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

IMPLICATIONS FOR AMP KINASE*
Open AccessPublished:October 07, 2016DOI:https://doi.org/10.1074/jbc.M116.744664
      Mammalian 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 Thr28, 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 (ΔΨm), 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, Thr28 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 Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

      Introduction

      Cytochrome c (Cytc)
      The abbreviations used are: Cytc
      cytochrome c
      ETC
      electron transport chain
      CcO
      cytochrome c oxidase
      ROS
      reactive oxygen species
      AMPK
      AMP-activated kinase
      CL
      cardiolipin
      TOCL
      tetralinoleyl-CL
      PDB
      Protein Data Bank
      RMSF
      root mean square fluctuation(s)
      Tricine
      N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
      DCIP
      2,6-dichloroindophenol
      ΔΨm
      mitochondrial membrane potential
      ESI
      electrospray ionization.
      is a small (12-kDa) globular nucleus-encoded mitochondrial protein containing a covalently attached heme group with multiple functions. In the electron transport chain (ETC), it functions as a single electron carrier between bc1 complex (complex III) and cytochrome c oxidase (CcO, complex IV) and is thus essential for aerobic energy production. The second important role of Cytc is seen under conditions of stress, when it functions as a crucial pro-apoptotic signal (
      • Green D.R.
      • Reed J.C.
      Mitochondria and apoptosis.
      ). During apoptosis, Cytc is released from mitochondria into the cytosol, where it interacts with Apaf-1 to form the apoptosome, which in turn activates caspase-9 and the downstream executioner caspase cascade. Furthermore, Cytc functions as a cardiolipin peroxidase during the early phase of apoptosis, when it oxidizes the mitochondrial membrane lipid cardiolipin, thereby facilitating its own release from the inner mitochondrial membrane (
      • Kagan V.E.
      • Tyurin V.A.
      • Jiang J.
      • Tyurina Y.Y.
      • Ritov V.B.
      • Amoscato A.A.
      • Osipov A.N.
      • Belikova N.A.
      • Kapralov A.A.
      • Kini V.
      • Vlasova I.I.
      • Zhao Q.
      • Zou M.
      • Di P.
      • Svistunenko D.A.
      • et al.
      Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors.
      ). In contrast, under healthy, non-apoptotic conditions, Cytc acts as a scavenger of reactive oxygen species (ROS) (
      • Pereverzev M.O.
      • Vygodina T.V.
      • Konstantinov A.A.
      • Skulachev V.P.
      Cytochrome c, an ideal antioxidant.
      ), and it takes part in other redox reactions inside mitochondria, including redox-coupled protein import (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins.
      ) and reduction of p66Shc, a protein that is implicated in the generation of ROS and apoptosis (
      • Giorgio M.
      • Migliaccio E.
      • Orsini F.
      • Paolucci D.
      • Moroni M.
      • Contursi C.
      • Pelliccia G.
      • Luzi L.
      • Minucci S.
      • Marcaccio M.
      • Pinton P.
      • Rizzuto R.
      • Bernardi P.
      • Paolucci F.
      • Pelicci P.G.
      Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis.
      ).
      Given the multiple functions of Cytc, it is not surprising that it is tightly regulated. Two regulatory mechanisms via expression of a somatic and testis-specific isoform pair and allosteric regulation through binding of ATP have been known for over 30 years (
      • Goldberg E.
      • Sberna D.
      • Wheat T.E.
      • Urbanski G.J.
      • Margoliash E.
      Cytochrome c: immunofluorescent localization of the testis-specific form.
      ,
      • Ferguson-Miller S.
      • Brautigan D.L.
      • Margoliash E.
      Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase.
      ). A third mechanism via reversible phosphorylation was discovered recently when we purified bovine Cytc from heart and liver tissue under conditions preserving the physiological phosphorylation status. The two proteins were phosphorylated on Tyr97 and Tyr48, respectively (
      • Lee I.
