Analysis of the pH-dependent thermodynamic stability, local motions, and microsecond folding kinetics of carbonmonoxycytochrome c

• Carbonmonoxycytochrome c has maximum thermodynamic stability at pH∼9.5.
• Between pH 7.0 and pH 11.9, ΔGD° of Cyt-CO varies by ∼10.1 kcal mol−1.
• Between pH 7.0 and pH 11.9, ΔGD°‡ of Cyt-CO varies by < 1.0 kcal mol−1.
• pH-dependent ionic-interactions greatly affect the global stability of protein.
• Electrostatic interactions exhibit a very small effect on the folding kinetics.

This paper analyzes the effect of pH on thermodynamic stability, low-frequency local motions and microsecond folding kinetics of carbonmonoxycytochrome c (Cyt-CO) all across the alkaline pH-unfolding transition of protein. Thermodynamic analysis of urea-induced unfolding transitions of Cyt-CO measured between pH 6 and pH 11.9 reveals that Cyt-CO is maximally stable at pH∼9.5. Dilution of unfolded Cyt-CO into refolding medium forms a native-like compact state (NCO-state), where Fe2+−CO interaction persists. Kinetic and thermodynamic parameters measured for slow thermally-driven CO dissociation (NCO→N+CO) and association (N+CO→NCO) reactions between pH 6.5 and pH 13 reveal that the thermal-motions of M80-containing Ω-loop are decreased in subdenaturing limit of alkaline pH. Laser photolysis of Fe2+-CO bond in NCO-state triggers the microsecond folding (NCO→N). The microsecond kinetics measured all across the alkaline pH-unfolding transition of Cyt-CO produce rate rollover in the refolding limb of chevron plot, which suggests a glass transition of NCO en route to N. Between pH 7 and pH 11.9, the natural logarithm of the microsecond folding rate varies by < 1.5 units while the natural logarithm of apparent equilibrium constant varies by 11.8 units. This finding indicates that the pH-dependent ionic-interactions greatly affect the global stability of protein but have very small effect on folding kinetics.

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