Cytochrome c oxidase (COX) is regulated through tissue-, development- or environment-controlled expression of subunit isoforms. The COX4 subunit is thought to optimize respiratory chain function according to oxygen-controlled expression of its isoforms COX4i1 and COX4i2. However, biochemical mechanisms of regulation by the two variants are only partly understood. We created an HEK293-based knock-out cellular model devoid of both isoforms (COX4i1/2 KO). Subsequent knock-in of COX4i1 or COX4i2 generated cells with exclusive expression of respective isoform. Both isoforms complemented the respiratory defect of COX4i1/2 KO. The content, composition, and incorporation of COX into supercomplexes were comparable in COX4i1- and COX4i2-expressing cells. Also, COX activity, cytochrome c affinity, and respiratory rates were undistinguishable in cells expressing either isoform. Analysis of energy metabolism and the redox state in intact cells uncovered modestly increased preference for mitochondrial ATP production, consistent with the increased NADH pool oxidation and lower ROS in COX4i2-expressing cells in normoxia. Most remarkable changes were uncovered in COX oxygen kinetics. The p50 (partial pressure of oxygen at half-maximal respiration) was increased twofold in COX4i2 versus COX4i1 cells, indicating decreased oxygen affinity of the COX4i2-containing enzyme. Our finding supports the key role of the COX4i2-containing enzyme in hypoxia-sensing pathways of energy metabolism.

Department of Bioenergetics Institute of Physiology Czech Academy of Sciences 14220 Prague 4 Czech Republic

Department of Cell Biology Faculty of Science Charles University 12000 Prague 2 Czech Republic

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Timon-Gomez A., Nyvltova E., Abriata L.A., Vila A.J., Hosler J., Barrientos A. Mitochondrial cytochrome c oxidase biogenesis: Recent developments. Semin Cell Dev. Biol. 2018;76:163–178. doi: 10.1016/j.semcdb.2017.08.055. PubMed DOI PMC

Villani G., Attardi G. In vivo control of respiration by cytochrome c oxidase in human cells. Free Radic. Biol. Med. 2000;29:202–210. doi: 10.1016/S0891-5849(00)00303-8. PubMed DOI

Pierron D., Wildman D.E., Huttemann M., Markondapatnaikuni G.C., Aras S., Grossman L.I. Cytochrome c oxidase: Evolution of control via nuclear subunit addition. Biochim. Biophys. Acta. 2012;1817:590–597. doi: 10.1016/j.bbabio.2011.07.007. PubMed DOI PMC

Huttemann M., Helling S., Sanderson T.H., Sinkler C., Samavati L., Mahapatra G., Varughese A., Lu G., Liu J., Ramzan R., et al. Regulation of mitochondrial respiration and apoptosis through cell signaling: Cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation. Biochim. Biophys. Acta. 2012;1817:598–609. doi: 10.1016/j.bbabio.2011.07.001. PubMed DOI PMC

Kadenbach B., Huttemann M., Arnold S., Lee I., Bender E. Mitochondrial energy metabolism is regulated via nuclear-coded subunits of cytochrome c oxidase. Free Radic. Biol. Med. 2000;29:211–221. doi: 10.1016/S0891-5849(00)00305-1. PubMed DOI

Cogliati S., Calvo E., Loureiro M., Guaras A.M., Nieto-Arellano R., Garcia-Poyatos C., Ezkurdia I., Mercader N., Vazquez J., Enriquez J.A. Mechanism of super-assembly of respiratory complexes III and IV. Nature. 2016;539:579–582. doi: 10.1038/nature20157. PubMed DOI

Huttemann M., Jaradat S., Grossman L.I. Cytochrome c oxidase of mammals contains a testes-specific isoform of subunit VIb--the counterpart to testes-specific cytochrome c? Mol. Reprod. Dev. 2003;66:8–16. doi: 10.1002/mrd.10327. PubMed DOI

Ludwig B., Bender E., Arnold S., Huttemann M., Lee I., Kadenbach B. Cytochrome C oxidase and the regulation of oxidative phosphorylation. Chembiochem. 2001;2:392–403. doi: 10.1002/1439-7633(20010601)2:6<392::AID-CBIC392>3.0.CO;2-N. PubMed DOI

Acin-Perez R., Gatti D.L., Bai Y., Manfredi G. Protein phosphorylation and prevention of cytochrome oxidase inhibition by ATP: Coupled mechanisms of energy metabolism regulation. Cell Metab. 2011;13:712–719. doi: 10.1016/j.cmet.2011.03.024. PubMed DOI PMC

Kwast K.E., Burke P.V., Poyton R.O. Oxygen sensing and the transcriptional regulation of oxygen-responsive genes in yeast. J. Exp. Biol. 1998;201:1177–1195. PubMed

Huttemann M., Kadenbach B., Grossman L.I. Mammalian subunit IV isoforms of cytochrome c oxidase. Gene. 2001;267:111–123. doi: 10.1016/S0378-1119(01)00385-7. PubMed DOI

