Far-UV time-resolved circular dichroism detection of electron-transfer-triggered cytochrome c folding

The early dynamics of reduced cytochrome c (cytc) folding Initiated by a ph otoinduced electron transfer reaction are studied using time-resolved circu lar dichroism spectroscopy: At 3.5 M GdnHCl oxidized cytc is partly unfolde d, whereas the reduced form is folded. Thus, under these conditions, rapid electron-injection into unfolded, oxidized cytc triggers folding. The kinet ics of secondary structure formation in reduced cytc occurs in two major ph ases. The earliest detection of reduction is accompanied by the appearance of 20% of the secondary structure within 5 mu s. From time-resolved absorpt ion and circular dichroism studies this rapid folding is ascribed to a subp opulation of unfolded protein molecules that have a structural tendency to form a native His 18-Fe(II)-Met80 ligation. Thereafter, almost no change in the secondary structure is observed until the CD signal starts to decrease in magnitude between 16 mu s and 1 ms. This "unfolding" phase (tau approxi mate to 180 mu s) is followed by a small increase in the magnitude of the C D signal (tau = 6 ms), forming 30% of the native secondary structure. It ap pears that a second subpopulation that is initially trapped by a His 18-Fe( II)-His26/33 non-native ligation slows down folding until His is displaced by the Met ligand (tau = 110 ms). Formation of 90-95% of the native reduced cytc secondary structure is then detected by similar to 320 ms.