Conformational change and human cytochrome c function: mutation of residue 41 modulates caspase activation and destabilizes Met-80 coordination

Josephs, Tracy M.; Liptak, Matthew D.; Hughes, Gillian; Lo, Alexandra; Smith, Rebecca; Wilbanks, Sigurd M.; Bren, Kara L.; Ledgerwood, Elizabeth C.

doi:10.1007/s00775-012-0973-1

Cited by 40 publications

(55 citation statements)

References 55 publications

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“…Rather, it would suggest that accumulation of Fe 2+ cyt c is required for Met 80 oxidation. Furthermore, it should be pointed out that conditions used in ex vivo mitochondrial respiration studies (low ATP) promote pentacoordination of cyt c [11]. This is consistent with previous findings that self-oxidation of Met 80 of cyt c occurs by its heme and molecular oxygen when Met 80 dissociates from the heme Fe in a reducing environment [38].…”

Section: Discussionsupporting

confidence: 87%

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

Birk

Chao

Liu

et al. 2015

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Background It was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met80 to Met80 sulfoxide (Met-O) in isolated hearts after ischemia-reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met80-Fe ligation of cyt c, forming pentacoordinated heme Fe2+, which inhibits electron transport (ET) and promotes oxygenase activity. Methods SS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of Met80-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy. Results Biochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met80-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20. Conclusion Our results show that ischemia disrupts the Met80-Fe ligation of cyt c resulting in formation of a globin-like pentacoordinated heme Fe2+ that inhibits ET, and converts cyt c into an oxygenase to cause CL peroxidation and proteolytic degradation of OPA1, resulting in cyt c release. General significance Cyt c heme structure represents a novel target for minimizing ischemic injury. SS-20, which we show to selectively target CL and protect the Met80-Fe ligation, minimizes ischemic injury and promotes ATP recovery.

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Section: Discussionsupporting

confidence: 87%

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

Birk

Chao

Liu

et al. 2015

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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“…The pK 695 values and thermodynamic parameters determined by chemical denaturation are for the ferric proteins. A significant decrease in the pK a value for the G41S mutant compared with WT is found and is in keeping with a recent report [39]. This indicates that the alkaline transition is accessible at a much lower pH for the G41S mutant compared with the WT protein ( Figure 2) and may have implications for peroxidase activity and CL binding at physiological pH values.…”

Section: Table 2 Physicochemical Properties Of Human Wt Cyt C and Mutsupporting

confidence: 90%

“…Protein Mass (Da) pK 695 E m histidine/methionine (mV compared with SHE) † G D-N H 2 O (kcal•mol − 1 ) m (kcal•mol − 1 •M − 1 (12263.5)* 7.6 (0.2) ‡ + 250 (5) ‡ 7.9 (0.3) 3.0 (0.3) 1.8 (0.1) Y46F 12217.63 (12217.5)* 8.4 (0.3) + 265 (5) 9.9 (0.4) 5.9 (0.2) 1.7 (0.3) Y48F 12216.74 (12217.5)* 8.9 (0.2) + 234 (5) 8.5 (0.4) 5.0 (0.2) 1.8 (0.2) Y46F/Y48F 12199.73 (12201.5)* 8.8 (0.2) No signals 8.7 (0.7) 5.0 (0.4) 1.8 (0.4) *Predicted mass of holo-protein. †Measured at pH 7.4.‡Similar values for the pK of the alkaline transition and E m (not determined by CV) have been reported in[39].…”

supporting

confidence: 62%

The hydrogen-peroxide-induced radical behaviour in human cytochrome c–phospholipid complexes: implications for the enhanced pro-apoptotic activity of the G41S mutant

