, Volume 29, Issue 4, pp 715–729 | Cite as

Effects of hydrogen sulfide on the heme coordination structure and catalytic activity of the globin-coupled oxygen sensor AfGcHK

  • Veronika Fojtikova
  • Martina Bartosova
  • Petr Man
  • Martin Stranava
  • Toru Shimizu
  • Marketa Martinkova


AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O2 or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H2S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O2, CO, and NO. To address this knowledge gap, we investigated the effects of H2S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O2-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na2S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na2S, the initially formed Fe(III)–SH heme complex was quickly converted into Fe(II) and Fe(II)–O2 complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)–SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)–OH complex), whereas in the case of EcDOS the changes in coordination caused by Na2S treatment led to remarkable increases in catalytic activity.


Hydrogen sulfide Heme-based oxygen sensor Autophosphorylation Histidine kinase Intramolecular catalytic regulation Two-component signal transduction 



A globin-coupled oxygen sensor histidine kinase from Anaeromyxobacter sp. Fw109-5


Escherichia coli direct oxygen sensor or heme-regulated phosphodiesterase from E. coli or EcDosP


Fe(III)–protoporphyrin IX complex, or hemin


Fe(II)–protoporphyrin IX complex


Globin-coupled oxygen sensor


Matrix-assisted laser desorption/ionization mass spectrometry


Matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance


An acronym derived from the words Per (Drosophila period clock protein)-Arnt (vertebrate aryl hydrocarbon receptor nuclear translocator)-Sim (Drosophila single-minded protein)


A globin-coupled diguanylate cyclase from E. coli or EcDosC


A sensor (globin) domain containing heme of YddV


Wild type



This work was supported in part by Charles University in Prague (UNCE 204025/2012), the Grant Agency of Charles University in Prague (362115) and the Grant Agency of the Czech Republic (Grant 15-19883S). The mass spectrometry facility used in this work was supported by the EU project CZ.1.05/1.1.00/02.0109. We are grateful to Dr. Kenichi Kitanishi for valuable discussion during the early stages of this project.

Supplementary material

10534_2016_9947_MOESM1_ESM.doc (488 kb)
Supplementary material 1 (DOC 488 kb)