Aras S., Pak O., Sommer N., Finley R., Jr., Huttemann M., Weissmann N., Grossman L.I. Oxygen-dependent expression of cytochrome c oxidase subunit 4-2 gene expression is mediated by transcription factors RBPJ, CXXC5 and CHCHD2. Nucleic Acids Res. 2013;41:2255–2266. doi: 10.1093/nar/gks1454. PubMed DOI PMC

Fukuda R., Zhang H., Kim J.W., Shimoda L., Dang C.V., Semenza G.L. HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. Cell. 2007;129:111–122. doi: 10.1016/j.cell.2007.01.047. PubMed DOI

Huttemann M., Lee I., Liu J., Grossman L.I. Transcription of mammalian cytochrome c oxidase subunit IV-2 is controlled by a novel conserved oxygen responsive element. FEBS J. 2007;274:5737–5748. doi: 10.1111/j.1742-4658.2007.06093.x. PubMed DOI

Huttemann M., Lee I., Gao X., Pecina P., Pecinova A., Liu J., Aras S., Sommer N., Sanderson T.H., Tost M., et al. Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology. FASEB J. 2012;26:3916–3930. doi: 10.1096/fj.11-203273. PubMed DOI PMC

Sommer N., Huttemann M., Pak O., Scheibe S., Knoepp F., Sinkler C., Malczyk M., Gierhardt M., Esfandiary A., Kraut S., et al. Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing. Circ. Res. 2017;121:424–438. doi: 10.1161/CIRCRESAHA.116.310482. PubMed DOI PMC

Gao L., Bonilla-Henao V., Garcia-Flores P., Arias-Mayenco I., Ortega-Saenz P., Lopez-Barneo J. Gene expression analyses reveal metabolic specifications in acute O2 -sensing chemoreceptor cells. J. Physiol. 2017;595:6091–6120. doi: 10.1113/JP274684. PubMed DOI PMC

Moreno-Dominguez A., Ortega-Saenz P., Gao L., Colinas O., Garcia-Flores P., Bonilla-Henao V., Aragones J., Huttemann M., Grossman L.I., Weissmann N., et al. Acute O2 sensing through HIF2alpha-dependent expression of atypical cytochrome oxidase subunits in arterial chemoreceptors. Sci. Signal. 2020;13 doi: 10.1126/scisignal.aay9452. PubMed DOI

Horvat S., Beyer C., Arnold S. Effect of hypoxia on the transcription pattern of subunit isoforms and the kinetics of cytochrome c oxidase in cortical astrocytes and cerebellar neurons. J. Neurochem. 2006;99:937–951. doi: 10.1111/j.1471-4159.2006.04134.x. PubMed DOI

Oliva C.R., Markert T., Gillespie G.Y., Griguer C.E. Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas. Oncotarget. 2015;6:4330–4344. doi: 10.18632/oncotarget.3015. PubMed DOI PMC

Ran F.A., Hsu P.D., Lin C.Y., Gootenberg J.S., Konermann S., Trevino A.E., Scott D.A., Inoue A., Matoba S., Zhang Y., et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154:1380–1389. doi: 10.1016/j.cell.2013.08.021. PubMed DOI PMC

Schagger H. Tricine-SDS-PAGE. Nat. Protoc. 2006;1:16–22. doi: 10.1038/nprot.2006.4. PubMed DOI

Wittig I., Braun H.P., Schagger H. Blue native PAGE. Nat. Protoc. 2006;1:418–428. doi: 10.1038/nprot.2006.62. PubMed DOI

Bentlage H.A., Wendel U., Schagger H., ter Laak H.J., Janssen A.J., Trijbels J.M. Lethal infantile mitochondrial disease with isolated complex I deficiency in fibroblasts but with combined complex I and IV deficiencies in muscle. Neurology. 1996;47:243–248. doi: 10.1212/WNL.47.1.243. PubMed DOI

Hartmannova H., Piherova L., Tauchmannova K., Kidd K., Acott P.D., Crocker J.F., Oussedik Y., Mallet M., Hodanova K., Stranecky V., et al. Acadian variant of Fanconi syndrome is caused by mitochondrial respiratory chain complex I deficiency due to a non-coding mutation in complex I assembly factor NDUFAF6. Hum. Mol. Genet. 2016;25:4062–4079. doi: 10.1093/hmg/ddw245. PubMed DOI

Cox J., Hein M.Y., Luber C.A., Paron I., Nagaraj N., Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell Proteomics. 2014;13:2513–2526. doi: 10.1074/mcp.M113.031591. PubMed DOI PMC

Tyanova S., Temu T., Sinitcyn P., Carlson A., Hein M.Y., Geiger T., Mann M., Cox J. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods. 2016;13:731–740. doi: 10.1038/nmeth.3901. PubMed DOI

