Nitration of tyrosine residues 368 and 345 in the β-subunit elicits FoF1-ATPase activity loss

Yasuko Fujisawa, Kazunobu Kato, Cecilia R Giulivi

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

Tyrosine nitration is a covalent post-translational protein modification associated with various diseases related to oxidative/nitrative stress. A role for nitration of tyrosine in protein inactivation has been proposed; however, few studies have established a direct link between this modification and loss of protein function. In the present study, we determined the effect of nitration of Tyr345 and Tyr368 in the β-subunit of the F1-ATPase using site-directed mutagenesis. Nitration of the β-subunit, achieved by using TNM (tetranitromethane), resulted in 66% ATPase activity loss. This treatment resulted in the modification of several asparagine, methionine and tyrosine residues. However, nitrated tyrosine and ATPase inactivation were decreased in reconstituted F1 with Y368F (54%), Y345F (28%) and Y345,368F (1%) β-subunits, indicating a clear link between nitration at these positions and activity loss, regardless of the presence of other modifications. Kinetic studies indicated that an F1 with one nitrated tyrosine residue (Tyr345 or Tyr368) or two Tyr368 residues was sufficient to grant inactivation. Tyr368 was four times more reactive to nitration due to its lower pKa. Inactivation was attributed mainly to steric hindrance caused by adding a bulky residue more than the presence of a charged group or change in the phenolic pKa due to the introduction of a nitro group. Nitration at this residue would be more relevant under conditions of low nitrative stress. Conversely, at high nitrative stress conditions, both tyrosine residues would contribute equally to ATPase inactivation.

Original languageEnglish (US)
Pages (from-to)219-231
Number of pages13
JournalBiochemical Journal
Volume423
Issue number2
DOIs
StatePublished - Oct 15 2009

Keywords

  • Aging
  • ATPase
  • Mitochondria
  • Nitrative stress
  • Structure-activity relationship
  • Tyrosine nitration

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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