DETERMINANTS OF PROTEIN HYDROGEN-EXCHANGE STUDIED IN EQUINE CYTOCHROME-C

The exchange of a large number of amide hydrogens in oxidized equine c ytochrome c was measured by NMR and compared with structural parameter s. Hydrogens known to exchange through local structural fluctuations a nd through larger unfolding reactions were separately considered. All hydrogens protected from exchange by factors greater than 10(3) are in defined H-bonds, and almost all H-bonded hydrogens including those at the protein surface were measured to exchange slowly. H-exchange rate s do not correlate with H-bond strength (length) or crystallographic B factors. It appears that the transient structural fluctuation necessa ry to bring an exchangeable hydrogen into H-bonding contact with the H -exchange catalyst (OH--ion) involves a fairly large separation of the H-bond donor and acceptor, several angstroms at least, and therefore depends on the relative resistance to distortion of immediately neighb oring structure. Accordingly, H-exchange by way of local fluctuational pathways tends to be very slow for hydrogens that are neighbored by t ightly anchored structure and for hydrogens that are well buried. The slowing of buried hydrogens may also reflect the need for additional m otions that allow solvent access once the protecting H-bond is separat ed, although it is noteworthy that burial in a protein like cytochrome c does not exceed 4 Angstrom. When local fluctuational pathways are v ery slow, exchange can become dominated by a different category of lar ger, cooperative, segmental unfolding reactions reaching up to global unfolding.