, Volume 28, Issue 4, pp 637–652 | Cite as

Catalytic enhancement of the heme-based oxygen-sensing phosphodiesterase EcDOS by hydrogen sulfide is caused by changes in heme coordination structure

  • Fang Yan
  • Veronika Fojtikova
  • Petr Man
  • Martin Stranava
  • Markéta Martínková
  • Yongming Du
  • Dongyang Huang
  • Toru Shimizu


EcDOS is a heme-based O2-sensing phosphodiesterase in which O2 binding to the heme iron complex in the N-terminal domain substantially enhances catalysis toward cyclic-di-GMP, which occurs in the C-terminal domain. Here, we found that hydrogen sulfide enhances the catalytic activity of full-length EcDOS, possibly owing to the admixture of 6-coordinated heme Fe(III)–SH and Fe(II)–O2 complexes generated during the reaction. Alanine substitution at Met95, the axial ligand for the heme Fe(II) complex, converted the heme Fe(III) complex into the heme Fe(III)–SH complex, but the addition of Na2S did not further reduce it to the heme Fe(II) complex of the Met95Ala mutant, and no subsequent formation of the heme Fe(II)–O2 complex was observed. In contrast, a Met95His mutant formed a stable heme Fe(II)–O2 complex in response to the same treatment. An Arg97Glu mutant, containing a glutamate substitution at the amino acid that interacts with O2 in the heme Fe(II)–O2 complex, formed a stable heme Fe(II) complex in response to Na2S, but this complex failed to bind O2. Interestingly, the addition of Na2S promoted formation of verdoheme (oxygen-incorporated, modified protoporphyrin IX) in an Arg97Ile mutant. Catalytic enhancement by Na2S was similar for Met95 mutants and the wild type, but significantly lower for the Arg97 mutants. Thus, this study shows the first isolation of spectrometrically separated, stable heme Fe(III)–SH, heme Fe(II) and heme Fe(II)–O2 complexes of full-length EcDOS with Na2S, and confirms that external-ligand–bound, 6-coordinated heme Fe(III)–SH or heme Fe(II)–O2 complexes critically contribute to the Na2S-induced catalytic enhancement of EcDOS.


Heme O2 sensor Phosphodiesterase c-di-GMP Hydrogen sulfide 



Cyclic dinucleotide-GMP, bis(3′,5′)-cyclic dimeric guanosine monophosphate


A heme-based O2-sensor phosphodiesterase from E. coli (also called DosP)

Heme Fe(II)

Protoporphyrin IX ferrous complex

Heme Fe(III)

Protoporphyrin IX ferric complex, hemin


High-pressure (performance) liquid chromatography


Linear dinucleotide-GMP (also called pGpG)


Matrix-assisted laser desorption/ionization


Fourier transform-ion cyclotron resonance




Cytochromes P450


A heme-based O2 sensor diguanylate cyclase from E. coli (also called DosC)



This work was supported in part by Grants-in-Aid from Shantou University Medical College, and Grants from the National Natural Science Foundation of China (NSFC) (31170736, 31371333), Guangdong Natural Science Foundation (S2012030006289), Charles University in Prague (UNCE 204025/2012), the Grant Agency of Charles University in Prague (756214) and the Grant Agency of the Czech Republic (Grant 15-19883S). Mass spectrometry facility was supported by the Operational Program Prague—Competitiveness, project CZ.2.16/3.1.00/24023. We are grateful to Profs. Jikai Jiang and Wenhong Luo for technical assistance of HPLC at the initial stage of this work.

Supplementary material

10534_2015_9847_MOESM1_ESM.pdf (102 kb)
(PDF 103 kb)


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Fang Yan
    • 1
  • Veronika Fojtikova
    • 2
  • Petr Man
    • 2
    • 3
  • Martin Stranava
    • 2
  • Markéta Martínková
    • 2
  • Yongming Du
    • 1
  • Dongyang Huang
    • 1
  • Toru Shimizu
    • 1
    • 2
  1. 1.Department of Cell Biology and Genetics and Key Laboratory of Molecular Biology in High Cancer Incidence Coastal Chaoshan Area of Guangdong Higher Education InstitutesShantou University Medical CollegeShantouChina
  2. 2.Department of Biochemistry, Faculty of ScienceCharles University in PraguePrague 2Czech Republic
  3. 3.Institute of MicrobiologyAcademy of Sciences of the Czech RepublicPrague 4Czech Republic

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