      • Salomon A.R.
      • Yu K.
      • Doan J.W.
      • Grossman L.I.
      • Hüttemann M.
      New prospects for an old enzyme: mammalian cytochrome c is tyrosine-phosphorylated in vivo.
      ,
      • Yu H.
      • Lee I.
      • Salomon A.R.
      • Yu K.
      • Hüttemann M.
      Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration.
      ). Both modifications lead to a partial inhibition of respiration. In addition, the phosphomimetic Y48E substitution abolished the capability of Cytc to trigger apoptosis, suggesting that Cytc phosphorylation regulates apoptosis at the level of the apoptosome (
      • Pecina P.
      • Borisenko G.G.
      • Belikova N.A.
      • Tyurina Y.Y.
      • Pecinova A.
      • Lee I.
      • Samhan-Arias A.K.
      • Przyklenk K.
      • Kagan V.E.
      • Hüttemann M.
      Phosphomimetic substitution of cytochrome c tyrosine 48 decreases respiration and binding to cardiolipin and abolishes ability to trigger downstream caspase activation.
      ).
      Here we report that Cytc purified from bovine and rat kidney tissues in the presence of phosphatase inhibitors is phosphorylated on Thr28. In vivo phosphorylated and T28E phosphomimetic Cytc led to an inhibition of respiration in the reaction with CcO. Introduction of WT and T28E phosphomimetic Cytc into Cytc knock-out cells showed that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels were reduced. This suggests that Cytc phosphorylation can regulate ETC flux, preventing ΔΨm hyperpolarization, a known trigger of ROS production and apoptosis (
      • Kadenbach B.
      • Arnold S.
      • Lee I.
      • Hüttemann M.
      The possible role of cytochrome c oxidase in stress-induced apoptosis and degenerative diseases.
      ). Finally, we provide evidence suggesting that phosphorylation of Thr28 is mediated by AMP kinase (AMPK), which co-localizes with Cytc in the mitochondrial intermembrane space. This is the first report of a mapped phosphorylation site on a mammalian oxidative phosphorylation component, together with functional and structural analyses and a kinase candidate mediating this site-specific modification.

      Discussion

      Little is known about the regulation of mitochondrial oxidative phosphorylation by cell signaling. We have previously reported two distinct tyrosine phosphorylation sites on Cytc from mammalian heart and liver tissue. The current report functionally characterizes a third tissue-specific phosphorylation site, Thr28, on Cytc purified from kidney. The same site was also mapped, but not further studied, in a high throughput mass spectrometry study using resting human skeletal muscle (
      • Zhao X.
      • Leon I.R.
      • Bak S.
      • Mogensen M.
      • Wrzesinski K.
      • Hojlund K.
      • Jensen O.N.
      Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes.
      ), suggesting that Thr28 can be targeted to regulate ETC function beyond kidney tissue. For the Cytc we isolated from kidney, the majority (between ∼60 and 80%, depending on the preparation) of the protein is phosphorylated at Thr28. In addition, we mutated Thr28 to phosphomimetic glutamate and non-phosphorylatable alanine to further characterize the effects of this phosphorylation in vitro and in cultured murine lung fibroblast cells lacking both Cytc isoforms.
      Bovine in vivo phosphorylated and unphosphorylated Cytc as well as overexpressed mouse WT, T28E, and T28A Cytc generated hyperbolic kinetics in the reaction with purified bovine liver CcO. Compared with WT, maximal turnover was reduced by 50 and 73% for in vivo phosphorylated and phosphomimetic Cytc, respectively. Furthermore, expression of phosphomimetic T28E Cytc led to a reduction of respiration in intact cells, suggesting for the first time that modification of the small electron carrier can control overall ETC flux. These findings are consistent with the concept that phosphorylation of mitochondrial proteins, in general, down-regulates whereas dephosphorylation activates mitochondrial function (
      • Hopper R.K.
      • Carroll S.
      • Aponte A.M.
