The passage of protons across membranes through F1Fo-ATP synthases spins their rotors and drives the synthesis of ATP. While the principle of torque generation by proton transfer is known, the mechanisms and routes of proton access and release and their evolution are not fully understood. Here, we show that the entry site and path of protons in the lumenal half channel of mitochondrial ATP synthases are largely defined by a short N-terminal α-helix of subunit-a. In Trypanosoma brucei and other Euglenozoa, the α-helix is part of another polypeptide chain that is a product of subunit-a gene fragmentation. This α-helix and other elements forming the proton pathway are widely conserved across eukaryotes and in Alphaproteobacteria, the closest extant relatives of mitochondria, but not in other bacteria. The α-helix blocks one of two proton routes found in Escherichia coli, resulting in a single proton entry site in mitochondrial and alphaproteobacterial ATP synthases. Thus, the shape of the access half channel predates eukaryotes and originated in the lineage from which mitochondria evolved by endosymbiosis.

Faculty of Science University of South Bohemia České Budějovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

See more in PubMed

Burki F, Roger AJ, Brown MW, Simpson AGB. 2020. The new tree of eukaryotes. Trends Ecol Evol. 35:43–55. PubMed

Cain BD, Simoni RD. 1988. Interaction between Glu-219 and His-245 within the a subunit of F1Fo-ATPase in Escherichia coli. J Biol Chem. 263:6606–6612. PubMed

Cain BD, Simoni RD. 1989. Proton translocation by the F1FOATPase of Escherichia coli. J Biol Chem. 264:3292–3300. PubMed

Demmer JK, Phillips BP, Uhrig OL, Filloux A, Allsopp LP, Bublitz M, Meier T. 2022. Structure of ATP synthase from ESKAPE pathogen Acinetobacter baumannii. Sci Adv. 8:eabl5966. PubMed PMC

Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32:1792–1797. PubMed PMC

Eya S, Maeda M, Futai M. 1991. Role of the carboxyl terminal region of H+-ATPase FoF1 a subunit from Escherichia coli. Arch Biochem Biophys. 284:71–77. PubMed

Feniouk BA, Kozlova MA, Knorre DA, Cherepanov DA, Mulkidjanian AY, Junge W. 2004. The proton-driven rotor of ATP synthase: ohmic conductance (10 fS), and absence of voltage gating. Biophys J. 86:4094–4109. PubMed PMC

Flygaard RK, Muhleip A, Tobiasson V, Amunts A. 2020. Type III ATP synthase is a symmetry-deviated dimer that induces membrane curvature through tetramerization. Nat Commun. 11:5342. PubMed PMC

Fu CJ, Sheikh S, Miao W, Andersson SG, Baldauf SL. 2014. Missing genes, multiple ORFs, and C-to-U type RNA editing in Acrasis kona (Heterolobosea, Excavata) mitochondrial DNA. Genome Biol Evol. 6:2240–2257. PubMed PMC

Gahura O, Hierro-Yap C, Zikova A. 2021. Redesigned and reversed: architectural and functional oddities of the trypanosomal ATP synthase. Parasitology 148:1151–1160. PubMed PMC

Gahura O, Muhleip A, Hierro-Yap C, Panicucci B, Jain M, Hollaus D, Slapnickova M, Zikova A, Amunts A. 2022. An ancestral interaction module promotes oligomerization in divergent mitochondrial ATP synthases. Nat Commun. 13:5989. PubMed PMC

Gahura O, Subrtova K, Vachova H, Panicucci B, Fearnley IM, Harbour ME, Walker JE, Zikova A. 2018. The F1-ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit. FEBS J. 285:614–628. PubMed

Goddard TD, Huang CC, Meng EC, Pettersen EF, Couch GS, Morris JH, Ferrin TE. 2018. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27:14–25. PubMed PMC

Guo H, Bueler SA, Rubinstein JL. 2017. Atomic model for the dimeric Fo region of mitochondrial ATP synthase. Science 358:936–940. PubMed PMC

Guo H, Courbon GM, Bueler SA, Mai J, Liu J, Rubinstein JL. 2021. Structure of mycobacterial ATP synthase bound to the tuberculosis drug bedaquiline. Nature 589:143–147. PubMed

Guo H, Rubinstein JL. 2022. Structure of ATP synthase under strain during catalysis. Nat Commun. 13:2232. PubMed PMC