  1. Andersson LA, Loehr TM, Lim AR, Mauk AG (1984) Sulfmyoglobin. Resonance Raman spectroscopic evidence for an iron-chlorin prosthetic group. J Biol Chem 259:15340–15349PubMedGoogle Scholar
  2. Banerjee R (2011) Hydrogen sulfide: redox metabolism and signaling. Antioxid Redox Signal 15:339–341. doi: 10.1089/ars.2011.3912 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Berzofsky JA, Peisach J, Blumberg WE (1971) Sulfheme proteins. I. Optical and magnetic properties of sulfmyoglobin and its derivatives. J Biol Chem 246:3367–3377PubMedGoogle Scholar
  4. Bostelaar T, Vitvitsky V, Kumutima J, Lewis BE, Yadav PK, Brunold TC, Filipovic M, Lehnert N, Stemmler TL, Banerjee R (2016) Hydrogen sulfide oxidation by myoglobin. J Am Chem Soc. doi: 10.1021/jacs.6b03456 PubMedGoogle Scholar
  5. Du Y, Liu G, Yan Y, Huang D, Luo W, Martinkova M, Man P, Shimizu T (2013) Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS). Biometals 26:839–852. doi: 10.1007/s10534-013-9640-4 CrossRefPubMedGoogle Scholar
  6. Fojtikova V, Stranava M, Vos MH, Liebl U, Hranicek J, Kitanishi K, Shimizu T, Martinkova M (2015) Kinetic analysis of a globin-coupled histidine kinase, AfGcHK: effects of the heme iron complex, response regulator, and metal cations on autophosphorylation activity. Biochemistry 54:5017–5029. doi: 10.1021/acs.biochem.5b00517 CrossRefPubMedGoogle Scholar
  7. Germani F, Moens L, Dewilde S (2013) Haem-based sensors: a still growing old superfamily. Adv Microb Physiol 63:1–47. doi: 10.1016/B978-0-12-407693-8.00001-7 CrossRefPubMedGoogle Scholar
  8. Gilles-Gonzalez M-A, Gonzalez G (2005) Heme-based sensors: defining characteristics, recent developments, and regulatory hypotheses. J Inorg Biochem 99:1–22. doi: 10.1016/j.jinorgbio.2004.11.006 CrossRefPubMedGoogle Scholar
  9. Girvan HM, Munro AW (2013) Heme sensor proteins. J Biol Chem 288:13194–13203. doi: 10.1074/jbc.R112.422642 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Green J, Crack JC, Thomson AJ, LeBrun NE (2009) Bacterial sensors of oxygen. Curr Opin Microbiol 12:145–151. doi: 10.1016/j.mib.2009.01.008 CrossRefPubMedGoogle Scholar
  11. Hirata S, Matsui T, Sasakura Y, Sugiyama S, Yoshimura T, Sagami I, Shimizu T (2003) Characterization of Met95 mutants of a heme-regulated phosphodiesterase from Escherichia coli. Optical absorption, magnetic circular dichroism, circular dichroism, and redox potentials. Eur J Biochem 270:4771–4779. doi: 10.1046/j.1432-1033.2003.03879.x CrossRefPubMedGoogle Scholar
  12. Igarashi J, Kitanishi K, Shimizu T (2011) Emerging role of heme as a signal and the gas-sensing site: heme-sensing and gas-sensing. In: Kadish KM, Smith KM, Guilard R (eds) Handbook of porphyrin science, vol 15. World Scientific Publishing, Hackensack (Chapter 73) Google Scholar
  13. Kimura H (2015) Signaling molecules: hydrogen sulfide and polysulfide. Antioxid Redox Signal 22:362–376. doi: 10.1089/ars.2014.5869 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Kimura Y, Toyofuku Y, Koike S, Shibuya N, Nagahara N, Lefer D, Ogasawara Y, Kimura H (2015) Identification of H2S3 and H2S produced by 3-mercaptopyruvate sulfurtransferase in the brain. Sci Rep 5:14774. doi: 10.1038/srep14774 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Kitanishi K, Kobayashi K, Kawamura Y, Ishigami I, Ogura T, Nakajima K, Igarashi J, Tanaka A, Shimizu T (2010) Important roles of Tyr43 at the putative heme distal side in the oxygen recognition and stability of the Fe(II)–O2 complex of YddV, a globin-coupled heme-based oxygen sensor diguanylate cyclase. Biochemistry 49:10381–10393. doi: 10.1021/bi100733q CrossRefPubMedGoogle Scholar
  16. Kitanishi K, Kobayashi K, Uchida T, Ishimori K, Igarashi J, Shimizu T (2011) Identification and functional and spectral characterization of a globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5. J Biol Chem 286:35522–35534. doi: 10.1074/jbc.M111.274811 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Lambry J-C, Stranava M, Lobato L, Martinkova M, Shimizu T, Liebl U, Vos MH (2016) Ultrafast spectroscopy evidence for picosecond ligand exchange at the binding site of a heme protein: heme-based sensor YddV. J Phys Chem Lett 7:69–74. doi: 10.1021/acs.jpclett.5b02517 CrossRefPubMedGoogle Scholar
  18. Martínková M, Kitanishi K, Shimizu T (2013) Heme-based globin-coupled oxygen sensors: linking oxygen binding to functional regulation of diguanylate cyclase, histidine kinase, and methyl-accepting chemotaxis. J Biol Chem 288:27702–27711. doi: 10.1074/jbc.R113.473249 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Mishanina TV, Libiad M, Banerjee R (2015) Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways. Nat Chem Biol 11:457–464. doi: 10.1038/nchembio.1834 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Muyzer G, Stams AJ (2008) The ecology and biotechnology of sulphate-reducing bacteria. Nat Rev Microbiol 6:441–454. doi: 10.1038/nrmicro1892 PubMedGoogle Scholar
  21. Nakajima K, Kitanishi K, Kobayashi K, Kobayashi N, Igarashi J, Shimizu T (2012) Leu65 in the heme distal side is critical for the stability of the Fe(II)–O2 complex of YddV, a globin-coupled oxygen sensor diguanylate cyclase. J Inorg Biochem 108:163–170. doi: 10.1016/j.jinorgbio.2011.09.019 CrossRefPubMedGoogle Scholar
