COUPLING NITROTYROSINE DEPROTONATION TO A PROTEIN CONFORMATIONAL CHANGE: THE CASE OF NITROCYTOCHROME C

RAFAEL RADI; DAIANA ANDREA CAPDEVILA; VERONICA DEMICHELI; VERONICA TORTORA; DANIEL MURGIDA

San Antonio

Congreso; Society for Free Radical Biology and Medicine 20th Annual Meeting; 2013

Society for Free Radical Biology and Medicine

Mammalian Cyt c contains four highly conserved Tyr residues atsequence positions 48, 67, 74 and 97. Treatment withperoxynitrite leads to preferential nitration of either Tyr74 orTyr97 which are solvent exposed. These modifications result ina conformational change which represents an early alkalinetransition with apparent pKa values of 7.3 and 8.7, respectivelythat contrast with the value of 9.4 determined for unmodified Cytc. NMR experiments confirmed that in all cases the transitionimplies replacement of Met80 by a Lys residue as axial ligand.Immunochemical evidence also indicates that these alternativeconformations exist in cells under peroxynitrite challenge. Themolecular basis by which nitration of a solvent exposed Tyr maytrigger an early alkaline transition is unknown. For this purpose,we performed acid-base titrations of ferric native Cyt c of themononitrated variants at positions 74 (NO2Tyr74-Cyt c) and 97(NO2Tyr97-Cyt c) using quasi-simultaneous UV-vis absorptionand resonance Raman (RR) detection. In the first case theevolution of the alkaline transition was monitored from the pH-dependent disappearance of the MetS→Fe charge transfer bandat 695 nm. RR titrations were performed under the Soret bandexcitation (413 nm laser line) to identify the redox state, spin andaxial ligands of the heme iron at the marker band region ca.1300-1700 cm-1. In addition, the RR spectra was obtained under458 nm laser excitation to explore the well-resolved vibrationalbands of both the heme group and the basic form of NO2-Tyrwith comparable intensities, allowing reliable simultaneoustitrations of the two chromophores in nitrocytochrome c. Notably,a one-to-one correlation was observed between NO2-Tyr74deprotonation and the alkaline transition. Functional studiesshowed that the conformational change and alternative ligationof the 6th coordination position resulted in a substantial loweringof the Cyt c redox potential (i.e. from 255 and -161 mV) and again-of-peroxidatic activity. To our knowledge, this is the firstreport where changes in the acid-base properties of the Tyrphenol side chain upon nitration are directly associated toconformational and functional changes in a protein.