Far-UV Time-Resolved Circular Dichroism Detection of Electron-Transfer-Triggered Cytochrome c Folding

The early dynamics of reduced cytochrome c (cyt c ) folding initiated by a photoinduced electron-transfer reaction are studied using time-resolved circular dichroism spectroscopy. At 3.5 M GdnHCl oxidized cyt c is partly unfolded, whereas the reduced form is folded. Thus, under these conditions, rapid electron injection into unfolded, oxidized cyt c triggers folding. The kinetics of secondary structure formation in reduced cyt c occurs in two major phases. The earliest detection of reduction is accompanied by the appearance of 20% of the secondary structure within 5 μs. From time-resolved absorption and circular dichroism studies this rapid folding is ascribed to a subpopulation of unfolded protein molecules that have a structural tendency to form a native His18-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 μs and 1 ms. This “unfolding” phase (τ ≈ 180 μs) is followed by a small increase in the magnitude of the CD signal (τ = 6 ms), forming 30% of the native secondary structure. It appears that a second subpopulation that is initially trapped by a His18-Fe(II)-His26/33 non-native ligation slows down folding until His is displaced by the Met ligand (τ = 110 ms). Formation of 90−95% of the native reduced cyt c secondary structure is then detected by ∼320 ms.