Cytochrome c Y¡§F mutants: a model to study the mechanisms and effects of tyrosine nitration

V. T¨®RTORA; F. SCANDROGLIO; L. BONILLA; M. MAR¨ªN; A. CASSINA; J.M. SOUZA; L. ABRIATA; A. VILA; D. ALVAREZ PAGGI; D. MURGIDA; L. CASTRO; R. RADI

San Francisco, USA

Congreso; Society for Free Radical Biology and Medicinefs 16th Annual Meeting; 2009

Society for Free Radical Biology and Medicine

Cytochrome c Y¡úF mutants: a model to study the mechanisms and effects of tyrosine nitration V.T¨®rtora1; F.Scandroglio1; L.Bonilla1; M.Mar¨ªn1; A.Cassina1; J.M.Souza1; L.Abriata2; A.Vila2; D.Alvarez3; D.Murgida3; L.Castro1 and R.Radi1. 1Center for Free Radical and Biomedical Research, Universidad de la Rep¨²blica, Uruguay; 2Universidad Nacional de Rosario and 3Universidad de Buenos Aires, Argentina. Cytocrome c is a hexacoordinated-heme protein that contains four conserved Y, two close to the heme (Y48, Y67) and two solvent-exposed (Y74, Y97). We have previously observed that cyt c is nitrated by ONOO- and NO2-+H2O2. Spectral analysis of nitro-cyt c showed an early ¡°alkaline-transition¡± by displacement of the heme-M80 coordination at pH=7.4, that leads to a gain of peroxidatic-activity and an incapacity to restore respiration in cyt c-depleted mitoplats. To better clarify cyt c nitration mechanisms and the effect of nitration in cyt c structure and function, we performed site-directed mutagenesis of each Y in horse heart cyt c and successfully expressed it in E. coli. In addition, we purified the M80A mutant as a model of penta-coordinated cyt c. The purified Y¡úF single mutants were stable proteins with spectra similar to wt. Spectrum of M80A showed a 2 nm blue-shift in the Soret band, and important differences in the 600-700 nm region. The redox potential of the Y¡úF mutants were quite similar to wt, but M80A cyt c was more negative. ONOO- nitrated all Y¡úF mutants with less efficiency than wt, and Y48F and Y67F also showed less formation of aggregates. Paramagnetic NMR analysis of all Y¡úF mutants and of pure nitro-cyt c species (nitro-Y74 or nitro-Y97) showed a heme microenvironment analogous to wt, in a low spin form. However, Y67F presented a delayed alkaline transition (pKa>10.6) respect to wt (pKa=8.9), while nitro-Y74 cyt c showed an early alkaline transition (pKa=7.2). M80A heme environment differed from wt and exists in a series of high- and low-spin forms.