Role of macromolecular crowding and salt ions on the structural-fluctuation of a highly compact configuration of carbonmonoxycytochrome c

Carbonmonoxycytochrome c refolds to a native-like compact state (NCO-state), where the non-native Fe^{2 +}-CO interaction persists. Structural and molecular properties extracted from CD, fluorescence and NMR experiments reveal that the NCO-state shows the generic properties of molten globules. Slow thermal-dissociation of CO transforms the NCO-state to native-state (N-state), where the native Fe^{2 +}-M80 bond recovers. To determine the role of crowding agents and salt ions on the structural-fluctuation of NCO, the kinetic and thermodynamic parameters for CO-dissociation from NCO (NCO → N + CO) were measured at varying concentrations of crowding agents (dextran 70, dextran 40, ficoll 70) and salt ions (anion: ClO₄^-, I^-, Br^-, NO₃^-, Cl^-; cation: NH₄⁺, K⁺, Na⁺). As [crowding agent] or [ion] is increased, the rate coefficient of CO-dissociation (k_diss) decreases exponentially. Furthermore, the extent of decrease in k_diss is found to be dependent on (i) size, charge density and charge dispersion of the ion, and (ii) size, shape, and viscosity of the crowding agent.