Photosynthesis Research

, Volume 139, Issue 1–3, pp 281–293 | Cite as

Properties and structure of a low-potential, penta-heme cytochrome c552 from a thermophilic purple sulfur photosynthetic bacterium Thermochromatium tepidum

  • Jing-Hua Chen
  • Long-Jiang Yu
  • Alain Boussac
  • Zheng-Yu Wang-Otomo
  • Tingyun Kuang
  • Jian-Ren ShenEmail author
Original Article


The thermophilic purple sulfur bacterium Thermochromatium tepidum possesses four main water-soluble redox proteins involved in the electron transfer behavior. Crystal structures have been reported for three of them: a high potential iron–sulfur protein, cytochrome c′, and one of two low-potential cytochrome c552 (which is a flavocytochrome c) have been determined. In this study, we purified another low-potential cytochrome c552 (LPC), determined its N-terminal amino acid sequence and the whole gene sequence, characterized it with absorption and electron paramagnetic spectroscopy, and solved its high-resolution crystal structure. This novel cytochrome was found to contain five c-type hemes. The overall fold of LPC consists of two distinct domains, one is the five heme-containing domain and the other one is an Ig-like domain. This provides a representative example for the structures of multiheme cytochromes containing an odd number of hemes, although the structures of multiheme cytochromes with an even number of hemes are frequently seen in the PDB database. Comparison of the sequence and structure of LPC with other proteins in the databases revealed several characteristic features which may be important for its functioning. Based on the results obtained, we discuss the possible intracellular function of this LPC in Tch. tepidum.


Purple sulfur bacteria Electron transfer Cytochrome c Multiheme Crystal structure Thermochromatium tepidum 



Ammonia monooxygenase




Chloride dismutase




Electron paramagnetic resonance


Hydroxylamine oxidoreductase


High potential iron–sulfur protein


Low-potential cytochrome


Multiheme cytochrome c


Nitrite reductase


Single wavelength anomalous diffraction





The authors thank Drs. M. Suga and Y. Umena for their help in the structural analysis of LPC, Dr. Y. Kashino for his help in identifying the LPC in the early stage of this work, Dr. M. T. Madigan for providing the Tch. tepidum strain, the staff members at beamlines BL41XU and BL26B1 of SPring-8 for their help in data collection, and the staff members of the Faculty of Science, Okayama University for their help in N-terminal sequencing and DNA sequencing experiments. This work was supported by the National Key R&D Program of China (2017YFA0503700), a Strategic Priority Research Program of CAS (XDB17000000), a CAS Key Research Project for Frontier Science (QYZDY-SSW-SMC003), and a JSPS KAKENHI Grant Number JP17H06433. JHC acknowledges the financial support provided by China Scholarship Council (CSC). AB was supported in part by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05. The sequences were deposited in GenBank under the Accession Number MH000336, and the coordinate and the structure factors were deposited in the Protein Data Bank under Accession Number 5ZE8.


