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

  • Chen E
  • Wittung-Stafshede P
  • Kliger D
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The early dynamics of reduced cytochrome c (cytc) folding Initiated by a photoinduced electron transfer reaction are studied using time-resolved circular dichroism spectroscopy: At 3.5 M GdnHCl oxidized cytc is partly unfolded, whereas the reduced form is folded. Thus, under these conditions, rapid electron-injection into unfolded, oxidized cytc triggers folding. The kinetics of secondary structure formation in reduced cytc occurs in two major phases. The earliest detection of reduction is accompanied by the appearance of 20% of the secondary structure within 5 mu s. 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 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 approximate to 180 mu s) is followed by a small increase in the magnitude of the CD signal (tau = 6 ms), forming 30% of the native secondary structure. It appears 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.

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  • Eefei Chen

  • Pernilla Wittung-Stafshede

  • David S. Kliger

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