bridge influence on the MP-11 dimer formations was measured using collision induced dissociation and showed a strong dependence with the type of salt bridge (i.e., a CE50 of 10.0, 11.5, 11.8 and 13.0 eV was observed for [2M + H]3+, [2M - H + NH4]3+, [2M + Na]3+ and [2M + K]3+, respectively). Measurements of the dimer equilibrium constant showed that the salt bridge interactions increase the binding strength of the dimeric species.