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

• Published in: Archives of biochemistry and biophysics 2019-04, Vol.665, p.96-106
• Main Authors: Oviedo-Rouco, Santiago, Castro, María A., Alvarez-Paggi, Damián, Spedalieri, Cecilia, Tortora, Verónica, Tomasina, Florencia, Radi, Rafael, Murgida, Daniel H.
• Format: Article
• Language: English
• Subjects: Alkalies - chemistry; Alkaline transition; Animals; Cytochrome c; Cytochromes c - metabolism; Horses; Hydrogen-Ion Concentration; Ligands; Molecular Dynamics Simulation; Nitrates - metabolism; Oxidation-Reduction; Protein electron transfer; Protein nitration; Protein spectroelectrochemistry; Static Electricity; Time-resolved SERR; Tyrosine - metabolism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2019.02.016

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 pKaalk 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 pKaalk 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 pKaalk downshift towards physiological values. Electrostatic interactions, on the other hand, result in strong acceleration of the backward ligand exchange reaction, which dominates the pKaalk upshift. The different physicochemical conditions found here to influence pKaalk 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. [Display omitted]