Hartzog PE, Cain BD. 1994. Second-site suppressor mutations at glycine 218 and histidine 245 in the alpha subunit of F1Fo ATP synthase in Escherichia coli. J Biol Chem. 269:32313–32317. PubMed

Hatch LP, Cox GB, Howitt SM. 1995. The essential arginine residue at position 210 in the alpha subunit of the Escherichia coli ATP synthase can be transferred to position 252 with partial retention of activity. J Biol Chem. 270:29407–29412. PubMed

Howitt SM, Lightowlers RN, Gibson F, Cox GB. 1990. Mutational analysis of the function of the a-subunit of the FoF1-ATPase of Escherichia coli. Biochim Biophys Acta. 1015:264–268. PubMed

Huet D, Rajendran E, van Dooren GG, Lourido S. 2018. Identification of cryptic subunits from an apicomplexan ATP synthase. Elife 7:e38097. PubMed PMC

Ivontsin L, Mashkovtseva E, Nartsissov Y. 2022. Insights on the proton translocation pathways in FoF1-ATP synthase using molecular dynamics simulations. Arch Biochem Biophys. 717:109135. PubMed

Jones DT, Taylor WR, Thornton JM. 1994. A model recognition approach to the prediction of all-helical membrane protein structure and topology. Biochemistry 33:3038–3049. PubMed

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Zidek A, Potapenko A, et al. . 2021. Highly accurate protein structure prediction with AlphaFold. Nature 596:583–589. PubMed PMC

Klusch N, Murphy BJ, Mills DJ, Yildiz O, Kuhlbrandt W. 2017. Structural basis of proton translocation and force generation in mitochondrial ATP synthase. Elife 6:e33274. PubMed PMC

Kuhlbrandt W. 2019. Structure and mechanisms of F-type ATP synthases. Annu Rev Biochem. 88:515–549. PubMed

Kyte J, Doolittle RF. 1982. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 157:105–132. PubMed

Lightowlers RN, Howitt SM, Hatch L, Gibson F, Cox G. 1988. The proton pore in the Escherichia coli FoF1-ATPase: substitution of glutamate by glutamine at position 219 of the alpha-subunit prevents Fo-mediated proton permeability. Biochim Biophys Acta. 933:241–248. PubMed

Montgomery MG, Gahura O, Leslie AGW, Zikova A, Walker JE. 2018. ATP Synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites. Proc Natl Acad Sci U S A. 115:2102–2107. PubMed PMC

Montgomery MG, Petri J, Spikes TE, Walker JE. 2021. Structure of the ATP synthase from Mycobacterium smegmatis provides targets for treating tuberculosis. Proc Natl Acad Sci U S A. 118:e2111899118. PubMed PMC

Muhleip A, Flygaard RK, Baradaran R, Haapanen O, Gruhl T, Tobiasson V, Marechal A, Sharma V, Amunts A. 2023. Structural basis of mitochondrial membrane bending by the I-II-III2-IV2 supercomplex. Nature 615:934–938. PubMed PMC

Muhleip A, Kock Flygaard R, Ovciarikova J, Lacombe A, Fernandes P, Sheiner L, Amunts A. 2021. ATP synthase hexamer assemblies shape cristae of Toxoplasma mitochondria. Nat Commun. 12:120. PubMed PMC

Muhleip A, McComas SE, Amunts A. 2019. Structure of a mitochondrial ATP synthase with bound native cardiolipin. Elife 8:e51179. PubMed PMC

Munoz-Gomez SA, Susko E, Williamson K, Eme L, Slamovits CH, Moreira D, Lopez-Garcia P, Roger AJ. 2022. Site-and-branch-heterogeneous analyses of an expanded dataset favour mitochondria as sister to known Alphaproteobacteria. Nat Ecol Evol. 6:253–262. PubMed

Murphy BJ, Klusch N, Langer J, Mills DJ, Yildiz O, Kuhlbrandt W. 2019. Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling. Science 364:eaaw9128. PubMed

Perez-Martinez X, Antaramian A, Vazquez-Acevedo M, Funes S, Tolkunova E, d’Alayer J, Claros MG, Davidson E, King MP, Gonzalez-Halphen D. 2001. Subunit II of cytochrome c oxidase in Chlamydomonad algae is a heterodimer encoded by two independent nuclear genes. J Biol Chem. 276:11302–11309. PubMed

