The study investigates the hydrogen exchange behavior of native cytochrome c under low concentrations of denaturants, revealing a sequence of metastable, partially unfolded forms that occupy free energy levels up to the fully unfolded state. These forms are identified as cooperative units, including entire omega loops and mutually stabilizing pairs of helices and loops. The hydrogen exchange experiments determine the hydrogens exposed in each higher energy form, their rates of exchange with solvent, and their sensitivity to denaturants and temperature. The results suggest that these partially unfolded forms represent the major intermediates in the reversible, dynamic unfolding reactions that occur even at native conditions, defining the major pathway for cytochrome c folding. The cooperative nature of protein structure is illuminated, and the unfolding and refolding pathways are defined, despite the intermediates being normally invisible to measurement. The study also discusses the implications for protein folding, suggesting a single linear pathway through a small number of discrete intermediates, each involving the unfolding of one cooperative unit at a time.The study investigates the hydrogen exchange behavior of native cytochrome c under low concentrations of denaturants, revealing a sequence of metastable, partially unfolded forms that occupy free energy levels up to the fully unfolded state. These forms are identified as cooperative units, including entire omega loops and mutually stabilizing pairs of helices and loops. The hydrogen exchange experiments determine the hydrogens exposed in each higher energy form, their rates of exchange with solvent, and their sensitivity to denaturants and temperature. The results suggest that these partially unfolded forms represent the major intermediates in the reversible, dynamic unfolding reactions that occur even at native conditions, defining the major pathway for cytochrome c folding. The cooperative nature of protein structure is illuminated, and the unfolding and refolding pathways are defined, despite the intermediates being normally invisible to measurement. The study also discusses the implications for protein folding, suggesting a single linear pathway through a small number of discrete intermediates, each involving the unfolding of one cooperative unit at a time.