New insight into the mechanism of the alkaline transition of cytochrome c

OVIEDO ROUCO, SANTIAGO; RADI, RAFAEL; SCOCOZZA, MAGALI; MURGIDA, DANIEL H.; TOMASINA, FLORENCIA

Florianopolis

Congreso; International Conference on Bio Inorganic Chemistry; 2017

The alkaline transition of cytochrome c (Cyt) is a pH-dependent conformational equilibrium (pKaapp ~ 9) that implies Met→Lys exchange of the heme axial ligand. Over the years, the structural and mechanistic aspects of this transition have been extensively investigated aiming to fully understand the process and its biological significance.1,2 The minimal mechanistic scheme includes a fast deprotonation of an unknown triggering group, followed by ligand exchange. Although simplified, this mechanism explains many experimental data, including the pKaapp value. Recently, it has been shown that some naturally occurring post-translational modifications, such as nitration at Tyr74 (Cyt-NO2-Tyr74), downshift the measured pKaapp, while other alterations such as Tyr67Phe mutation have the opposite effect. The molecular basis of these modulation effects remain largely elusive. Here we present a novel experimental strategy for investigating the dynamics of the alkaline transition at constant pH. The method takes advantage of the redox state dependence of the equilibrium, which at pH > pKaapp is fully displaced towards the alkaline form for Cyt3+ and towards the native form for Cyt2+. Thus, the method couples potential jumps at constant pH to trigger the alkaline transition with time resolved surface-enhanced resonance Raman monitoring. We utilized this methodology for studying the mechanistic differences between the alkaline transitions of WT Cyt, Cyt-NO2-Tyr74 and the Tyr67Phe mutant. Compared to WT Cyt, nitration of Tyr74 causes a downshift of both the pKH for the deprotonation of the triggering group, and the overall measured pKaapp. Moreover, the obtained pKH for the triggering group is identical to the pKa of phenolic group of the NO2-Tyr74, thus suggesting that Tyr74 might play the role of triggering group. On the other hand, equilibrium and potential jump experiments performed on the point mutant Tyr74Phe yield results identical to those obtained for WT Cyt, thus suggesting the opposite. Additional experiments on the Tyr67Phe mutant and the double-modified variant Cyt-NO2-Tyr74/,Tyr67Phe show that the effect on the determined pKaapp produced by Tyr74 nitration vanishes when the Tyr67 residue is replaced. Thus, we propose that Tyr67 participates in the mechanism of alkaline transition, probably as deprotonable triggering group whose signal transmitting mechanism involves the disruption of the Tyr67/Met80 hydrogen bond and the consequent loosening of the ligand loop, which in turn includes Tyr74 and, therefore, is expected to be affected upon nitration.