Nitration of Solvent-exposed Tyrosine 74 on Cytochrome c Triggers Heme Iron-Methionine 80 Bond Disruption NUCLEAR MAGNETIC RESONANCE AND OPTICAL SPECTROSCOPY STUDIES*

LUCIANO A. ABRIATA, ADRIANA CASSINA, VERÓNICA TÓRTORA, MÓNICA MARÍN, JOSÉ M. SOUZA, LAURA CASTRO, ALEJANDRO J. VILA, AND RAFAEL RADI

JOURNAL OF BIOLOGICAL CHEMISTRY

ASBMB

Lugar: Washington, USA; Año: 2008

0021-9258

Cytochrome c, a mitochondrial electron transfer protein containinga hexacoordinated heme, is involved in other physiologicallyrelevant events, such as the triggering of apoptosis, and theactivation of a peroxidatic activity. The latter occurs secondaryto interactions with cardiolipin and/or post-translational modifications,including tyrosine nitration by peroxynitrite andother nitric oxide-derived oxidants. The gain of peroxidaticactivity in nitrated cytochrome c has been related to a heme sitetransition in the physiological pH region, which normallyoccurs at alkaline pH in the native protein. Herein, we report aspectroscopic characterization of two nitrated variants of horseheart cytochrome c by using optical spectroscopy studies andNMR. Highly pure nitrated cytochrome c species modified atsolvent-exposed Tyr-74 or Tyr-97 were generated after treatmentwith a flux of peroxynitrite, separated, purified by preparativehigh pressure liquid chromatography, and characterizedby mass spectrometry-based peptide mapping. It is shown thatnitration of Tyr-74 elicits an early alkaline transition with apKa
7.2, resulting in the displacement of the sixth and axialiron ligand Met-80 and replacement by a weaker Lys ligand toyield an alternative low spin conformation. Based on the study ofsite-specific Tyr to Phe mutants in the four conserved Tyr residues,we also show that this transition is not due to deprotonationof nitro-Tyr-74, but instead we propose a destabilizingsteric effect of the nitro group in the mobile -loop of cytochromec, which is transmitted to the iron center via the nearbyTyr-67. The key role of Tyr-67 in promoting the transitionthrough interactions with Met-80 was further substantiated inthe Y67F mutant. These results therefore provide new insightsinto how a remote post-translational modification in cytochromec such as tyrosine nitration triggers profound structuralchanges in the heme ligation and microenvironment andimpacts in protein function.