Nitration of Solvent-exposed Tyrosine 74 on Cytochrome c Triggers Heme Iron-Methionine 80 Bond Disruption

Abstract

Cytochrome c, a mitochondrial electron transfer protein containing a hexacoordinated heme, is involved in other physiologically relevant events, such as the triggering of apoptosis, and the activation of a peroxidatic activity. The latter occurs secondary to interactions with cardiolipin and/or post-translational modifications, including tyrosine nitration by peroxynitrite and other nitric oxide-derived oxidants. The gain of peroxidatic activity in nitrated cytochrome c has been related to a heme site transition in the physiological pH region, which normally occurs at alkaline pH in the native protein. Herein, we report a spectroscopic characterization of two nitrated variants of horse heart cytochrome c by using optical spectroscopy studies and NMR. Highly pure nitrated cytochrome c species modified at solvent-exposed Tyr-74 or Tyr-97 were generated after treatment with a flux of peroxynitrite, separated, purified by preparative high pressure liquid chromatography, and characterized by mass spectrometry-based peptide mapping. It is shown that nitration of Tyr-74 elicits an early alkaline transition with a pK a ‫؍‬ 7.2, resulting in the displacement of the sixth and axial iron ligand Met-80 and replacement by a weaker Lys ligand to yield an alternative low spin conformation. Based on the study of site-specific Tyr to Phe mutants in the four conserved Tyr residues, we also show that this transition is not due to deprotonation of nitro-Tyr-74, but instead we propose a destabilizing steric effect of the nitro group in the mobile ⍀-loop of cytochrome c, which is transmitted to the iron center via the nearby Tyr-67. The key role of Tyr-67 in promoting the transition through interactions with Met-80 was further substantiated in the Y67F mutant. These results therefore provide new insights into how a remote post-translational modification in cytochrome c such as tyrosine nitration triggers profound structural changes in the heme ligation and microenvironment and impacts in protein function.

Peroxynitrite, a Stealthy Biological Oxidant

“…Recent evidence points to cytochrome c as an intramitochondrial heme peroxidase after a conformational change triggered by binding to cardiolipin and indicates that this process is related to apoptotic signaling (68). We have found that the preferential nitration of cytochrome by peroxynitrite-derived radicals in one of the solvent-exposed tyrosine residues (Tyr-74) leads to a conformational change that causes the displacement of the sixth ligand to the heme (Met-80) and a gain in peroxidase activity (67); the conformational change induced by nitration in cells also facilitates its translocation to the cytosol (even in non-apoptotic cells) (69). The biological significance of these events is currently under investigation.…”

“…Other oxidizing and nitrating species tend to react with tyrosines other than Tyr-34 and do not lead to enzyme inactivation. Efforts have been also directed to understand how peroxynitrite-dependent nitration affects the redox properties of cytochrome c, an abundant mitochondrial protein and key partner of the electron transport chain (67). Recent evidence points to cytochrome c as an intramitochondrial heme peroxidase after a conformational change triggered by binding to cardiolipin and indicates that this process is related to apoptotic signaling (68).…”

Cardiolipin drives cytochrome c proapoptotic and antiapoptotic actions

“…However, it has also been shown that peroxynitrite very slowly induces the nitration of the solvent-exposed Tyr74 residue of hhcytc in the absence of CL (the process takes 10 to 30 min). This leads to the cleavage of the Fe-Met80 bond, which is substituted by a weak Fe-Lys72 heme distal ligation (44).…”

A Non‐damaging Method to Analyze the Configuration and Dynamics of Nitrotyrosines in Proteins

“…[9] Thus, recent reports on the effects of protein nitration rely on the treatment of isolated proteins with this chemical. [10][11][12][13][14][15][16] In general, phenol rings are preferentially nitrated at ortho positions (e carbons in Tyr residues) relative to the hydroxyl group. Still, nitration could also take place at meta carbons, although the yield in this case is lower.…”