Dissociation of Heme from Myoglobin and Cytochrome b5: Comparison of Behavior in Solution and the Gas Phase†,‡

The relationship of the structure of a protein in solution to the structure of a gas-phase protein ion and the manner in which gas-phase protein ions bind small molecules noncovalently are topics of current debate. To address these issues, the stability of heme binding to wild-type and variant forms of apomyoglobin and apocytochrome b5 has been studied in the gas phase by electrospray mass spectrometry (ES-MS) and compared with the stability of heme binding to the same proteins in solution. The voltage required to dissociate ions of the heme−protein complexes in the orifice−skimmer region of an electrospray mass spectrometer, a measure of the complex stability, is found to be correlated with the activation energy for dissociation of the complexes in solution across a series of proteins in which the number of hydrogen bonds between the heme propionate groups and surface residues is systematically reduced. However, variants in which the hydrogen bonds to the proximal histidine have been removed are destabil...