Journal of Molecular Evolution

, Volume 82, Issue 6, pp 279–290 | Cite as

Evolution of the SOUL Heme-Binding Protein Superfamily Across Eukarya

  • Antonio Emidio FortunatoEmail author
  • Paolo Sordino
  • Nikos AndreakisEmail author
Original Article


SOUL homologs constitute a heme-binding protein superfamily putatively involved in heme and tetrapyrrole metabolisms associated with a number of physiological processes. Despite their omnipresence across the tree of life and the biochemical characterization of many SOUL members, their functional role and the evolutionary events leading to such remarkable protein repertoire still remain cryptic. To explore SOUL evolution, we apply a computational phylogenetic approach, including a relevant number of SOUL homologs, to identify paralog forms and reconstruct their genealogy across the tree of life and within species. In animal lineages, multiple gene duplication or loss events and paralog functional specializations underlie SOUL evolution from the dawn of ancestral echinoderm and mollusc SOUL forms. In photosynthetic organisms, SOUL evolution is linked to the endosymbiosis events leading to plastid acquisition in eukaryotes. Derivative features, such as the F2L peptide and BH3 domain, evolved in vertebrates and provided innovative functionality to support immune response and apoptosis. The evolution of elements such as the N-terminal protein domain DUF2358, the His42 residue, or the tetrapyrrole heme-binding site is modern, and their functional implications still unresolved. This study represents the first in-depth analysis of SOUL protein evolution and provides novel insights in the understanding of their obscure physiological role.


SOUL Evolution Heme binding Tetrapyrrole metabolism Light sensing Apoptosis 



This research was supported by an SZN-Funded University of Palermo PhD Program and a Travel Grant “Premio Brancaccio 2009” from Lions Club “Megaride” (Napoli, Italy) to AEF. PS is supported by a MIUR PON Grant (PONa3_00239) and a FIRB Grant (RBFR12QW4I). NA is funded through the National Environmental Research Program (NERP), an Australian Government Initiative supporting world class, public good research. The NERP Marine Biodiversity Hub is a collaborative partnership between the University of Tasmania, CSIRO Wealth from Oceans Flagship, Geoscience Australia, Australian Institute of Marine Science, Museum Victoria, Charles Darwin University, and the University of Western Australia (

Compliance with Ethical Standards

Conflict of interest

The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

Supplementary material

239_2016_9745_MOESM1_ESM.pdf (310 kb)
Supplementary material 1 (PDF 309 kb) Genealogy of all SOUL homologs identified in this study. Unrooted ML phylogenetic hypothesis of SOUL protein sequences from metazoan, non-metazoan, and photosynthetic organisms. A1 and A2 refer to animal branches. Clusters A1 and A2 include all the sequences used to compute the phylogenetic hypotheses shown in Figs. 1, 2, S2 and S3. P denotes SOUL from primary endosymbionts (green branches, Archaeplastida). S indicates SOUL proteins from both primary and secondary endosymbionts (brown branches). Clusters P and S include all the sequences used for the phylogenetics hypothesis shown in Fig. 3. The two long black branches include the eight bacterial SOUL isoforms reported in Table 1
239_2016_9745_MOESM2_ESM.pdf (533 kb)
Supplementary material 2 (PDF 533 kb) SOUL genealogy inferred from vertebrates and selected invertebrate sequences. Maximum likelihood phylogenetic hypothesis of SOUL vertebrate and selected invertebrate homologous protein sequences. The positions of Ciona and Amphioxus are highlighted in gray. Color gradients denote bootstrap support distributed across the topology. A1 and A2 refer to animal branches
239_2016_9745_MOESM3_ESM.pdf (368 kb)
Supplementary material 3 (PDF 367 kb) SOUL genealogy in invertebrates. Maximum likelihood genealogical hypothesis of SOUL invertebrate homologous protein sequences. Color gradients denote bootstrap support distributed across the phylogeny. Cnidarian SOUL orthologs were used as outgroup
239_2016_9745_MOESM4_ESM.pdf (431 kb)
Supplementary material 4 (PDF 430 kb) Functional domain mapping in vertebrate SOUL proteins. Mapping the heme-binding site, His42, signal peptide, BH3 domain, and F2L peptide in selected vertebrate SOUL proteins based on the phylogenetic tree in Fig. 1. Filled and empty circles indicate the presence or absence, respectively, of a specific feature following the legend
239_2016_9745_MOESM5_ESM.pdf (509 kb)
Supplementary material 5 (PDF 509 kb) Functional domain mapping in SOUL mined from photosynthetic organisms. Mapping the domain DUF2458, the signal peptide, and the putative localization in selected SOUL proteins from photosynthetic organisms based on the phylogenetic tree in Fig. 3. Filled and empty circles indicate the presence or absence, respectively, of a specific feature following the legend
239_2016_9745_MOESM6_ESM.fas (66 kb)
Supplementary material 6 (FAS 67 kb) SOUL sequences alignment used for the “Global” tree
239_2016_9745_MOESM7_ESM.fas (27 kb)
Supplementary material 7 (FAS 27 kb) SOUL sequences alignment used for the “Vertebrate” tree
239_2016_9745_MOESM8_ESM.txt (10 kb)
Supplementary material 8 (TXT 10 kb) SOUL sequences alignment used for the “Invertebrate” tree
239_2016_9745_MOESM9_ESM.fas (29 kb)
Supplementary material 9 (FAS 30 kb) SOUL sequences alignment used for the “Vertebrate + Invertebrate” tree
239_2016_9745_MOESM10_ESM.txt (14 kb)
Supplementary material 10 (TXT 14 kb) SOUL sequences alignment used for the “Invertebrate + Vertebrate” tree
239_2016_9745_MOESM11_ESM.fas (33 kb)
Supplementary material 11 (FAS 34 kb) SOUL sequences alignment used for the “Photosynthetic organisms” tree
239_2016_9745_MOESM12_ESM.pdf (156 kb)
Supplementary material 12 (PDF 155 kb) SOUL sequences and their accession numbers used in this work