  1. Allen JWA, Daltrop O, Stevens JM, Ferguson SJ (2003) C-type cytochromes: diverse structures and biogenesis systems pose evolutionary problems. Philos Trans R Soc B 358:255–266CrossRefGoogle Scholar
  2. Bamford VA, Angove HC, Seward HE, Thomson AJ, Cole JA, Butt JN, Hemmings AM, Richardson DJ (2002a) Structure and spectroscopy of the periplasmic cytochrome c nitrite reductase from Escherichia coli. Biochemistry 41:2921–2931CrossRefGoogle Scholar
  3. Bamford VA, Bruno S, Rasmussen T, Appia-Ayme C, Cheesman MR, Berks BC, Hemmings AM (2002b) Structural basis for the oxidation of thiosulfate by a sulfur cycle enzyme. EMBO J 21:5599–5610CrossRefGoogle Scholar
  4. Beinert H (2000) A tribute to sulfur. Eur J Biochem 267:5657–5664CrossRefGoogle Scholar
  5. Bergmann DJ, Hooper AB, Klotz MG (2005) Structure and sequence conservation of hao cluster genes of autotrophic ammonia-oxidizing bacteria: evidence for their evolutionary history. Appl Environ Microbiol 71:5371–5382CrossRefGoogle Scholar
  6. Bertini I, Cavallaro G, Rosato A (2006) Cytochrome c: occurrence and functions. Chem Rev 106:90–115CrossRefGoogle Scholar
  7. Bewley KD, Ellis KE, Firer-Sherwood MA, Elliott SJ (2013) Multi-heme proteins: nature’s electronic multi-purpose tool. Biochim Biophys Acta 1827:938–948CrossRefGoogle Scholar
  8. Blanc B, Mayfield JA, McDonald CA, Lukat-Rodgers GS, Rodgers KR, DuBois JL (2012) Understanding how the distal environment directs reactivity in chlorite dismutase: spectroscopy and reactivity of Arg183 mutants. Biochemistry 51:1895–1910CrossRefGoogle Scholar
  9. Bork P, Holm L, Sander C (1994) The Immunoglobulin fold—structural classification, sequence patterns and common core. J Mol Biol 242:309–320Google Scholar
  10. Brummendorf T, Rathjen FG (1995) Cell-adhesion molecules 1: immunoglobulin superfamily. Protein Profile 2:963–1108Google Scholar
  11. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D 66:12–21CrossRefGoogle Scholar
  12. Costa C, Moura JJG, Moura I, Liu MY, Peck HD Jr, LeGall J, Wang Y, Huynh BH (1990) Hexaheme nitrite reductase from Desulfovibrio desulfuricans: Mössbauer and EPR characterization of the heme groups. J Biol Chem 265:143132–114387Google Scholar
  13. Cowtan K (2006) The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr D 62:1002–1011CrossRefGoogle Scholar
  14. Cunha CA, Macieira S, Dias JM, Almeida G, Goncalves LL, Costa C, Lampreia J, Huber R, Moura JJG, Moura I, Romao MJ (2003) Cytochrome c nitrite reductase from Desulfovibrio desulfuricans ATCC 27774—the relevance of the two calcium sites in the structure of the catalytic subunit (NrfA). J Biol Chem 278:17455–17465CrossRefGoogle Scholar
  15. Dam P, Olman V, Harris K, Su ZC, Xu Y (2007) Operon prediction using both genome-specific and general genomic information. Nucleic Acids Res 35:288–298CrossRefGoogle Scholar
  16. DeLano WL (2002) The PyMOL molecular graphics system. DeLano Scientific, San Carlos.
  17. Einsle O, Messerschmidt A, Stach P, Bourenkov GP, Bartunik HD, Huber R, Kroneck PMH (1999) Structure of cytochrome c nitrite reductase. Nature 400:476–480CrossRefGoogle Scholar
  18. Einsle O, Stach P, Messerschmidt A, Simon J, Kroger A, Huber R, Kroneck PMH (2000) Cytochrome c nitrite reductase from Wolinella succinogenes—structure at 1.6 angstrom resolution, inhibitor binding, and heme-packing motifs. J Biol Chem 275:39608–39616CrossRefGoogle Scholar
  19. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D 60:2126–2132CrossRefGoogle Scholar