      • Johnson D.T.
      • French S.
      • Shen R.F.
      • Witzmann F.A.
      • Harris R.A.
      • Balaban R.S.
      Mitochondrial matrix phosphoproteome: effect of extra mitochondrial calcium.
      ). Reduced respiration rates both in vitro with purified CcO and in intact cells expressing T28E Cytc may be a result of the observed change in the redox midpoint potential as well as of structural changes in the protein. Thr28 lies in the center of an unusual structural element termed the “negative classical γ turn,” which is composed of residues 27–29 and is important for the stability of Cytc (
      • Sanishvili R.
      • Volz K.W.
      • Westbrook E.M.
      • Margoliash E.
      The low ionic strength crystal structure of horse cytochrome c at 2.1 Å resolution and comparison with its high ionic strength counterpart.
      ). Thr28 is a surface residue close to the solvent exposed and accessible tip of the heme group that mediates electron transfer to CcO. It is located on the frontal right side of the molecule in the conventional view (Fig. 3A), which is part of the circular, positively charged epitope surrounding the heme crevice with which Cytc binds to the corresponding negatively charged epitope on CcO (
      • Roberts V.A.
      • Pique M.E.
      Definition of the interaction domain for cytochrome c on cytochrome c oxidase. III. Prediction of the docked complex by a complete, systematic search.
      ). Interestingly, in this computational Cytc-CcO docking model, Thr28 is located at the interface of catalytic subunits I and II and nuclear encoded subunit VIIc, with closest distances of <6 Å to Lys47 of subunit VIIc and of <7 Å to Asp50 of subunit I and Trp104 and Ser202 of subunit II (supplemental Fig. 4G). Two of the four CcO residues are particularly noteworthy. Asp50 is one of only a handful of residues in the entire CcO complex that have noticeably different geometries between the reduced and oxidized state in the crystal structure (
      • Sugitani R.
      • Stuchebrukhov A.A.
      Molecular dynamics simulation of water in cytochrome c oxidase reveals two water exit pathways and the mechanism of transport.
      ). In addition, Asp50 is located next to another flexible amino acid, Asp51, which, when mutated to Asn, blocks proton pumping of the enzyme and was proposed to be the proton ejection site of CcO (
      • Tsukihara T.
      • Shimokata K.
      • Katayama Y.
      • Shimada H.
      • Muramoto K.
      • Aoyama H.
      • Mochizuki M.
      • Shinzawa-Itoh K.
      • Yamashita E.
      • Yao M.
      • Ishimura Y.
      • Yoshikawa S.
      The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process.
      ). Phosphorylation of Thr28 may interfere with the outward movement of the Asp50-Asp51 region and thus the opening of the proposed proton exit channel during reduction of CcO. This would lead to inhibition of CcO by inhibiting electron transfer-coupled proton pumping. Alternatively, repulsion between phospho-Thr28 and Asp50 may result in suboptimal binding to CcO, causing reduced respiration rates. The second interesting interaction site on CcO is Trp104, which is the site on CcO where electrons from Cytc enter before reaching the first metal center, the binuclear CuA site. Spatial interference upon Cytc Thr28 phosphorylation with Trp104, which is essential for catalysis, could also explain reduced activity.
      We have previously shown that phosphomimetic Y48E Cytc is incapable of downstream caspase activation (
      • Pecina P.
      • Borisenko G.G.
      • Belikova N.A.
      • Tyurina Y.Y.
      • Pecinova A.
      • Lee I.
      • Samhan-Arias A.K.
      • Przyklenk K.
      • Kagan V.E.
      • Hüttemann M.
      Phosphomimetic substitution of cytochrome c tyrosine 48 decreases respiration and binding to cardiolipin and abolishes ability to trigger downstream caspase activation.
      ). In contrast, phosphorylation of Cytc Thr28 or its phosphomimetic substitution does not interfere with the ability of Cytc to trigger downstream caspase activation. This difference agrees with the observation that the Tyr48 epitope is directly involved in the interaction of Cytc with Apaf-1 whereas the Thr28 epitope is not (
      • Zhou M.
