The alkaline transition of cytochrome c revisited: Effects of electrostatic interactions and tyrosine nitration on the reaction dynamics.

Abstract Here we investigated the effect of electrostatic interactions and of protein tyrosine nitration of mammalian cytochrome c on the dynamics of the so-called alkaline transition, a pH- and redox-triggered conformational change that implies replacement of the axial ligand Met80 by a Lys residue. Using a combination of electrochemical, time-resolved SERR spectroelectrochemical experiments and molecular dynamics simulations we showed that in all cases the reaction can be described in terms of a two steps minimal reaction mechanism consisting of deprotonation of a triggering group followed by ligand exchange. The p K a a l k values of the transition are strongly modulated by these perturbations, with a drastic downshift upon nitration and an important upshift upon establishing electrostatic interactions with a negatively charged model surface. The value of p K a a l k is determined by the interplay between the acidity of a triggering group and the kinetic constants for the forward and backward ligand exchange processes. Nitration of Tyr74 results in a change of the triggering group from Lys73 in WT Cyt to Tyr74 in the nitrated protein, which dominates the p K a a l k downshift towards physiological values. Electrostatic interactions, on the other hand, result in strong acceleration of the backward ligand exchange reaction, which dominates the p K a a l k upshift. The different physicochemical conditions found here to influence p K a a l k are expected to vary depending on cellular conditions and subcellular localization of the protein, thus determining the existence of alternative conformations of Cyt in vivo. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]