A further clue to understanding the mobility of mitochondrial yeast cytochrome c -: A 15N T1ρ investigation of the oxidized and reduced species

A new approach was developed to overproduce N-15-enriched yeast iso-1-cytochrome c in the periplasm of Escherichia coli in order to perform a study of the motions in the ms-mus time scale on the oxidized and reduced forms through rotating frame N-15 relaxation rates and proton/deuterium exchange studies. It is confirmed that the reduced protein is rather rigid whereas the oxidized species is more flexible. The regions of the protein that display increased internal mobility upon oxidation are easily identified by the number of residues experiencing conformational equilibria and by their exchange rates. These data complement the information already available in the literature and provide a comprehensive picture of the mobility in the protein. In particular, oxidation mobilizes the loop containing Met80 and, through specific contacts, affects the mobility of helix 3 and possibly of helix 5, and of a section of protein connecting the heme propionates to helix 2. The relevance of internal motions to molecular recognition and to the early steps of the unfolding process of the oxidized species is also discussed. In agreement with the reported data, subnanosecond mobility is found to be less informative than the ms-mus with respect to redox dependent properties.