Non-two-state thermal denaturation of ferricytochrome c at neutral and slightly acidic pH values

•Thermal denaturation of cyt c at pH > 5 has a non-two-state nature.•Thermal transition of cyt c at pH > 5 proceeds through non-native low-spin states.•Local conformational transition resembles alkaline-like transition.•Final state of global conformational transition resembles bis-histidinyl state.•Thermal transition of cyt c at pH < 5 is a one step process from low to high-spin states.

Thermal denaturation of ferricytochrome c (cyt c) has been methodically studied by absorbance, fluorescence, circular dichroism spectroscopy, viscosimetry and differential scanning calorimetry in pH range from pH 3.5 to 7.5. Thermal transitions have been monitored by intrinsic local probes of heme region such as absorbance at Soret, 620 nm and 695 nm bands and circular dichroism signals at 417 nm. Global conformational changes were analyzed by circular dichroism signal at 222 nm, fluorescence of the single tryptophan, reduced viscosity and differential scanning calorimetry. We show that cyt c thermal denaturation above pH ~5 can be described by an apparent two-step transition in which the heme iron stays in a low-spin state. The thermal denaturations of cyt c below pH ~5 proceed in one step to an unfolded highly compact form with a high-spin state of the heme iron. Cyt c conformational plasticity is discussed in regard to its physiological functions.

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