Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

Proc Natl Acad Sci U S A. 2014 Dec 9;111(49):17468-73. doi: 10.1073/pnas.1409692111. Epub 2014 Nov 24.

Abstract

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification.

Keywords: catalase A; cytochrome c peroxidase; heme transfer; mitochondrial H2O2; reverse translocation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalase / chemistry*
  • Cell Respiration
  • Cytochrome-c Peroxidase / chemistry*
  • Green Fluorescent Proteins / chemistry
  • Heme / chemistry*
  • Hydrogen Peroxide / chemistry
  • Intracellular Membranes / metabolism
  • Iron / chemistry
  • Ligands
  • Microscopy, Fluorescence
  • Mitochondria / enzymology*
  • Oxidation-Reduction
  • Oxidative Stress
  • Oxygen / chemistry
  • Saccharomyces cerevisiae / metabolism
  • Signal Transduction
  • Sulfhydryl Compounds

Substances

  • Ligands
  • Sulfhydryl Compounds
  • Green Fluorescent Proteins
  • Heme
  • Hydrogen Peroxide
  • Iron
  • Cytochrome-c Peroxidase
  • Catalase
  • Oxygen