Properties of modified cytochromes. I. Equilibrium and kinetics of the pH-dependent transition in carboxymethylated horse heart cytochrome c.

Reduced (Fe+2) carboxymethylated cytochrome c, Cm-cyt. c, undergoes a reversible pH-dependent transition with a pK of 7.16 at 20°. This pK is found to be nearly temperature-independent indicating that the over-all enthalpy for the transition is close to zero. The kinetics of this transition have been investigated by the temperature jump technique. A single well resolved relaxation process (in the millisecond time range) is observed over the pH region of the static titrations. The amplitude of this relaxation at different wave lengths fits the statically derived difference spectrum between the alkaline and acid forms of the protein. Both the amplitude and the relaxation time τ are pH dependent; the over-all enthalpy of the process is estimated to be ∼ +1 Cals per mole. The observed behavior may be accounted for by a model in which a proton-linked conformational change in the protein is responsible for the spectral changes. It is suggested that the deprotonation of an e-amino group of lysine (possibly that of lysine 79) is followed by the binding of this group to the ferrous iron to fill the vacant sixth coordination position. The observed spectral changes are attributed to the binding of a nitrogen atom to the iron. The thermodynamic parameters governing the conformational part of the reaction are calculated on the basis of the above model and values of ΔH = -10 Cals per mole and ΔS = -20 e.u. are found. These values are discussed in the context of the binding of protein residue to the iron and the consequent changes in the crevice structure