      • Li Y.
      • Hu Q.
      • Bai X.C.
      • Huang W.
      • Yan C.
      • Scheres S.H.
      • Shi Y.
      Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.
      ). This ability to similarly inhibit CcO activity but differently affect caspase activation is an example of tissue-specific signaling, which in this case may result from AMPK isoform expression in a tissue-specific manner.
      A recent study compared 285 Cytc sequences across all phyla from humans to bacteria (
      • Zaidi S.
      • Hassan M.I.
      • Islam A.
      • Ahmad F.
      The role of key residues in structure, function, and stability of cytochrome-c.
      ). Overall, threonine is the most common amino acid at position 28 and is conserved in mammals. However, in some non-mammalian organisms, five other amino acids can also be found, namely Gln, Val, Ile, Ser, and even the phosphomimetic Glu, which is present in several plants, including potatoes and tomatoes. Interestingly, alanine is not among the residues evolutionarily tolerated in this position. Another study using T28A mutant Cytc also reported decreased respiration rates with CcO (
      • Guerra-Castellano A.
      • Díaz-Moreno I.
      • Velázquez-Campoy A.
      • De la Rosa M.A.
      • Díaz-Quintana A.
      Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47.
      ), confirming our results. We found that T28A Cytc has a higher ability to activate downstream caspases and unfolds more easily, which increases CL oxidation. Its instability compared with WT and T28E Cytc is also suggested in the respective circular dichroism spectra (supplemental Fig. 1C, see lower wavelength range). In addition, T28A Cytc is most rapidly oxidized and degraded in the presence of H2O2. Structural analysis of all three Cytc variants shows that the amino acids comprising the negative classical γ turn element display by far the highest root mean square deviation values compared with any other Cytc sequence, suggesting that the Thr28 epitope is the most flexible element of the entire molecule. A plot of the average temperature factors, which for these high resolution structures are a reasonable measure of local mobility, also showed, other than at the termini, that the highest relative values occurred at the 22–30 loop for all three structures. Furthermore, molecular dynamics simulations of crystalized T28A Cytc produce a structure in which the Thr28 Cα atom is moved by 11.9 Å, approximately twice as far compared with WT, T28E, and modeled phospho-Thr28 Cytc (Fig. 3). These findings collectively suggest that alanine was evolutionarily selected against because it introduces additional flexibility at a site near the heme crevice, probably due to its small size, leading to a detrimental reduction of protein stability and interference with its multiple functions. This may also at least in part account for the finding of lower Cytc protein levels in cells expressing this mutant. Another evolutionarily forbidden substitution, T28D, which introduces a negative charge, also generates “rogue” functional changes in Cytc, as seen in the reaction with CcO, that are even opposite (
      • Guerra-Castellano A.
      • Díaz-Moreno I.
      • Velázquez-Campoy A.
      • De la Rosa M.A.
      • Díaz-Quintana A.
      Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47.
      ) of what we report for in vivo phosphorylated Cytc, whereas glutamate replacement, as used here, produces the same functional effects as phosphorylated Cytc.
      Our studies suggest that AMPK targets Cytc for Thr28 phosphorylation within the mitochondria. Future work using a genetic approach should be conducted to confirm the role of AMPK in Cytc phosphorylation. However, because kidneys express both the AMPK-α1 and -α2 catalytic isoforms and because their double knock-out results in embryonic lethality (
      • Viollet B.
      • Athea Y.
      • Mounier R.
      • Guigas B.
      • Zarrinpashneh E.
      • Horman S.
      • Lantier L.
      • Hebrard S.
      • Devin-Leclerc J.
      • Beauloye C.
      • Foretz M.
      • Andreelli F.
      • Ventura-Clapier R.
      • Bertrand L.
      AMPK: lessons from transgenic and knockout animals.
