Time resolved calorimetry of photo-induced folding in horse heart cytochrome c at high pH

Abstract

Here the molar volume and enthalpy changes associated with the early events in the folding of ferrocytochrome c (Cc) at high pH have been examined using time resolved photoacoustic calorimetry (PAC). The data reveal an overall volume change of 1.3 ± 0.3 mL mol−1 and an enthalpy change of 13 ± 7 kcal mol −1 occurring subsequent to photodissociation of the unfolded CO bound Cc species in <∼20 ns. Two additional kinetic phases are observed that are associated with non-native His binding (ΔH and ΔV of 2 ± 4 kcal mol−1 and -0.5 mL mol−1, τ ∼ 2.5 μs ) and Met binding (ΔH and ΔV -0.4 ± 2 kcal mol−1 and -0.1 ± 0.1 mL mol−1, τ∼ 660 ns). Considering only protein conformational changes (excluding volume and enthalpies associated with heme ligation events) the initial conformational event exhibits a ΔH and ΔV of 6 ± 3 kcal mol−1 and -3±0.1 mL mol−1, respectively, that are attributed to a small contraction of the unfolded protein. The corresponding enthalpy associated with both native and non-native ligand binding are found to be −5±4 kcal mol−1 (Fe-Met) and +20 ± 4 kcal mol−1 (Fe-His) with the change in volume for both phases being essential negligible. This would indicate that non-native ligand binding likely occurs from an already collapsed conformation.