The role of molecular crowding in the gain of alternative peroxidatic function of Cytochrome c

SCOCOZZA, MAGALI; OVIEDO ROUCO, SANTIAGO; MURGIDA, DANIEL H.

Congreso; BioTrans 2019; 2019

The role of molecular crowding in the gain of alternative peroxidaticfunction of Cytochrome cScocozza M. F.; Oviedo-Rouco, S. and Murgida D. H.Departamento de Química Inorganica, Analítica y Química Física?INQUIMAE, Facultad de CienciasExactas y Naturales, Universidad de Buenos Aires?CONICET, Buenos Aires, ArgentinaCytochrome C (Cyt) is a multifunctional soluble hemoprotein: it may alternate betweenworking as an electron carrier and as a peroxidase enzyme. The gain of peroxidaseactivity is associated to the induction of alternative conformations that differ in the axialcoordination of the heme iron. One of these conformers is the product of the so-calledalkaline transition, which is a pH-dependent conformational equilibrium associated to aMet → Lys ligand exchange (pKa ̴ 9.4).Here we report on the effect of molecular crowding on the thermodynamical and kineticparameters of the alkaline transition. The concentrations of the crowding agents(PEG4000 and sucrose) were adjusted to produce viscosities up to 40 cp. Theconformational change was characterized by stationary and time-resolved surface-enhanced resonance Raman experiments. Soret excitation (407 nm laser line) allowed usto monitor the redox state, spin and axial ligands of the heme iron.We observed that the ligand exchange occurs at high viscosities as well as in dilutedsolutions. However, thermodynamical and kinetic parameters of the alkaline transitionare affected: we observed a decrease in pKa as well as a decrease in the kineticsconstants of the process.This work highlights the need of explicitly considering molecular crowding effects indynamics of Cyt. Studying alkaline transition allows us to understand the mechanism toachieve peroxidase activity both in vitro as well as in biological environments.