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

- NCO-state is a highly compact configuration of ferrocytochrome c.
- Size, shape and viscosity of crowders affect the structural fluctuation of Ω-loop.
- Anions and cations have favorable interaction sites on ferrocytochrome c.
- Size and charge density of ions affect the structural fluctuation of Ω-loop.
- Enthalpic effect is more dominated than the entropic effect on mobility of Ω-loop.

Carbonmonoxycytochrome c refolds to a native-like compact state (NCO-state), where the non-native Fe2 +-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 Fe2 +-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 (kdiss) decreases exponentially. Furthermore, the extent of decrease in kdiss 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.