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

Rajagopal, Badri S.; Edzuma, Ann N.; Hough, Michael A.; Blundell, Katie L. I. M.; Kagan, Valerian E.; Kapralov, Alexandr A.; Fraser, Lewis A.; Butt, Julea N.; Silkstone, Gary; Wilson, Michael T.; Svistunenko, Dimitri A.; Worrall, J.A.R.

doi:10.1042/bj20130758

Cited by 83 publications

(142 citation statements)

References 47 publications

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“…The overall quality of the 20 lowest TF ensembles was good, according to the PROCHECK G factor (38), MolProbity clashscores (39), and other structural quality indicators (SI Appendix, Table S2). Most residues were in the Ramachandran plot favored regions, whereas Cys14, Cys17, His18, Val20, Lys37, and Asn70 were in the generously allowed ones, as also observed for the solution structure of WT Cc (33,40). Cys14, Cys17, and His18 are covalently bonded to the heme moiety, thereby straining their backbone conformation, as already described for c-type cytochromes (35,36).…”

mentioning

confidence: 68%

“…In addition, CL interacts with Cc in a two-step binding reaction (65): In the first step, the so-called A site at the Cc surface makes transient electrostatic contacts with the membrane; in the second step, hydrophobic forces drive the formation of a tight and stable Cc-CL complex, with one of the CL acyl chains entering the hydrophobic groove of Cc (termed the C site) (66-68). C-site binding then triggers Cc-regulated CL peroxidation under oxidative stress and induces early apoptosis (40). Interestingly, several residues of Cc (Lys22, Lys27, and His33) become protonated at low pH to form an extra binding site-the so-called L site-that facilitates the electrostatic interaction of Cc with mitochondrial membranes (69).…”

Section: Phosphorylation Of Tyr48 Enhances Internal Mobility In Cytocmentioning

confidence: 99%

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Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

Moreno‐Beltrán

Guerra‐Castellano

Díaz-Quintana

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-l-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects.

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mentioning

confidence: 68%

Section: Phosphorylation Of Tyr48 Enhances Internal Mobility In Cytocmentioning

confidence: 99%

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

Moreno‐Beltrán

Guerra‐Castellano

Díaz-Quintana

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…The distance between Glu 28 C␣ and Glu 28 * C␣ is 7.2 Å. I, equivalent to C for T28E Cytc. The RMSF values for Ala 44 are of average size and similar for both halves of the simulation. The RMSF values for the loop containing Glu 28 are higher than average but similar for both halves of the simulation.…”

mentioning

confidence: 82%

“…The RMSF values for Ala 44 are lower in the second 100 ns, but those for Lys 27 are significantly higher in the second 100 ns, in fact the highest seen in all four RMSF plots. G, chain A from the T28E crystal structure (PDB entry 5DF5) before (green) and after (cyan) 200 ns of molecular dynamics.…”

mentioning

confidence: 91%

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney

Mahapatra

Varughese

et al. 2017

Journal of Biological Chemistry

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Edited by Dennis R. VoelkerMammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr 28 , leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (⌬⌿ m ), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr 28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr 28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr 28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissuespecific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ⌬⌿ m hyperpolarization, a known cause of ROS and trigger of apoptosis.

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“…1a) (23) and bovine CcO (3AG2, chains A-M, Fig. 1b) (24) were used as initial geometries for molecular docking simulations because of their high resolution without any amino acid mutations.…”

Section: Methodsmentioning

confidence: 99%

Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation

Sato

Hitaoka

Inoue

et al. 2016

Journal of Biological Chemistry

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Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 12271-12276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions that were almost cancelled out by large favorable (Columbic) electrostatic interactions but resulted in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, whereas the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO. The electron transfer (ET)3 reactions in mitochondrial and bacterial respiratory chains are essential processes for energy transduction in cells. A series of ET reactions is terminated at cytochrome c oxidase (CcO), where molecular oxygen is reduced to water. Associated with the reduction of molecular oxygen, CcO functions as a proton pump across the membrane, and the proton gradient is the primary driving force for the generation of ATP (1-5).In the respiratory chain of mitochondria, the electrons to reduce molecular oxygen at CcO are donated from a small hemoprotein, cytochrome c (Cyt c), and Cyt c is thought to form an ET complex with CcO to promote the ET reaction from the heme iron in Cyt c to the Cu A site in CcO (6, 7). Although the reduction of molecular oxygen to water molecules requires four electrons, Cyt c can carry only one electron, implying that Cyt c repetitively associates with and dissociates from CcO and suggesting that the specific interprotein interactions between Cyt c and CcO regulate the binding affinity and the ET rate from Cyt c to CcO.The amino acid sequence and isoelectric point of Cyt c suggest that many positively charged residues are located on the protein surface (6, 7), as confirmed by solving the three-dimensional structures of Cyt c (8), allowing...

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

Cited by 83 publications

References 47 publications

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney

Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation

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