  1. Abascal F, Zardoya R, Posada D (2005) ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21:2104CrossRefPubMedGoogle Scholar
  2. Altekar G, Dwarkadas S, Huelsenbeck JP, Ronquist F (2004) Parallel Metropolis coupled Markov chain Monte Carlo for Bayesian phylogenetic inference. Bioinformatics 20:407CrossRefPubMedGoogle Scholar
  3. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389CrossRefPubMedPubMedCentralGoogle Scholar
  4. Ambrosi E, Capaldi S, Bovi M, Saccomani G, Perduca M, Monaco HL (2011) Structural changes in the BH3 domain of SOUL protein upon interaction with the anti-apoptotic protein Bcl-xL. Biochem J 438:291CrossRefPubMedPubMedCentralGoogle Scholar
  5. Ascenzi P, Fasano M (2007) Heme-hemopexin: a ‘chronosteric’ heme-protein. IUBMB Life 59:700CrossRefPubMedGoogle Scholar
  6. Ascenzi P, Fasano M (2009) Serum heme-albumin: an allosteric protein. IUBMB Life 61:1118CrossRefPubMedGoogle Scholar
  7. Babusiak M, Man P, Sutak R, Petrak J, Vyoral D (2005) Identification of heme binding protein complexes in murine erythroleukemic cells: study by a novel two-dimensional native separation—liquid chromatography and electrophoresis. Proteomics 5:340CrossRefPubMedGoogle Scholar
  8. Blackmon BJ, Dailey TA, Xiao LC, Dailey HA (2002) Characterization of a human and mouse tetrapyrrole-binding protein. Arch Biochem Biophys 407:196CrossRefGoogle Scholar
  9. Darriba D, Taboada GL, Doallo R, Posada D (2011) ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27:1164CrossRefPubMedGoogle Scholar
  10. Davidi L, Levin Y, Ben-Dor S, Pick U (2015) Proteome analysis of cytoplasmatic and plastidic beta-carotene lipid droplets in Dunaliella bardawil. Plant Physiol 167:60CrossRefPubMedGoogle Scholar
  11. Dias JS, Macedo AL, Ferreira GC, Peterson FC, Volkman BF, Goodfellow BJ (2006) The first structure from the SOUL/HBP family of heme-binding proteins, murine P22HBP. J Biol Chem 281:31553CrossRefPubMedGoogle Scholar
  12. Faller M, Matsunaga M, Yin S, Loo JA, Guo F (2007) Heme is involved in microRNA processing. Nat Struct Mol Biol 14:23CrossRefPubMedGoogle Scholar
  13. Furuyama K, Kaneko K, Vargas PD (2007) Heme as a magnificent molecule with multiple missions: heme determines its own fate and governs cellular homeostasis. Tohoku J Exp Med 213:1CrossRefPubMedGoogle Scholar
  14. Gao JL, Guillabert A, Hu JY, Le YY, Urizar E, Seligman E, Fang KJ, Yuan X, Imbault V, Communi D, Wang JM, Parmentier M, Murphy PM, Migeotte F (2007) F2L, a peptide derived from heme-binding protein, chemoattracts mouse neutrophils by specifically activating Fpr2, the low-affinity N-formylpeptide receptor. J Immunol 178:1450CrossRefPubMedGoogle Scholar
  15. Gell DA, Westman BJ, Gorman D, Liew C, Welch JJ, Weiss MJ, Mackay JP (2006) A novel haem-binding interface in the 22 kDa haem-binding protein p22HBP. J Mol Biol 362:287CrossRefPubMedGoogle Scholar
  16. Gotoh S, Ohgari Y, Nakamura T, Osumi T, Taketani S (2008) Heme-binding to the nuclear receptor retinoid X receptor alpha (RXRalpha) leads to the inhibition of the transcriptional activity. Gene 423:207CrossRefPubMedGoogle Scholar
  17. Guindon S, Delsuc F, Dufayard JF, Gascuel O (2009) Estimating maximum likelihood phylogenies with PhyML. Methods Mol Biol 537:113CrossRefPubMedGoogle Scholar