  22. Nicoletti FP, Comandini A, Bonamore A, Boechi L, Boubeta FM, Feis A, Smulevich G, Boffi A (2010) Sulfide binding properties of truncated hemoglobins. Biochemistry 49:2269–2278. doi: 10.1021/bi901671d CrossRefPubMedGoogle Scholar
  23. Paul BD, Snyder SH (2015) H2S: a novel gasotransmitter that signals by sulfhydration. Trends Biochem Sci 40:687–700. doi: 10.1016/j.tibs.2015.08.007 CrossRefPubMedGoogle Scholar
  24. Pietri R, Román-Morales E, López-Garriga J (2011) Hydrogen sulfide and hemeproteins: knowledge and mysteries. Antioxid Redox Signal 15:393–404. doi: 10.1089/ars.2010.3698 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Poulos TL (2014) Heme enzyme structure and function. Chem Rev 114:3919–3962. doi: 10.1021/cr400415k CrossRefPubMedPubMedCentralGoogle Scholar
  26. Ramos-Alvarez C, Yoo B-K, Pietri R, Lamarre I, Martin J-L, Lopez-Garriga J, Negrerie M (2013) Reactivity and dynamics of H2S, NO, and O2 interacting with hemoglobins from Lucina pectinata. Biochemistry 52:7007–7021. doi: 10.1021/bi400745a CrossRefPubMedGoogle Scholar
  27. Rey FE, Gonzalez MD, Cheng J, Wu M, Ahern PP, Gordon JI (2013) Metabolic niche of a prominent sulfate-reducing human gut bacterium. Proc Natl Acad Sci USA 110:13582–13587. doi: 10.1073/pnas.1312524110 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Ríos-González BB, Román-Morales EM, Pietri R, López-Garriga J (2014) Hydrogen sulfide activation in hemeproteins: the sulfheme scenario. J Inorg Biochem 133:78–86. doi: 10.1016/j.jinorgbio.2014.01.013 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Sakamoto H, Omata Y, Adachi Y, Palmer G, Noguchi M (2000) Separation and identification of the regioisomers of verdoheme by reversed-phase ion-pair high-performance liquid chromatography, and characterization of their complexes with heme oxygenase. J Inorg Biochem 82:113–121CrossRefPubMedGoogle Scholar
  30. Shimizu T (2013) Revisit of the interactions between hydrogen sulfide and heme proteins. Curr Chem Biol 7:207–212CrossRefGoogle Scholar
  31. Shimizu T, Huang D, Yan F, Stranava M, Bartosova M, Fojtíková V, Martínková M (2015) Gaseous O2, NO, and CO in signal transduction: structure and function relationships of heme-based gas sensors and heme-redox sensors. Chem Rev 115:6491–6533. doi: 10.1021/acs.chemrev.5b00018 CrossRefPubMedGoogle Scholar
  32. Stranava M, Martínková M, Stiborová M, Man P, Kitanishi K, Muchová L, Vítek L, Martínek V, Shimizu T (2014) Introduction of water into the heme distal side by Leu65 mutations of an oxygen sensor, YddV, generates verdoheme and carbon monoxide, exerting the heme oxygenase reaction. J Inorg Biochem 140:29–38. doi: 10.1016/j.jinorgbio.2014.06.010 CrossRefPubMedGoogle Scholar
  33. Takahashi H, Sekimoto M, Tanaka M, Tanaka A, Igarashi J, Shimizu T (2012) Hydrogen sulfide stimulates the catalytic activity of a heme-regulated phosphodiesterase from Escherichia coli (Ec DOS). J Inorg Biochem 109:66–71. doi: 10.1016/j.jinorgbio.2012.01.001 CrossRefPubMedGoogle Scholar
  34. Tarnawski M, Barends TRM, Schlichting I (2015) Structural analysis of an oxygen-regulated diguanylate cyclase. Acta Crystallogr D Biol Crystallogr 71:2158–2177. doi: 10.1107/S139900471501545X CrossRefPubMedGoogle Scholar
  35. Tuckerman JR, Gonzalez G, Sousa EHS, Wan X, Saito JA, Alam M, Gilles-Gonzalez M-A (2009) An oxygen-sensing diguanylate cyclase and phosphodiesterase couple for c-di-GMP control. Biochemistry 48:9764–9774. doi: 10.1021/bi901409g CrossRefPubMedGoogle Scholar
  36. Uchida T, Kitagawa T (2005) Mechanism for transduction of the ligand-binding signal in heme-based gas sensory proteins revealed by resonance Raman spectroscopy. Acc Chem Res 38:662–670. doi: 10.1021/ar030267d CrossRefPubMedGoogle Scholar
  37. Voet D, Voet J (2011) Hemoglobin: Protein function in microcosm. Biochemistry, 4th edn. Wiley, New York, pp 323–358Google Scholar
  38. Washio J, Sato T, Koseki T, Takahashi N (2005) Hydrogen sulfide-producing bacteria in tongue biofilm and their relationship with oral malodour. J Med Microbiol 54:889–895. doi: 10.1099/jmm.0.46118-0 CrossRefPubMedGoogle Scholar
  39. Yan F, Fojtikova V, Man P, Stranava M, Martínková M, Du Y, Huang D, Shimizu T (2015) Catalytic enhancement of the heme-based oxygen-sensing phosphodiesterase EcDOS by hydrogen sulfide is caused by changes in heme coordination structure. Biometals 28:637–652. doi: 10.1007/s10534-015-9847-7 CrossRefPubMedGoogle Scholar
  40. Zhang W, Phillips GN (2003) Structure of the oxygen sensor in Bacillus subtilis: signal transduction of chemotaxis by control of symmetry. Structure 11:1097–1110. doi: 10.1016/S0969-2126(03)00169-2 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Veronika Fojtikova
    • 1
  • Martina Bartosova
    • 1
  • Petr Man
    • 1
    • 2
  • Martin Stranava
    • 1
  • Toru Shimizu
    • 1
  • Marketa Martinkova
    • 1
  1. 1.Department of Biochemistry, Faculty of ScienceCharles University in PraguePrague 2Czech Republic
  2. 2.Biotechnology and Biomedicine Centre (BioCeV)Institute of Microbiology of the Czech Academy of Science, v.v.i.VestecCzech Republic

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