Conformational dynamics of a biologically active three-fragment complex of horse cytochrome c.

The conformational dynamics of a biologically active noncovalent complex containing three fragments, ferroheme fragment (1-25)H and apofragments (28-38) and [3H](56-104) [or [3H](39-104)], of horse cytochrome c has been studied with respect to kinetics and thermodynamics of dissociation. The rate of unfolding of the two-fragment complex ferro(1-25)H . (56-104) was also estimated. The results indicate that the ferrous three-fragment complex exhibits a higher frequency of dissociation-association with fragment (28-38) and a lower frequency of overall unfolding-folding at pH 7.0. In the presence of an excess of free (28-38) and below 30 degrees C, unfolding of the ferrous three-fragment complex appears to occur by activation to the transitional state without a large change in conformation, followed by virtually simultaneous dissociation of all three of the fragments [without going through the complex (1-25)H . (56-104), which is a major intermediate for folding]. Above 30 degrees C unfolding via the complex (1-25)H . (56-104) becomes detectable because the equilibrium between the two- and the three-fragment complex is highly temperature dependent. Thus, the relative probabilities of these two different ways of transition for unfolding are modulated by temperature. The observations suggest that the mode of activation of protein and hence the pathway for unfolding may vary depending on temperature. It is also suggested that the interatomic interactions binding the three fragments together in the ordered complex are linked to strengthen each other in the ground state.