  20. Fathir I, Tanaka K, Yoza K, Kojima A, Kobayashi M, Wang ZY, Lottspeich F, Nozawa T (1997) The genes coding for the L, M and cytochrome subunits of the photosynthetic reaction center from the thermophilic purple sulfur bacterium Chromatium tepidum. Photosynth Res 51:71–82CrossRefGoogle Scholar
  21. Gilch S, Meyer O, Schmidt I (2009) A soluble form of ammonia monooxygenase in nitrosomonas europaea. Biol Chem 390:863–873CrossRefGoogle Scholar
  22. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  23. Henriksen A, Smith AT, Gajhede M (1999) The structures of the horseradish peroxidase c-ferulic acid complex and the ternary complex with cyanide suggest how peroxidases oxidize small phenolic substrates. J Biol Chem 274:35005–35011CrossRefGoogle Scholar
  24. Holm L, Rosenström P (2010) DALI server: conservation mapping in 3D. Nucleic Acids Res 38:545–549CrossRefGoogle Scholar
  25. Igarashi N, Moriyama H, Fujiwara T, Fukumori Y, Tanaka N (1997) The 2.8 angstrom structure of hydroxylamine oxidoreductase from a nitrifying chemoautotrophic bacterium, Nitrosomonas europaea. Nat Struct Biol 4:276–284CrossRefGoogle Scholar
  26. Iverson TM, Arciero DM, Hsu BT, Logan MSP, Hooper AB, Rees DC (1998) Heme packing motifs revealed by the crystal structure of the tetra-heme cytochrome c 554 from Nitrosomonas europaea. Nat Struct Biol 5:1005–1012CrossRefGoogle Scholar
  27. Iverson TM, Arciero DM, Hooper AB, Rees DC (2001) High-resolution structures of the oxidized and reduced states of cytochrome c 554 from Nitrosomonas europaea. J Biol Inorg Chem 6:390–397CrossRefGoogle Scholar
  28. Kabsch W (2010) XDS. Acta Crystallogr D 66:125–132CrossRefGoogle Scholar
  29. Kilmartin JR, Maher MJ, Krusong K, Noble CJ, Hanson GR, Bernhardt PV, Riley MJ, Kappler U (2011) Insights into structure and function of the active site of SoxAX cytochromes. J Biol Chem 286:24872–24881CrossRefGoogle Scholar
  30. Kim HJ, Zatsman A, Upadhyay AK, Whittaker M, Bergmann D, Hendrich MP, Hooper AB (2008) Membrane tetraheme cytochrome c m552 of the ammonia-oxidizing Nitrosomonas europaea: a ubiquinone reductase. Biochemistry 47:6539–6551CrossRefGoogle Scholar
  31. Klimmek O, Stein T, Pisa R, Simon J, Kroger A (1999) The single cysteine residue of the Sud protein is required for its function as a polysulfide-sulfur transferase in Wolinella succinogenes. Eur J Biochem 263:79–84CrossRefGoogle Scholar
  32. Kobayashi M, Saito T, Takahashi K, Wang ZY, Nozawa T (2005) Electronic properties and thermal stability of soluble redox proteins from a thermophilic purple sulfur photosynthetic bacterium, Thermochromatium tepidum. Bull Chem Soc Jpn 78:2164–2170CrossRefGoogle Scholar
  33. Lottspeich F (1985) Microscale isocratic separation of phenylthiohydantoin amino-acid derivatives. J Chromatogr 326:321–327CrossRefGoogle Scholar
  34. Maalcke WJ, Reimann J, de Vries S, Butt JN, Dietl A, Kip N, Mersdorf U, Barends TRM, Jetten MSM, Keltjens JT, Kartal B (2016) Characterization of anammox hydrazine dehydrogenase, a key N2-producing enzyme in the global nitrogen cycle. J Biol Chem 291:17077–17092CrossRefGoogle Scholar
  35. Mao FL, Dam P, Chou J, Olman V, Xu Y (2009) DOOR: a database for prokaryotic operons. Nucl Acids Res 37:459–463CrossRefGoogle Scholar
  36. Meyer TE, Vorkink WP, Tollin G, Cusanovich MA (1985) Chromatium flavocytochrome c: kinetics of reduction of the heme subunit, and the flavocytochrome c-mitochondrial cytochrome c complex. Arch Biochem Biophys 236:52–58CrossRefGoogle Scholar
  37. Mouncey NJ, Choudhary M, Kaplan S (1997) Characterization of genes encoding dimethyl sulfoxide reductase of Rhodobacter sphaeroides 2.4.1T: an essential metabolic gene function encoded on chromosome II. J Bacteriol 179:7617–7624CrossRefGoogle Scholar
