Nature of the fast and slow refolding reactions of iron(III) cytochrome c

Abstract

The fast and slow refolding reactions of [horse heart] iron (III) cytochrome c (Fe(III) cyt c), previously studied by Ikai et al. were reinvestigated. The fast reaction has the major amplitude (78%) and is 100-fold faster than the slow reaction in these conditions (pH 7.2, 25.degree. C, 1.75 M guanidine hydrochloride). Native cyt c is the product formed in the fast reaction as well as in the slow reaction. Two probes were used to test for formation of native cyt c; absorbance in the 695 nm band and rate of reduction by L-ascorbate. Different unfolded species (UF, US) give rise to the fast and slow refolding reactions, as shown by refolding assays at different times after unfolding (double-jump experiments) and by the formation of native cyt c in each of the fast and slow refolding reactions. Thus the fast refolding reaction is UF .fwdarw. N and the slow refolding reaction is US .fwdarw. N, where N is native cyt c and there is a US .tautm. UF equilibrium in unfolded cyt c. The results are consistent with the UF .tautm. US reaction being proline isomerization but this has not yet been tested in detail. Folding intermediates were detected in both reactions. In the UF .fwdarw. N reactions, the Soret absorbance change precedes the recovery of the native 695 nm band spectrum, showing that Soret absorbance monitors the formation of a folding intermediate. In the US .fwdarw. N reaction an ascorbate-reducible intermediate was found at an early stage in folding and the Soret absorbance change occurs together with the change at 695 nm as N is formed in the final stage of folding.