  18. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307CrossRefPubMedGoogle Scholar
  19. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp 41:95Google Scholar
  20. Han SW, Ching YC, Hammes SL, Rousseau DL (1991) Vibrational structure of the formyl group on heme-a—implications on the properties of cytochrome-c-oxidase. Biophys J 60:45CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hira S, Tomita T, Matsui T, Igarashi K, Ikeda-Saito M (2007) Bach1, a heme-dependent transcription factor, reveals presence of multiple heme binding sites with distinct coordination structure. IUBMB Life 59:542CrossRefPubMedGoogle Scholar
  22. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754CrossRefPubMedGoogle Scholar
  23. Jaquinod M, Villiers F, Kieffer-Jaquinod S, Hugouvieux V, Bruley C, Garin J, Bourguignon J (2007) A proteomics approach highlights a myriad of transporters in the Arabidopsis thaliana vacuolar membrane. Plant Signal Behav 2:413CrossRefPubMedPubMedCentralGoogle Scholar
  24. Keeling PJ (2013) The number, speed, and impact of plastid endosymbioses in eukaryotic evolution. Annu Rev Plant Biol 64:583CrossRefPubMedGoogle Scholar
  25. Khanna R, Shen Y, Toledo-Ortiz G, Kikis EA, Johannesson H, Hwang YS, Quail PH (2006) Functional profiling reveals that only a small number of phytochrome-regulated early-response genes in Arabidopsis are necessary for optimal deetiolation. Plant Cell 18:2157CrossRefPubMedPubMedCentralGoogle Scholar
  26. Krebs J, Saremaslani P, Caduff R (2002) ALG-2: a Ca2+-binding modulator protein involved in cell proliferation and in cell death. Biochim Biophys Acta Proteins Proteomics 1600:68CrossRefGoogle Scholar
  27. Kumar S, Bandyopadhyay U (2005) Free heme toxicity and its detoxification systems in human. Toxicol Lett 157:175CrossRefPubMedGoogle Scholar
  28. Lee SY, Lee MS, Lee HY, Kim SD, Shim JW, Jo SH, Lee JW, Kim JY, Choi YW, Baek SH, Ryu SH, Bae YS (2008) F2L, a peptide derived from heme-binding protein, inhibits LL-37-induced cell proliferation and tube formation in human umbilical vein endothelial cells. FEBS Lett 582:273CrossRefPubMedGoogle Scholar
  29. Lee HJ, Mochizuki N, Masuda T, Buckhout TJ (2012) Disrupting the bimolecular binding of the haem-binding protein 5 (AtHBP5) to haem oxygenase 1 (HY1) leads to oxidative stress in Arabidopsis. J Exp Bot 63:5967CrossRefPubMedPubMedCentralGoogle Scholar
  30. Mauk MR, Rosell FI, Mauk AG (2007) Chromatographically distinguishable heme insertion isoforms of human hemopexin. Biochemistry 46:15033CrossRefPubMedGoogle Scholar
  31. Migeotte I, Riboldi E, Franssen JD, Gregoire F, Loison U, Wittamer V, Detheux M, Robberecht P, Costagliola S, Vassart G, Sozzani S, Parmentier M, Communi D (2005) Identification and characterization of an endogenous chemotactic ligand specific for FPRL2. J Exp Med 201:83CrossRefPubMedPubMedCentralGoogle Scholar
  32. Mochizuki N, Tanaka R, Grimm B, Masuda T, Moulin M, Smith AG, Tanaka A, Terry MJ (2010) The cell biology of tetrapyrroles: a life and death struggle. Trends Plant Sci 15:488CrossRefPubMedGoogle Scholar
  33. Moustafa A, Beszteri B, Maier UG, Bowler C, Valentin K, Bhattacharya D (2009) Genomic footprints of a cryptic plastid endosymbiosis in diatoms. Science 324:1724CrossRefPubMedGoogle Scholar