Rajagopal

Edzuma

Hough

et al. 2013

Biochemical Journal

135

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We have investigated whether the pro-apoptotic properties of the G41S mutant of human cytochrome c can be explained by a higher than wild-type peroxidase activity triggered by phospholipid binding. A key complex in mitochondrial apoptosis involves cytochrome c and the phospholipid cardiolipin. In this complex cytochrome c has its native axial Met(80) ligand dissociated from the haem-iron, considerably augmenting the peroxidase capability of the haem group upon H2O2 binding. By EPR spectroscopy we reveal that the magnitude of changes in the paramagnetic haem states, as well as the yield of protein-bound free radical, is dependent on the phospholipid used and is considerably greater in the G41S mutant. A high-resolution X-ray crystal structure of human cytochrome c was determined and, in combination with the radical EPR signal analysis, two tyrosine residues, Tyr(46) and Tyr(48), have been rationalized to be putative radical sites. Subsequent single and double tyrosine-to-phenylalanine mutations revealed that the EPR signal of the radical, found to be similar in all variants, including G41S and wild-type, originates not from a single tyrosine residue, but is instead a superimposition of multiple EPR signals from different radical sites. We propose a mechanism of multiple radical formations in the cytochrome c-phospholipid complexes under H2O2 treatment, consistent with the stabilization of the radical in the G41S mutant, which elicits a greater peroxidase activity from cytochrome c and thus has implications in mitochondrial apoptosis.

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“…47 The Met-to-Lys ligand switch also takes place at near neutral pH in some mutants 46,48–53 of cyt c and in the presence of denaturant urea. 54 Lysine ligation to the heme iron at near neutral pH has also been observed upon carboxymethylation 55 and tyrosine nitration of cyt c 56 and also upon binding to CL.…”

Section: Resultsmentioning

confidence: 99%

Ligation and Reactivity of Methionine-Oxidized Cytochrome c

Zhong

Pletneva

2018

Inorg. Chem.

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Met80, one of the heme iron ligands in cytochrome c (cyt c) is readily oxidized to Met sulfoxide (Met-SO) by several biologically-relevant oxidants. The modification has been suggested to impact both the electron-transfer (ET) and apoptotic functions of this metalloprotein. The coordination of the heme iron in Met-oxidized cyt c (Met-SO cyt c) is critical for both of these functions but has remained poorly defined. We present electronic absorption, NMR, and EPR spectroscopic investigations as well kinetic studies and mutational analyses to identify the heme iron ligands in yeast iso-1 Met-SO cyt c. Similar to the alkaline form of native cyt c, Lys73 and Lys79 ligate to the ferric heme iron in the Met80-oxidized protein but this coordination takes place at much lower pH. The ferrous heme iron is ligated by Met-SO implying the redox-linked ligand switch in the modified protein. Binding studies with a model peptide microperoxidase-8 provide a rationale for alterations in ligation and for the role of the polypeptide packing in native and Met-SO cyt c. Imidazole binding experiments have revealed that Lys dissociation from the ferric heme in K73A/K79G/M80K (M80K#) and Met-SO is more than three orders of magnitude slower than the opening of the heme pocket that limits Met80 replacement in native cyt c. The Lys-to-Met-SO ligand substitution gates ET of ferric Met-SO cyt c with Co(terpy)22+. Owing to the slow Lys dissociation step, ET reaction is slow but possible, which is not the case for non-switchable M80A and M80K#. Acidic conditions cause Lys replacement by a water ligand in Met-SO cyt c (pKa=6.3±0.1) increasing intrinsic peroxidase activity of the protein. This pH-driven ligand switch may be a mechanism to boost peroxidase function of cyt c specifically in apoptotic cells.

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Conformational change and human cytochrome c function: mutation of residue 41 modulates caspase activation and destabilizes Met-80 coordination

Cited by 40 publications

References 55 publications

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

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

The hydrogen-peroxide-induced radical behaviour in human cytochrome c–phospholipid complexes: implications for the enhanced pro-apoptotic activity of the G41S mutant

Ligation and Reactivity of Methionine-Oxidized Cytochrome c

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