Expression and purification of cytochrome c mutants to unravel mechanisms and role of heme-ligand coordination on tyrosine nitration and its impact in cytochrome c functions

VERÓNICA TÓRTORA, MÓNICA MARÍN, LUCÍA BONILLA, JOSÉ M. SOUZA, LUCIANO ABRIATA, ALEJANDRO VILA, RAFAEL RADI AND LAURA CASTRO

Montevideo-Uruguay

Congreso; Free Radicals in Montevideo; 2007

ytochrome c (Cytc) is a small globular protein localized in the intermembrane space side of the inner mitochondrial membrane and participates in mitochondrial electron transport. The release of Cytc from mitochondria to the cytosol via a Bcl-2-inhibitable mechanism constitutes an early event in apoptosis. The Cytc heme group is covalently bound toC14 and C17, with the 5th and 6th coordination positions of the heme-Fe interacting with H18 and M80, respectively. In addition to these four invariant amino acids, there are others that have been conserved during evolution, e.g. four tyrosine residues (Y48, Y67, Y74 and Y97 and nine lysine residues that give the protein its characteristically high isoelectric point (pI 9.5).We have shown that low peroxynitrite (ONOO-) fluxes yield mononitrated Cytc species (Y97 and Y74) whereas at higher concentrations heme-adjacentY67 was preferentially nitrated and well-represented in dinitrated species. Herein, we show that high yields of pure forms of nitro-Cytc could be obtained using a cation-exchange DEAE-5-PW preparative column.High purity of different nitro- Cytc was confirmed by MALDI-TOF-MS.Spectral analysis of nitroY97-Cytc showed a loss in 695 nm band at pH 7.4 reflecting the disappearance of heme-M80 coordination and consistent with an early “alkaline transition”. NMR spectra of nitroY97-Cytc recorded at 318K at different pH (from 4.18 to 12.5) showed that the signals in the upfield region of the spectra corresponding to the axial M80 disappeared at a lower alkaline pH, with an estimated pka of  7.9, compared to the 9.4 value in native Cytc. At alkaline pH, the appearance an additional signal could be attributed to an axial K73 ligand replacement of M80. NitroY97-Cytc exhibited a 5-fold increase in peroxidase activity at pH 7.4. We also assayed the respiratory function of purified nitro-Cytc in succinate-dependent oxygen consumption of cytochrome c-depleted mitochondria. None of the nitrated cytc was capable of fully restoring oxygen consumption rates.Interestingly, nitration of solvent-exposed Y97, even though distant from the Cytc heme group, induced structural changes in cytochrome c which have an impact in its biochemical properties and, potentially, in its cellular functions.