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

Abstract

Significance We provide to our knowledge the first in vivo and in vitro evidence for H 2 O 2 -triggered heme transfer between proteins. Specifically, H 2 O 2 binds to and labilizes cytochrome c peroxidase (Ccp1)’s heme by oxidizing the proximal Fe ligand (His175), which activates Ccp1 to transfer its heme to apoCta1, and apoCcp1 subsequently escapes from mitochondria. This sequence of H 2 O 2 -activated heme labilization, heme transfer between proteins, and protein relocalization defines a previously undefined mechanism of H 2 O 2 signaling in cells. In contrast, established H 2 O 2 signaling mechanisms are dominated by thiol-based redox changes.

LC-MS/MS suggests that hole hopping in cytochrome c peroxidase protects its heme from oxidative modification by excess H2O2

“…Recombinant Ccp1 with MI at positions –2 and –1 of the mature protein was overexpressed as the apoprotein in BL21(DE3) cells, purified and reconstituted with hemin as described previously. 14 Suppliers of (bio)chemicals were as follows: Coomassie (MP Biomedicals); hemin chloride, HPLC grade acetonitrile, diethylenetriamine-pentaacetic acid (DTPA) (Sigma Aldrich); and 30% hydrogen peroxide (Fisher Scientific).…”

“…32 The CCP activity of oxidized Ccp1 was determined by monitoring the oxidation of horse heart Cyc II by H 2 O 2 as reported. 14 …”

“…1), is extensively oxidized in Ccp1 isolated from respiring yeast. 14 Thus, labilization of Ccp1's heme on H175 oxidation enables its transfer to apoCta1, converting the latter into a powerful catalytic H 2 O 2 scavenger.…”

Characterization of two key enzymes for aromatic amino acid biosynthesis in symbiotic archaea

“…Tryptic peptides were obtained by resolving TyrA preparations by SDS-PAGE, excising appropriate regions from the gel and then performing trypsin proteolysis on each sample as outlined previously (Shevchenko et al 1996). The resulting peptides were resuspended in 15-50 µL of solubilization solution containing 2 % acetonitrile (ACN) and 1 % of formic acid (FA) and processed by LC-MS/MS as described previously (Kathiresan et al 2014). MS/MS fragments were analyzed using the Thermo Proteome Discoverer software (v1.3) with the SEQUEST search engine with mass filters for the oxidation of methionine (+15.995 Da) and for the alkylation of cysteine (+57.021 Da).…”

Peroxynitrite and hydrogen peroxide elicit similar cellular stress responses mediated by the Ccp1 sensor protein

“…Very recently, we reported that signaling by endogenous, respiration-derived H 2 O 2 involves transfer of Ccp1's heme to mitochondrial/peroxisomal catalase A [23,59]. The strength of this signaling regulates life span by limiting the protective H 2 O 2 -induced mitohormesis response of the cell [47].…”