Cytochrome c reconstituted from two peptide fragments displays native-likeredox properties

Recombination of two fragments of horse cytochrome c (the heme-containing N -fragment, residues 1-56, and the C-fragment, residues 57-104), which are s ubstantially unstructured at neutral pH, gives rise to a 1 : 1 fragment com plex with a compact conformation, in which the alpha helical structure and the native Met80-Fe(III) axial bond are recovered. With respect to the nati ve protein, the ferric complex shows a less rigid atomic packing and a decr eased stability [Delta(DeltaG(o))(D) = 14.7 kJ.mol(-1)], ascribed to pertur bations involving the Trp59 microenvironment and, to a lower extent, the he me pocket region. The redox potential, E-1/2 = 234 +/- 5 mV vs. normal hydr ogen electrode at 25 degreesC, is close to that of the intact protein, cons istent with recovery of the native Met80-heme Fe(III) axial bond. Furthermo re, the fragment complex shows reactivity similar to intact cytochrome c, i n the reaction with cytochrome c oxidase. We conclude that the absence in t he complex of some native cross-links and interlocked packing important for protein rigidity and stability is not as relevant for maintaining the nati ve redox properties of the protein, provided that some structural requireme nts (i.e. recovering of the native-like alpha helical structure) are fulfil led and coordination of Met80 to the heme-iron is restored.