      ), a more advanced approach would be necessary, such as a tissue-specific knock-out.
      AMPK is one of the most important and evolutionarily oldest metabolic sensors and regulators (
      • Hardie D.G.
      AMPK–sensing energy while talking to other signaling pathways.
      ). It is implicated in human disease, including diabetes, where its activity is impaired in several organs, including the kidneys (
      • Dugan L.L.
      • You Y.H.
      • Ali S.S.
      • Diamond-Stanic M.
      • Miyamoto S.
      • DeCleves A.E.
      • Andreyev A.
      • Quach T.
      • Ly S.
      • Shekhtman G.
      • Nguyen W.
      • Chepetan A.
      • Le T.P.
      • Wang L.
      • Xu M.
      • et al.
      AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function.
      ). Generally, AMPK promotes catabolic processes, and it is activated by phosphorylation and allosterically under conditions when ATP levels drop and AMP levels increase. However, our understanding of the role of AMPK specifically in the kidney is in its infancy (
      • Ix J.H.
      • Sharma K.
      Mechanisms linking obesity, chronic kidney disease, and fatty liver disease: the roles of fetuin-A, adiponectin, and AMPK.
      ), and there are reports suggesting that it operates differently in this organ compared with other tissues. For example, AMPK is already active in the kidney under basal conditions and shows a paradoxical decrease in activity in a rat kidney ablation and infarction model (
      • Satriano J.
      • Sharma K.
      • Blantz R.C.
      • Deng A.
      Induction of AMPK activity corrects early pathophysiological alterations in the subtotal nephrectomy model of chronic kidney disease.
      ), a condition when energy depletion and thus demand is maximal. In cells from patients with hereditary leiomyomatosis renal cell cancer, the Krebs cycle is inhibited, which also leads to a paradoxical decrease of AMPK activity (
      • Tong W.H.
      • Sourbier C.
      • Kovtunovych G.
      • Jeong S.Y.
      • Vira M.
      • Ghosh M.
      • Romero V.V.
      • Sougrat R.
      • Vaulont S.
      • Viollet B.
      • Kim Y.S.
      • Lee S.
      • Trepel J.
      • Srinivasan R.
      • Bratslavsky G.
      • et al.
      The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels.
      ). The high basal activity of AMPK also observed in this study (Fig. 6, B and C) may be due to the fact that kidneys are always active and rely heavily on oxidative phosphorylation (
      • Dickerson L.
      Biochemical oxygen demand.
      ). Consistent with our findings that AMPK-mediated Cytc phosphorylation partially suppresses mitochondrial respiration, it was shown in human renal proximal tubular epithelial cells that additional activation of AMPK with metformin results in a significant reduction of cellular respiration (
      • Takiyama Y.
      • Harumi T.
      • Watanabe J.
      • Fujita Y.
      • Honjo J.
      • Shimizu N.
      • Makino Y.
      • Haneda M.
      Tubular injury in a rat model of type 2 diabetes is prevented by metformin: a possible role of HIF-1α expression and oxygen metabolism.
      ).
      The strong periodicity of kidney function (
      • Zuber A.M.
      • Centeno G.
      • Pradervand S.
      • Nikolaeva S.
      • Maquelin L.
      • Cardinaux L.
      • Bonny O.
      • Firsov D.
      Molecular clock is involved in predictive circadian adjustment of renal function.
      ,
      • Firsov D.
      • Bonny O.
      Circadian regulation of renal function.
      ) (i.e. activity oscillations controlled by circadian rhythm) provides a potential rationale for the buffering against rapid increase in ETC activity, with attendant ROS increase, provided by the paradoxical AMPK response found in kidney. We thus propose a model, shown in Fig. 6F, in which under physiological conditions a central role of Cytc Thr28 phosphorylation in kidney is to maintain optimal intermediate ΔΨm levels, which allow efficient energy production but prevent ROS generation because ROS are produced at high ΔΨm levels (reviewed in Ref.
