Abstract
Cytochrome c (cyt c) is a small hemoprotein involved in electron shuttling in the mitochondrial respiratory chain and is now also recognized as an important mediator of apoptotic cell death. Its role in inducing programmed cell death is closely associated with the formation of a complex with the mitochondrion-specific phospholipid cardiolipin (CL), leading to a gain of peroxidase activity. However, the molecular mechanisms behind this gain and eventual cyt c autoinactivation via its release from mitochondrial membranes remain largely unknown. Here, we examined the kinetics of the H2O2-mediated peroxidase activity of cyt c both in the presence and absence of tetraoleoyl cardiolipin (TOCL)- and tetralinoleoyl cardiolipin (TLCL)-containing liposomes to evaluate the role of cyt c-CL complex formation in the induction and stimulation of cyt c peroxidase activity. Moreover, we examined peroxide-mediated cyt c heme degradation to gain insights into the mechanisms by which cyt c self-limits its peroxidase activity. Bottom-up proteomics revealed >50 oxidative modifications on cyt c upon peroxide reduction. Of note, one of these by-products was the Tyr-based "cofactor" trihydroxyphenylalanine quinone (TPQ) capable of inducing deamination of Lys ϵ-amino groups and formation of the carbonylated product aminoadipic semialdehyde. In view of these results, we propose that autoinduced carbonylation, and thus removal of a positive charge in Lys, abrogates binding of cyt c to negatively charged CL. The proposed mechanism may be responsible for release of cyt c from mitochondrial membranes and ensuing inactivation of its peroxidase activity.
Highlights
Taking into account the presence of several oxidation states for cyt c Tyr residues accompanied by a high number of carbonylated Lys residues, we speculated that Lys deamination to carbonyl-containing aminoadipic semialdehyde via in situ produced trihydroxyphenylalanine quinone (TPQ) “cofactor,” similar to the enzymatic deamination of primary amines described for copper amine oxidase [28], might occur in cyt c under oxidative environment accompanied by the release of a free iron (Fig. S8)
Cyt c has received a lot of scientific attention due its role in apoptosis associated with cyt c release in cytoplasm and caspase activation
Cyt c forms a complex with the mitochondrion-specific lipid cardiolipin, which, upon oxidation, leads to the detachment of the protein from the inner mitochondrial membrane, permeabilization of the outer membrane, and subsequent release of cyt c to the cytoplasm [29]
Summary
Cytochrome c (cyt c) is a small hemoprotein involved in electron shuttling in the mitochondrial respiratory chain and is recognized as an important mediator of apoptotic cell death. Cyt c binds to cardiolipin (CL), a mitochondrion-specific phospholipid, which redistributes from the inner to the outer mitochondrial membrane upon apoptosis onset (8 –10) This complex formation results in structural modifications of the protein that trigger a strong peroxidase activity in the presence of H2O2 [11, 12], leading to a specific oxidation of the polyunsaturated fatty acyl chains in CL [7, 12, 13], causing permeabilization of the mitochondrial membrane and the release of cyt c and other proapoptotic factors [8, 13]. Over 50 oxidative modifications of cyt c resulting from the enzymatic activity of cyt c–CL complexes were studied using a bottom-up proteomics approach, and a new mechanism of autocatalyzed cyt c carbonylation via Tyr-derived cofactor leading to the reduction of protein positive charge and dissociation of cyt c–CL complex was proposed
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