Self-oxidation of cytochrome c at methionine80 with molecular oxygen induced by cleavage of the Met–heme iron bond

Abstract

Met80 of cytochrome c (cyt c) has been shown to dissociate from its heme iron when cyt c interacts with cardiolipin (CL), which triggers the release of cyt c into the cytosol initiating apoptosis. We found that the mass of human cyt c increases by 16 Da in the Met80-Lys86 region by reaction with molecular oxygen in the presence of CL-containing liposomes and dithiothreitol (DTT). To investigate the effect of Met80 dissociation on the reaction of cyt c with molecular oxygen without affecting its secondary structures, a human cyt c mutant (Δ8384 cyt c) was constructed by removing two amino acids (Val83 and Gly84) from the loop containing Met80. According to MALDI-TOF-MS and tandem mass measurements, Met80 of Δ8384 cyt c was modified site-specifically to methionine sulfoxide when purified in the presence of molecular oxygen, whereas Met80 was not modified in the absence of molecular oxygen. A red-shift of the Soret band from 406 to 412 nm and absorption increase at ∼536 and ∼568 nm were observed for Δ8384 cyt c when it reacted with DTT and molecular oxygen, followed by a further red-shift of the Soret band to 416 nm and absorption increase at ∼620 and ∼650 nm. These results indicate that Met80 of cyt c is oxidized site-specifically by formation of the oxy and subsequent compound I-like species when Met80 dissociates from the heme iron, where the Met80 modification may affect its peroxidase activity related to apoptosis.

Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia

“…Since Met 80 can only undergo self-oxidation when it is not coordinating the heme Fe [19], these results support ex vivo formation of pentacoordinated cyt c during ischemia-reperfusion. Using normal isolated mitochondria, these investigators proposed that electron flow through cyt c , coupled with excessive ROS production, is required for the oxidation of Met 80 [24].…”

“…These changes can account for the inhibition of mitochondrial respiration when ischemic mitochondria are exposed to reperfusion conditions. Displacement of Met 80 from the heme Fe 2+ converts cyt c from an electron carrier to an oxygenase that can oxidize cardiolipin, resulting in cristae degradation and detachment of cyt c from the IMM [17–19]. The ultimate release of cyt c from the ICS occurs when OPA1 is degraded during ischemia, as OPA1 plays a major role in establishing cristae junctions and keeping the cyt c within the ICS [41].…”

“…In addition, the restoration of Fe 3+ cyt c upon reperfusion can be oxidized by H 2 O 2 to oxyferryl heme, forming the so-called peroxidase compound I-type intermediate, a highly reactive oxidant that can react with proteins and lipids on the IMM and in the IMS [38, 49]. Peroxidase activity and oxidized CL can be readily detected with [cyt c /CL] complexes in vitro and with isolated mitochondria [18, 19]. Cyt c oxygenase or peroxidase activity has not been demonstrated in vivo , but the presence of Met-O cyt c supports the existence of oxygenase/peroxidase activity, which can account for cristae degradation.…”

“…Cyt c oxygenase or peroxidase activity has not been demonstrated in vivo , but the presence of Met-O cyt c supports the existence of oxygenase/peroxidase activity, which can account for cristae degradation. Degeneration of mitochondrial morphology, especially cristaeolysis, is commonly observed in ischemic mitochondria [3, 19, 24]. Peroxidized cardiolipin can further elicit a free radical chain reaction even without further ROS production.…”

“…Electrostatic interaction between cyt c and CL anchors cyt c to the IMM and optimizes ET between complex III and complex IV [13]. Interestingly, low ATP conditions favor hydrophobic interaction between cyt c and CL [14, 15], which is known to disrupt the Met 80 -Fe ligation, resulting in a pentacoordinated heme Fe [16–19]. The loss of the Met 80 -Fe ligation inhibits ET and converts cyt c from an electron carrier to an oxygenase/peroxidase that can cause CL peroxidation [12, 20–23].…”