Lee I., Pecinova A., Pecina P., Neel B.G., Araki T., Kucherlapati R., Roberts A.E., Huttemann M. A suggested role for mitochondria in Noonan syndrome. Biochim. Biophys. Acta. 2010;1802:275–283. doi: 10.1016/j.bbadis.2009.10.005. PubMed DOI PMC

Pecina P., Houstkova H., Mracek T., Pecinova A., Nuskova H., Tesarova M., Hansikova H., Janota J., Zeman J., Houstek J. Noninvasive diagnostics of mitochondrial disorders in isolated lymphocytes with high resolution respirometry. BBA Clin. 2014;2:62–71. doi: 10.1016/j.bbacli.2014.09.003. PubMed DOI PMC

Pecina P., Gnaiger E., Zeman J., Pronicka E., Houstek J. Decreased affinity for oxygen of cytochrome-c oxidase in Leigh syndrome caused by SURF1 mutations. Am. J. Physiol. Cell Physiol. 2004;287:C1384–C1388. doi: 10.1152/ajpcell.00286.2004. PubMed DOI

Gnaiger E., Steinlechner-Maran R., Mendez G., Eberl T., Margreiter R. Control of mitochondrial and cellular respiration by oxygen. J. Bioenerg. Biomembr. 1995;27:583–596. doi: 10.1007/BF02111656. PubMed DOI

Boukalova S., Stursa J., Werner L., Ezrova Z., Cerny J., Bezawork-Geleta A., Pecinova A., Dong L., Drahota Z., Neuzil J. Mitochondrial Targeting of Metformin Enhances Its Activity against Pancreatic Cancer. Mol. Cancer Ther. 2016;15:2875–2886. doi: 10.1158/1535-7163.MCT-15-1021. PubMed DOI

Mracek T., Pecina P., Vojtiskova A., Kalous M., Sebesta O., Houstek J. Two components in pathogenic mechanism of mitochondrial ATPase deficiency: Energy deprivation and ROS production. Exp. Gerontol. 2006;41:683–687. doi: 10.1016/j.exger.2006.02.009. PubMed DOI

Hermanova I., Arruabarrena-Aristorena A., Valis K., Nuskova H., Alberich-Jorda M., Fiser K., Fernandez-Ruiz S., Kavan D., Pecinova A., Niso-Santano M., et al. Pharmacological inhibition of fatty-acid oxidation synergistically enhances the effect of l-asparaginase in childhood ALL cells. Leukemia. 2016;30:209–218. doi: 10.1038/leu.2015.213. PubMed DOI

Scandurra F.M., Gnaiger E. Cell respiration under hypoxia: Facts and artefacts in mitochondrial oxygen kinetics. Adv. Exp. Med. Biol. 2010;662:7–25. doi: 10.1007/978-1-4419-1241-1_2. PubMed DOI

Gnaiger E. Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv. Exp. Med. Biol. 2003;543:39–55. doi: 10.1007/978-1-4419-8997-0_4. PubMed DOI

Taylor C.T., Moncada S. Nitric oxide, cytochrome C oxidase, and the cellular response to hypoxia. Arterioscler. Thromb. Vasc. Biol. 2010;30:643–647. doi: 10.1161/ATVBAHA.108.181628. PubMed DOI

Sinkler C.A., Kalpage H., Shay J., Lee I., Malek M.H., Grossman L.I., Huttemann M. Tissue- and Condition-Specific Isoforms of Mammalian Cytochrome c Oxidase Subunits: From Function to Human Disease. Oxid Med. Cell Longev. 2017 doi: 10.1155/2017/1534056. PubMed DOI PMC

Tsukihara T., Aoyama H., Yamashita E., Tomizaki T., Yamaguchi H., Shinzawa-Itoh K., Nakashima R., Yaono R., Yoshikawa S. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science. 1996;272:1136–1144. doi: 10.1126/science.272.5265.1136. PubMed DOI

Allen L.A., Zhao X.J., Caughey W., Poyton R.O. Isoforms of yeast cytochrome c oxidase subunit V affect the binuclear reaction center and alter the kinetics of interaction with the isoforms of yeast cytochrome c. J. Biol. Chem. 1995;270:110–118. doi: 10.1074/jbc.270.1.110. PubMed DOI

Waypa G.B., Marks J.D., Guzy R.D., Mungai P.T., Schriewer J.M., Dokic D., Ball M.K., Schumacker P.T. Superoxide generated at mitochondrial complex III triggers acute responses to hypoxia in the pulmonary circulation. Am. J. Respir. Crit. Care Med. 2013;187:424–432. doi: 10.1164/rccm.201207-1294OC. PubMed DOI PMC

Hoffman D.L., Salter J.D., Brookes P.S. Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: Implications for hypoxic cell signaling. Am. J. Physiol. Heart Circ. Physiol. 2007;292:101–108. doi: 10.1152/ajpheart.00699.2006. PubMed DOI

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