  38. Murshudov GN, Skubak P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Long F, Vagin AA (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D 67:355–367CrossRefGoogle Scholar
  39. Nicholas KB, Nicholas HB, Deerfield DW (1997) GeneDoc: analysis and visualization of genetic variation. EMBnet News 4:14–18Google Scholar
  40. Osyczka A, Yoshida M, Nagashima KVP, Shimada K, Matsuura K (1997) Electron transfer from high-potential iron-sulfur protein and low-potential cytochrome c-551 to the primary donor of Rubrivivax gelatinosus reaction center mutationally devoid of the bound cytochrome subunit. Biochim Biophys Acta 1321:93–99CrossRefGoogle Scholar
  41. Pereira IAC, Pacheco I, Liu M-Y, LeGall J, Xavier AV, Texeira M (1997) Multiheme cytochromes from the sulfur-reducing bacterium Desulfuromonas acetoxidans. Eur J Biochem 248:323–328CrossRefGoogle Scholar
  42. Pereira IAC, LeGall J, Xavier AV, Teixeira M (2000) Characterization of a heme c nitrite reductase from a non-ammonifying microorganism, Desulfovibrio vulgaris Hildenborough. Biochim Biophys Acta 1481:119–130CrossRefGoogle Scholar
  43. Sharma S, Cavallaro G, Rosato A (2010) A systematic investigation of multiheme c-type cytochromes in prokaryotes. J Biol Inorg Chem 15:559–571CrossRefGoogle Scholar
  44. Shaw AL, Hanson GR, McEwan AG (1996) Cloning and sequence analysis of the dimethylsulfoxide reductase structural gene from Rhodobacter capsulatus. Biochim Biophys Acta 1276:176–180CrossRefGoogle Scholar
  45. Sheldrick GM (2010) Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr D 66:479–485CrossRefGoogle Scholar
  46. Smith LJ, Kahraman A, Thornton JM (2010) Heme proteins—diversity in structural characteristics, function, and folding. Proteins 78:2349–2368CrossRefGoogle Scholar
  47. Suga M, Lai TL, Sugiura M, Shen J-R, Boussac A (2013) Crystal structure at 1.5 angstrom resolution of the PsbV2 cytochrome from the cyanobacterium Thermosynechococcus elongates. FEBS Lett 587:3267–3272CrossRefGoogle Scholar
  48. Upadhyay AK, Petasis DT, Arciero DM, Hooper AB, Hendrich MP (2003) Spectroscopic characterization and assignment of reduction potentials in the tetraheme cytochrome c 554 from Nitrosomonas europaea. J Am Chem Soc 125:1738–1747CrossRefGoogle Scholar
  49. Urich T, Gomes CM, Kletzin A, Frazao C (2006) X-ray structure of a self-compartmentalizing sulfur cycle metalloenzyme. Science 311:996–1000CrossRefGoogle Scholar
  50. Walker FA (2006) The heme environment of mouse neuroglobin: histidine imidazole plane orientations obtained from solution NMR and EPR spectroscopy as compared with X-ray crystallography. J Biol Inorg Chem 11:391–397CrossRefGoogle Scholar
  51. Williams AF, Barclay AN (1988) The immunoglobulin superfamily—domains for cell-surface recognition. Ann Rev Immun 6:381–405CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Jing-Hua Chen
    • 1
    • 2
    • 3
  • Long-Jiang Yu
    • 2
  • Alain Boussac
    • 4
  • Zheng-Yu Wang-Otomo
    • 5
  • Tingyun Kuang
    • 1
  • Jian-Ren Shen
    • 1
    • 2
    Email author
  1. 1.Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of BotanyChinese Academy of SciencesBeijingChina
  2. 2.Research Institute for Interdisciplinary Science, Graduate School of Natural Science and TechnologyOkayama UniversityOkayamaJapan
  3. 3.University of Chinese Academy of SciencesBeijingChina
  4. 4.I2BC, SB2SM, CNRS UMR 9198, CEA SaclayGif-sur-YvetteFrance
  5. 5.Faculty of ScienceIbaraki UniversityMitoJapan

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