Pinke G, Zhou L, Sazanov LA. 2020. Cryo-EM structure of the entire mammalian F-type ATP synthase. Nat Struct Mol Biol. 27:1077–1085. PubMed

Potter SC, Luciani A, Eddy SR, Park Y, Lopez R, Finn RD. 2018. HMMER Web server: 2018 update. Nucleic Acids Res. 46:W200–W204. PubMed PMC

Rodriguez-Salinas E, Riveros-Rosas H, Li Z, Fucikova K, Brand JJ, Lewis LA, Gonzalez-Halphen D. 2012. Lineage-specific fragmentation and nuclear relocation of the mitochondrial cox2 gene in chlorophycean green algae (Chlorophyta). Mol Phylogenet Evol. 64:166–176. PubMed

Sinha SD, Wideman JG. 2023. The persistent homology of mitochondrial ATP synthases. iScience 26:106700. PubMed PMC

Sobti M, Ishmukhametov R, Bouwer JC, Ayer A, Suarna C, Smith NJ, Christie M, Stocker R, Duncan TM, Stewart AG. 2019. Cryo-EM reveals distinct conformations of E. coli ATP synthase on exposure to ATP. Elife 8:e43864. PubMed PMC

Sobti M, Walshe JL, Wu D, Ishmukhametov R, Zeng YC, Robinson CV, Berry RM, Stewart AG. 2020. Cryo-EM structures provide insight into how E. coli F1Fo ATP synthase accommodates symmetry mismatch. Nat Commun. 11:2615. PubMed PMC

Spikes TE, Montgomery MG, Walker JE. 2020. Structure of the dimeric ATP synthase from bovine mitochondria. Proc Natl Acad Sci U S A. 117:23519–23526. PubMed PMC

Swart EC, Nowacki M, Shum J, Stiles H, Higgins BP, Doak TG, Schotanus K, Magrini VJ, Minx P, Mardis ER, et al. . 2012. The Oxytricha trifallax mitochondrial genome. Genome Biol Evol. 4:136–154. PubMed PMC

Szafranski P. 2017a. Evolutionarily recent, insertional fission of mitochondrial cox2 into complementary genes in bilaterian Metazoa. BMC Genomics 18:269. PubMed PMC

Szafranski P. 2017b. Intercompartmental piecewise gene transfer. Genes (Basel) 8:260. PubMed PMC

Tikhonenkov DV, Mikhailov KV, Gawryluk RMR, Belyaev AO, Mathur V, Karpov SA, Zagumyonnyi DG, Borodina AS, Prokina KI, Mylnikov AP, et al. . 2022. Microbial predators form a new supergroup of eukaryotes. Nature 612:714–719. PubMed

Tikhonenkov DV, Strassert JFH, Janouskovec J, Mylnikov AP, Aleoshin VV, Burki F, Keeling PJ. 2020. Predatory colponemids are the sister group to all other alveolates. Mol Phylogenet Evol. 149:106839. PubMed

Tobiasson V, Amunts A. 2020. Ciliate mitoribosome illuminates evolutionary steps of mitochondrial translation. Elife 9:e59264. PubMed PMC

Tobiasson V, Berzina I, Amunts A. 2022. Structure of a mitochondrial ribosome with fragmented rRNA in complex with membrane-targeting elements. Nat Commun. 13:6132. PubMed PMC

Tusnady GE, Simon I. 2001. The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850. PubMed

Vik SB, Cain BD, Chun KT, Simoni RD. 1988. Mutagenesis of the alpha subunit of the F1Fo-ATPase from Escherichia coli. Mutations at Glu-196, Pro-190, and Ser-199. J Biol Chem. 263:6599–6605. PubMed

Walker JE. 2017. Structure, mechanism and regulation of ATP synthases. In: Wikstrom M, editor. Mechanisms of primary energy transduction in biology. Cambridge (UK): The Royal Society of Chemistry. p. 338.–; .

Waller RF, Keeling PJ. 2006. Alveolate and chlorophycean mitochondrial cox2 genes split twice independently. Gene 383:33–37. PubMed

Yanagisawa S, Frasch WD. 2021. pH-dependent 11 degrees F1Fo ATP synthase sub-steps reveal insight into the Fo torque generating mechanism. Elife 10:e70016. PubMed PMC

Zhou L, Maldonado M, Padavannil A, Guo F, Letts JA. 2022. Structures of Tetrahymena's respiratory chain reveal the diversity of eukaryotic core metabolism. Science 376:831–839. PubMed PMC