  34. Nakasone K, Nagahama Y, Okubo K (2013) hebp3, a novel member of the heme-binding protein gene family, is expressed in the medaka meninges with higher abundance in females due to a direct stimulating action of ovarian estrogens. Endocrinology 154:920CrossRefPubMedGoogle Scholar
  35. Natt D, Lindqvist N, Stranneheim H, Lundeberg J, Torjesen PA, Jensen P (2009) Inheritance of acquired behaviour adaptations and brain gene expression in chickens. PLoS One 4:e6405CrossRefPubMedPubMedCentralGoogle Scholar
  36. Oliveira PL, Kawooya JK, Ribeiro JM, Meyer T, Poorman R, Alves EW, Walker FA, Machado EA, Nussenzveig RH, Padovan GJ et al (1995) A heme-binding protein from hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus. Isolation and characterization. J Biol Chem 270:10897CrossRefPubMedGoogle Scholar
  37. Paiva-Silva GO, Sorgine MH, Benedetti CE, Meneghini R, Almeida IC, Machado EA, Dansa-Petretski M, Yepiz-Plascencia G, Law JH, Oliveira PL, Masuda H (2002) On the biosynthesis of Rhodnius prolixus heme-binding protein. Insect Biochem Mol Biol 32:1533CrossRefPubMedGoogle Scholar
  38. Peltier JB, Ytterberg AJ, Sun Q, van Wijk KJ (2004) New functions of the thylakoid membrane proteome of Arabidopsis thaliana revealed by a simple, fast, and versatile fractionation strategy. J Biol Chem 279:49367CrossRefPubMedGoogle Scholar
  39. Peltier JB, Cai Y, Sun Q, Zabrouskov V, Giacomelli L, Rudella A, Ytterberg AJ, Rutschow H, van Wijk KJ (2006) The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts. Mol Cell Proteomics 5:114CrossRefPubMedGoogle Scholar
  40. Rafie-Kolpin M, Han AP, Chen JJ (2003) Autophosphorylation of threonine 485 in the activation loop is essential for attaining eIF2alpha kinase activity of HRI. Biochemistry 42:6536CrossRefPubMedGoogle Scholar
  41. Rambaut A, Drummond AJ (2007) Tracer v1.4.
  42. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572CrossRefPubMedGoogle Scholar
  43. Rossi B, Mariotto G, Ambrosi E, Monaco HL (2009) Raman scattering investigation of selenomethionine replacement in protein SOUL crystals. J Raman Spectrosc 40:1844CrossRefGoogle Scholar
  44. Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li SM, Albala JS, Lim JH, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP, Vidal M (2005) Towards a proteome-scale map of the human protein–protein interaction network. Nature 437:1173CrossRefPubMedGoogle Scholar
  45. Sato H, Hayashi T, Ando T, Hisaeda Y, Ueno T, Watanabe Y (2004) Hybridization of modified-heme reconstitution and distal histidine mutation to functionalize sperm whale-myoglobin. J Am Chem Soc 126:436CrossRefPubMedGoogle Scholar
  46. Schulze T, Schreiber S, Iliev D, Boesger J, Trippens J, Kreimer G, Mittag M (2013) The heme-binding protein SOUL3 of Chlamydomonas reinhardtii influences size and position of the eyespot. Mol Plant 6:931CrossRefPubMedGoogle Scholar
  47. Severance S, Hamza I (2009) Trafficking of heme and porphyrins in metazoa. Chem Rev 109:4596CrossRefPubMedPubMedCentralGoogle Scholar
  48. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312CrossRefPubMedPubMedCentralGoogle Scholar
