Comparison of cytochrome c and apocytochrome c structures by hydrogen exchange as monitored by NMR

Abstract

When in contact with phospholipid vesicle, in acidic pH, a short C-terminal stretch of cytochrome c is protected from the tryptic digestion while a longer C-terminal segment of apocytochrome c is protected from the digestion. We investigated the structural difference of the fragment contained tryptophan-59 residue in two protein. Flexibility of the fragment was measured by hydrogen exchange rate of imino proton of tryptophan, using 300 MHz NMR. The imino proton of Trp-59 in apocytochrome c was assigned newly in this report by NOE experiment. The imino proton of Trp is labile, exchangable with solvent, proton. The exchange rate of imino proton with solvent depends on solvent accessibility and hydrogen bonding. It was observed that the fragment contained Trp-59 in apocytochrome c is about $10^6$ times as flexible as the fragment in cytochrome c. And cytochrome c was more flexible at pH 5 than at pH 7. UV/VIS sepctroscopy method was performed to measure the rate of oxido-reduction of cytochrome c depending on pH. Reduced cytochrome c is more stable at neutral pH than acidic pH. From these results, we concluded that flexible structure of two proteins at acidic pH is important to interact with vesicles, to act as a cofactor of electron transfort systems.