Nuclear magnetic resonance studies of slowly exchanging peptide protons in cytochrome c in aqueous solution.

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Abstract
The slowly exchanging protons in oxidized and reduced horse heart cytochrome c (D2O uncorrected pH meter reading 6.5, room temperature) were monitored by recording the 270 and 360 MHz proton NMR spectra of the reduced protein between 5-11 ppm downfield from 2,2-dimethyl-2-silapentane-5-sulfonate. There are about 12 well-resolved exchangeable resonances between 7.9-10.1 ppm, and the experimental chemical shift data suggest that they originated from the same residues in both oxidation states. These resonances correspond to 19-22 protons in the reduced protein and a lesser number due to partial exchange in the oxidized protein. The base catalysis of the exchange in both oxidation states was used to demonstrate the sequential exchange of these well-resolved resonances in D2O. Alternatively, the temperature dependence of the exchange of a fixed pH was used to differentiate and monitor separately the slower and the slowest exchanging resonances as a function of time. The different rates of exchange among the slowly exchanging peptide protons provide evidence against a resistant cluster of protons that exchange together.