59 - Peroxide-Regulated Heme Transfer: A Novel Redox Signaling Mechanism

Abstract

Using high-performance mass spectrometry (MS), we have uncovered an unprecedented mechanism of redox signaling by H2O2. Remarkably, many cycles of sequential binding and reduction of peroxide can occur in vitro at the heme of cytochrome c peroxidase (Ccp1) in the absence of its reducing substrate, cytochrome c. MS reveals that ~20 oxygen atoms are incorporated into Ccp1, predominantly at methionine and tryptophan residues. Additionally, extensive intramolecular dityrosine formation involving neighboring tyrosines (Y36/Y39, Y36/Y42 and Y67/Y71) was uncovered on MS sequencing of the crosslinked peptides. A total of 24 residues are oxidized by repeated hole-hopping from the heme in Ccp1 treated with a 10-fold excess of H2O2. Oxidation of the catalytic distal H52 shuts down H2O2 reduction but not until the proximal heme ligand (H175) is oxidized, which labilizes the heme. Notably, no irreversible heme modification is detected by MS, indicating that Ccp1 preserves the integrity of its heme by directing the oxidizing equivalents from H2O2 onto its polypeptide. These properties of Ccp1 in vitro are consistent with our observation that it acts as a heme chaperone for apoCta1, a heme-dependent catalase found in yeast mitochondria. When levels of H2O2 rise during the cell’s switch to respiratory metabolism, maturation of Cta1, as monitored by the rise in its catalase activity, coincides with apoCcp1 exit from mitochrondria (1). MS analysis reveals that extramitochondrial Ccp1 is ~80% oxidized at H175, which confirms that its heme has been labilized by mitochondrial H2O2 to trigger heme transfer to apoCta1. This sequence of events constitutes the first example of H2O2 regulating its own intracellular concentration by turning on catalase activity post-translationally via heme transfer. M. Kathiresan, D. Martins and A.M. English Proc. Nat. Acad. Sci., USA 2014, 111, 17468–17473.