  49. Szigeti A, Bellyei S, Gasz B, Boronkai A, Hocsak E, Minik O, Bognar Z, Varbiro G, Sumegi B, Gallyas F (2006) Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL. FEBS Lett 580:6447CrossRefPubMedGoogle Scholar
  50. Szigeti A, Hocsak E, Rapolti E, Racz B, Boronkai A, Pozsgai E, Debreceni B, Bognar Z, Bellyei S, Sumegi B, Gallyas F Jr (2010) Facilitation of mitochondrial outer and inner membrane permeabilization and cell death in oxidative stress by a novel Bcl-2 homology 3 domain protein. J Biol Chem 285:2140CrossRefPubMedGoogle Scholar
  51. Takahashi S, Ogawa T, Inoue K, Masuda T (2008) Characterization of cytosolic tetrapyrrole-binding proteins in Arabidopsis thaliana. Photochem Photobiol Sci 7:1216CrossRefPubMedGoogle Scholar
  52. Taketani S (2005) Aquisition, mobilization and utilization of cellular iron and heme: endless findings and growing evidence of tight regulation. Tohoku J Exp Med 205:297CrossRefPubMedGoogle Scholar
  53. Taketani S, Adachi Y, Kohno H, Ikehara S, Tokunaga R, Ishii T (1998) Molecular characterization of a newly identified heme-binding protein induced during differentiation of urine erythroleukemia cells. J Biol Chem 273:31388CrossRefPubMedGoogle Scholar
  54. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731CrossRefPubMedPubMedCentralGoogle Scholar
  55. Vanhee C, Zapotoczny G, Masquelier D, Ghislain M, Batoko H (2011) The Arabidopsis multistress regulator TSPO is a heme binding membrane protein and a potential scavenger of porphyrins via an autophagy-dependent degradation mechanism. Plant Cell 23:785CrossRefPubMedPubMedCentralGoogle Scholar
  56. Wagner V, Gessner G, Heiland I, Kaminski M, Hawat S, Scheffler K, Mittag M (2006) Analysis of the phosphoproteome of Chlamydomonas reinhardtii provides new insights into various cellular pathways. Eukaryot Cell 5:457CrossRefPubMedPubMedCentralGoogle Scholar
  57. Wagner V, Ullmann K, Mollwo A, Kaminski M, Mittag M, Kreimer G (2008) The phosphoproteome of a Chlamydomonas reinhardtii eyespot fraction includes key proteins of the light signaling pathway. Plant Physiol 146:772CrossRefPubMedPubMedCentralGoogle Scholar
  58. Wakao S, Chin BL, Ledford HK, Dent RM, Casero D, Pellegrini M, Merchant SS, Niyogi KK (2014) Phosphoprotein SAK1 is a regulator of acclimation to singlet oxygen in Chlamydomonas reinhardtii. Elife 3:e02286CrossRefPubMedPubMedCentralGoogle Scholar
  59. Welch JJ, Watts JA, Vakoc CR, Yao Y, Wang H, Hardison RC, Blobel GA, Chodosh LA, Weiss MJ (2004) Global regulation of erythroid gene expression by transcription factor GATA-1. Blood 104:3136CrossRefPubMedGoogle Scholar
  60. Zylka MJ, Reppert SM (1999) Discovery of a putative heme-binding protein family (SOUL/HBP) by two-tissue suppression subtractive hybridization and database searches. Brain Res Mol Brain Res 74:175CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Sorbonne Universités, UPMCInstitut de Biologie Paris-Seine, CNRS, Laboratoire de Biologie Computationnelle et Quantitative UMR 7238ParisFrance
  2. 2.Biology and Evolution of Marine OrganismsStazione Zoologica Anton DohrnNaplesItaly
  3. 3.Australian Institute of Marine ScienceTownsville MCAustralia
  4. 4.College of Marine and Environmental SciencesJames Cook UniversityTownsvilleAustralia

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