Ligand Binding Subsequent to NO Photolyses of Partially Unfolded Cytochrome c

Here we report folding studies of horse heart ferricytochrome c in the presence of NO (NO‐cyt c ). The midpoint concentration of guanidine hydrochloride (GuHCl) for unfolding was measured to be 2.8 M in the presence of NO and is only moderately altered relative to native cyt c under similar conditions (i.e., 2.6 M GuHCl). Recombination of NO to cyt c in the presence of 2.8 M GuHCl subsequent to photolysis is biphasic with rate constants of (4.24 ± 0.15) × 10 3 s −1 and 197.7 ± 3.3 s −1 with the fast and slow phase representing ˜60% and 40% relative amplitudes, respectively. NO rebinding to unfolded cyt c (in the presence of 4 M GuHCl) exhibits two phases with rate constants similar to those observed in the presence of 2.8 M GuHCl with the relative amplitude of the fast phase increasing to roughly 90%. These data suggest that NO rebinds to two populations of cyt c with one population containing a five coordinate heme and the other having a six‐coordinate heme with bis‐His axial ligation. These results suggest that the mis‐ligated bis‐His conformation forms rapidly during the refolding of cyt. c 3+ , although a population exists that can refold down a native pathway.