Photoacoustic Calorimetry Studies of Ferric Cytochrome-C Folding using an No Photo-Trigger

Abstract

In this study, NO is utilized as a photo-trigger together with photoacoustic calorimetry to probe the kinetics, enthalpy and molar volume changes associated with the earliest folding events in ferric Cytochrome-c (Cc3+). The ferric heme protein was examined under different denaturing conditions including guanidine hydrochloride (2.8M GdnHCl) and Sodium dodecyl sulfate (SDS 0.4mM) (both in 50 mM Hepes buffer, pH ∼7.5) along with NO resulting in the disruption of the axial heme Methionine-80 heme bond, triggering the partial unfolding of the complex. Under these conditions photo-dissociation of NO leaves the protein in a conformational state that favors refolding of the protein. The PAC data reveals three kinetic phases taking place subsequent to photolysis regardless of the denaturant environment. Specifically, in the presence of 2.8 M GdnHCl photolysis gave rise to kinetic events with lifetimes of <20 ns, ∼860 ns, and ∼6 μs that were associated with ΔH/ΔV = −25 kcal mol-1/-9 mL mol-1, 26 kcal mol-1/15mL mol-1, and 9 kcal mol-1/-26 mL mol-1, respectively. In the presence of 0.4 mM SDS, kinetic events were observed with lifetimes of <20 ns, ∼640 ns, and ∼8 μs with corresponding ΔH/ΔV of −11 kcal mol-1/-1 mL mol-1, 5 kcal mol-1/3 mL mol-1, and 30 kcal mol-1/-9 mL mol-1, respectively. The data suggests a uniform mechanism for the early folding events occurring in the folding of Cc3+ complex subsequent to NO photo-dissociation which are attributed to NO dissociation from the heme, followed by reorganization of the distal pocket (i.e hydrogen bond formation/breakage, NO solvation, etc.) and potentially a intermolecular binding of Methionine (80 or 65) or Histidine (23 or 33) to the heme iron upon folding.