Gauging of Cytochrome c Structural Fluctuation by Time-Resolved Proton Pulse

A brief proton pulse technique, based on photo-excitation of pyranine, was employed for measuring the protonation dynamics of cytochrome c in aqueous solutions. Time-resolved monitoring of the protonation state of the pyranine anion yielded detailed information from which the temporal state protonation of the surface carboxylates and histidine residues were deduced. The surface groups were found to be coupled by electrostatic interaction where the state of protonation of one affects the pK of the others. In the same sense, altering the charge distribution of a protein by a redox reaction affects the reactivity of the surface groups with protons in the bulk. The surface groups can exchange protons among themselves at a very high rate. The velocities of these reactions are functions of the connectivity between the proton binding sites. A single proton exchange reaction between the surface groups could be identified as the proton exchange between H26 and E44. The reaction is fast and affected by the redox state of the protein. It is proposed that the enhanced rate of this reaction is coupled with transient conformational changes that are not noticeable in time-averaging measurements such as X-ray diffraction or NMR.