      • Hüttemann M.
      • Lee I.
      • Grossman L.I.
      • Doan J.W.
      • Sanderson T.H.
      Phosphorylation of mammalian cytochrome c and cytochrome c oxidase in the regulation of cell destiny: respiration, apoptosis, and human disease.
      ). In line with our concept, it was shown in cultured mouse proximal tubular cells subjected to various forms of metabolic stress that genetic or pharmacologic inhibition of AMPK caused apoptosis. Furthermore, in rats subjected to renal ischemia/reperfusion injury, application of a high dose of AMPK activator AICAR shortly before ischemia significantly improves cell survival (
      • Lempiäinen J.
      • Finckenberg P.
      • Levijoki J.
      • Mervaala E.
      AMPK activator AICAR ameliorates ischaemia reperfusion injury in the rat kidney.
      ). Cytc purified from ischemic kidney is dephosphorylated (not shown), similar to Cytc isolated from ischemic brain (
      • Sanderson T.H.
      • Mahapatra G.
      • Pecina P.
      • Ji Q.
      • Yu K.
      • Sinkler C.
      • Varughese A.
      • Kumar R.
      • Bukowski M.J.
      • Tousignant R.N.
      • Salomon A.R.
      • Lee I.
      • Hüttemann M.
      Cytochrome c is tyrosine 97 phosphorylated by neuroprotective insulin treatment.
      ). This would allow maximal ETC flux, ΔΨm hyperpolarization, and ROS production during reperfusion when ETC function is reinstated due to reintroduction of oxygen. We have shown that neuroprotective insulin treatment before brain ischemia/reperfusion leads to Cytc Tyr97 phosphorylation, which also decreases respiration, resulting in suppression of the release of Cytc from the mitochondria and a 50% reduction of neuronal death (
      • Sanderson T.H.
      • Mahapatra G.
      • Pecina P.
      • Ji Q.
      • Yu K.
      • Sinkler C.
      • Varughese A.
      • Kumar R.
      • Bukowski M.J.
      • Tousignant R.N.
      • Salomon A.R.
      • Lee I.
      • Hüttemann M.
      Cytochrome c is tyrosine 97 phosphorylated by neuroprotective insulin treatment.
      ). It will be interesting to see in future studies whether AICAR treatment maintains Cytc Thr28 phosphorylation during ischemia in the kidney, alleviating ETC hyperactivation during reperfusion.
      In conclusion, all three functionally studied Cytc phosphorylations to date (Tyr97, Tyr48, and Thr28) lead to a partial inhibition in the reaction with CcO, or “controlled respiration” (Fig. 6F). We propose that this mechanism provides a basis for the maintenance of “healthy” intermediate ΔΨm levels under normal conditions. This, in turn, prevents excessive ROS production that occurs at high ΔΨm levels under conditions of stress when mitochondrial proteins become dephosphorylated, allowing maximal ETC flux, ΔΨm hyperpolarization, excessive ROS, and cell death. Although all three known Cytc phosphorylations limit respiration, the choice of site in a particular tissue may depend on tissue-specific metabolic differences or on additional effects of the phosphorylation, such as regulation of apoptosis.

      Author Contributions

      G. M., Q. J., I. L., J. L., A. A. K., A. Varughese, C. S., and M. H. designed and performed experiments. A. Vaishnav, J. S. B., and B. E. determined the crystal structures. C. T. M., T. H. S., L. I. G., T. L. S., V. E. K., A. R. S., B. F. P. E., and M. H. assisted with the experimental design and supervised the project. G. M. and M. H. wrote the manuscript. All authors edited and approved the final version of the manuscript.

      Acknowledgments

      This research used resources of the Advanced Photon Source, a United States Department of Energy Office of Science User Facility operated for the Department of Energy Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation, the Michigan Technology Tri-Corridor (Grant 085P1000817), and Wayne State University Office of the Vice-President for Research.

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