Urea-induced modification of cytochrome c flexibility as probed by cyanide binding.

Cyanide binding to cytochrome c was monitored by absorption spectroscopy from neutral to acidic pH in the presence of urea. These results were compared with acid-induced unfolding at corresponding urea concentration monitored by absorption spectroscopy and circular dichroism. The association rate constant ka increased 20-fold when the concentration of urea was raised from 0M to 6M at neutral pH. However, the secondary structure of the protein was not affected, i.e. there was no striking conformational change in these urea concentrations at neutral pH. At the pH that was very close to the pK of acid-induced unfolding, the ka value reached its maximum (ka,max) in all urea concentrations. Interestingly, the ka,max value increased exponentially with increasing urea concentrations. These results are interpreted in terms of a change in the flexibility of the least stable part of the cyt c structure that is responsible for the Fe-S(Met80) bond disruption and for ligand binding to heme iron.