This study investigates the hydrogen exchange behavior of native cytochrome c in low concentrations of denaturant, revealing a sequence of metastable, partially unfolded forms that occupy free energy levels up to the fully unfolded state. These forms represent major intermediates in the reversible, dynamic unfolding and refolding reactions of cytochrome c under native conditions. The study shows that hydrogen exchange experiments can determine the structure, free energy, and surface exposure of each protein form by analyzing the hydrogens exposed in different energy states. The hydrogen exchange behavior of cytochrome c reveals a small sequence of distinct partially unfolded forms with increasing free energy and degree of unfolding. These forms are likely the major intermediates in the unfolding and refolding pathways of cytochrome c. The study also discusses the theory of hydrogen exchange, showing that the exchange rate of a hydrogen is determined by its chemical exchange rate in the open form and the equilibrium opening constant. The energy levels and occupation of conformationally excited states can be manipulated by denaturants and temperature. The study identifies cooperative units in cytochrome c, including entire omega loops and mutually stabilizing pairs of helices and loops. These units are involved in the unfolding and refolding processes. The results show that the hydrogen exchange isotherms reveal the global unfolding reaction and define its free energy and m values. The study also discusses the residual structure in unfolded states, showing that some residues remain protected against exchange even in the fully unfolded state. The study identifies several cooperative units in cytochrome c, including the 60's helix, the 20 to 35 loop, and the 70 to 85 loop. These units are involved in the unfolding and refolding processes. The study also discusses the cooperative unit relations, showing that the hydrogen exchange isotherms can be used to identify the cooperative units that are unfolded or folded. The study concludes that the results specify all the cooperative unit boundaries within one residue, with the exception of the boundary at the 32 to 35 beta turn. The cooperative units are entire omega loops and mutually stabilizing pairs of whole helices and loops, with a segment size of about 15 residues. The study also discusses the implications of the results for protein folding, showing that proteins cannot fold in one undifferentiated, whole molecule, search-dependent reaction but must move to the native state through intermediate forms. The results support a folding strategy that carries cytochrome c through a single linear pathway, consisting of a small number of discrete intermediates, in steps that fold one cooperative unit at a time. The study also discusses the implications for the biological evolution of contemporary protein structures.This study investigates the hydrogen exchange behavior of native cytochrome c in low concentrations of denaturant, revealing a sequence of metastable, partially unfolded forms that occupy free energy levels up to the fully unfolded state. These forms represent major intermediates in the reversible, dynamic unfolding and refolding reactions of cytochrome c under native conditions. The study shows that hydrogen exchange experiments can determine the structure, free energy, and surface exposure of each protein form by analyzing the hydrogens exposed in different energy states. The hydrogen exchange behavior of cytochrome c reveals a small sequence of distinct partially unfolded forms with increasing free energy and degree of unfolding. These forms are likely the major intermediates in the unfolding and refolding pathways of cytochrome c. The study also discusses the theory of hydrogen exchange, showing that the exchange rate of a hydrogen is determined by its chemical exchange rate in the open form and the equilibrium opening constant. The energy levels and occupation of conformationally excited states can be manipulated by denaturants and temperature. The study identifies cooperative units in cytochrome c, including entire omega loops and mutually stabilizing pairs of helices and loops. These units are involved in the unfolding and refolding processes. The results show that the hydrogen exchange isotherms reveal the global unfolding reaction and define its free energy and m values. The study also discusses the residual structure in unfolded states, showing that some residues remain protected against exchange even in the fully unfolded state. The study identifies several cooperative units in cytochrome c, including the 60's helix, the 20 to 35 loop, and the 70 to 85 loop. These units are involved in the unfolding and refolding processes. The study also discusses the cooperative unit relations, showing that the hydrogen exchange isotherms can be used to identify the cooperative units that are unfolded or folded. The study concludes that the results specify all the cooperative unit boundaries within one residue, with the exception of the boundary at the 32 to 35 beta turn. The cooperative units are entire omega loops and mutually stabilizing pairs of whole helices and loops, with a segment size of about 15 residues. The study also discusses the implications of the results for protein folding, showing that proteins cannot fold in one undifferentiated, whole molecule, search-dependent reaction but must move to the native state through intermediate forms. The results support a folding strategy that carries cytochrome c through a single linear pathway, consisting of a small number of discrete intermediates, in steps that fold one cooperative unit at a time. The study also discusses the implications for the biological evolution of contemporary protein structures.