Studies of structure, dynamics and interactions of ferricytochrome c by hydrogen exchange, nuclear magnetic resonance, and related techniques

Hydrogen exchange (HX), nuclear magnetic resonance (NMR), and related techniques were used to characterize the structure and stability of oxidized equine cytochrome c (cyt c) at acid pH, and to study binding sites and dynamics of cyt c with its partners, cytochrome c peroxidase (ccp) and cytochrome b$\sb5$ (cyt b$\sb5$). HX and NMR studies under acid and high salt condition (1.5 M NaCl, pD 2.2) show that cyt c exists in a globular conformation (A state) with properties resembling those of the molten globule state described for other proteins. A detailed comparison of H-bonded secondary and tertiary structure was done between the A state and the native state. The HX results show that the major helices of cyt c and their common hydrophobic domain are largely preserved in the globular acidic form while the loop regions of the native structure are partially disordered. Structure, dynamics, and stability of cyt c at low pH were studied. As NaCl concentration is decreased below 0.05 M, cyt c expands due to the deshielding of electrostatic repulsions, attaining a linear extent perhaps double that of the native protein (viscosity, fluorescence). In this extended form, tertiary structural hydrogen bonds are largely broken (HX data), some normally buried parts of the protein are exposed to water (fluorescence), and many of the native side-chain contacts must be lost. Nevertheless, almost all of the helical content is retained (CD). The helices involve the same amino acid residues that are helical in the native state (HX labeling monitored by 2D NMR) with equilibrium constant 10 (0.02 M NaCl, pD 2.2), even though the isolated helices have little or no structure. The results indicate that the native-like helical segments in the expanded nonglobular form of cyt c involve the separation of submolecular folding units that possess significant equilibrium stability. The binding sites of cyt c with its partners, ccp and cyt b$\sb5$, were studied by HX and 2D NMR. The results agree with the computer models. The flexibility of binding shown in both cyt c-ccp and cyt c-cyt b$\sb5$ complexes may indicate that complementary charge interaction may not support a sufficient recognition between electron donors and acceptors