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A novel insight into the heme and NO/CO binding mechanism of the alpha subunit of human soluble guanylate cyclase

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Abstract

Human soluble guanylate cyclase (sGC), a critical heme-containing enzyme in the NO-signaling pathway of eukaryotes, is an αβ heterodimeric hemoprotein. Upon the binding of NO to the heme, sGC catalyzes the conversion of GTP to cyclic GMP, playing a crucial role in many physiological processes. However, the specific contribution of the α and β subunits of sGC in the intact heme binding remained intangible. The recombinant human sGC α1 subunit has been expressed in Escherichia coli and characterized for the first time. The heme binding and related NO/CO binding properties of both the α1 subunit and the β1 subunit were investigated via heme reconstitution, UV–vis spectroscopy, EPR spectroscopy, stopped-flow kinetics, and homology modeling. These results indicated that the α1 subunit of human sGC, lacking the conserved axial ligand, is likely to interact with heme noncovalently. On the basis of the equilibrium and kinetics of CO binding to sGC, one possible CO binding model was proposed. CO binds to human sGCβ195 by simple one-step binding, whereas CO binds to human sGCα259, possibly from both axial positions through a more complex process. The kinetics of NO dissociation from human sGC indicated that the NO dissociation from sGC was complex, with at least two release phases, and human sGCα259 has a smaller k 1 but a larger k 2. Additionally, the role of the cavity of the α1 subunit of human sGC was explored, and the results indicate that the cavity likely accommodates heme. These results are beneficial for understanding the overall structure of the heme binding site of the human sGC and the NO/CO signaling mechanism.

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Abbreviations

cDNA:

Complementary DNA

cGMP:

Guanosine 3′,5′-cyclic monophosphate

DEA/NO:

Diethylammonium (Z)-1-(N,N-diethylamino)-diazen-1-ium-1,2-diolate

DTT:

Dithiothreitol

H-NOX domain:

Heme–nitric oxide/oxygen binding domain

hsGC:

Human soluble guanylate cyclase

hsGCα259:

N-terminus truncated human soluble guanylate cyclase α1 subunit with residues 65–259

hsGCα385:

N-terminus truncated human soluble guanylate cyclase α1 subunit with residues 65–385

hsGCα690:

Full-length human soluble guanylate cyclase α1 subunit with residues 1–690

sGC:

Soluble guanylate cyclase

YC-1:

5-[1-(Phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol

References

  1. Chen KJ, Pittman RN, Popel AS (2008) Antioxid Redox Signal 10:1185–1198

    Article  PubMed  CAS  Google Scholar 

  2. Denninger JW, Marletta MA (1999) Biochim Biophys Acta 1411:334–350

    Article  PubMed  CAS  Google Scholar 

  3. Evgenov OV, Pacher P, Schmidt PM, Hasko G, Schmidt HH, Stasch JP (2006) Nat Rev Drug Discov 5:755–768

    Article  PubMed  CAS  Google Scholar 

  4. Wang-Rosenke Y, Neumayer HH, Peters H (2008) Curr Med Chem 15:1396–1406

    Article  PubMed  CAS  Google Scholar 

  5. Pellicena P, Karow DS, Boon EM, Marletta MA, Kuriyan J (2004) Proc Natl Acad Sci USA 101:12854–12859

    Article  PubMed  CAS  Google Scholar 

  6. Schmidt PM, Schramm M, Schroder H, Wunder F, Stasch JP (2004) J Biol Chem 279:3025–3032

    Article  PubMed  CAS  Google Scholar 

  7. Stone JR, Marletta MA (1994) Biochemistry (Mosc) 33:5636–5640

    Article  CAS  Google Scholar 

  8. Foerster J, Harteneck C, Malkewitz J, Schultz G, Koesling D (1996) Eur J Biochem 240:380–386

    Article  PubMed  CAS  Google Scholar 

  9. Stone JR, Marletta MA (1995) Biochemistry (Mosc) 34:14668–14674

    Article  CAS  Google Scholar 

  10. Tomita T, Tsuyama S, Imai Y, Kitagawa T (1997) J Biochem 122:531–536

    PubMed  CAS  Google Scholar 

  11. Pal B, Kitagawa T (2010) Biochem Biophys Res Commun 397:375–379

    Article  PubMed  CAS  Google Scholar 

  12. Hu X, Murata LB, Weichsel A, Brailey JL, Roberts SA, Nighorn A, Montfort WR (2008) J Biol Chem 283:20968–20977

    Article  PubMed  CAS  Google Scholar 

  13. Zhong F, Wang H, Ying T, Huang ZX, Tan X (2010) Amino Acids 39:399–408

    Article  PubMed  CAS  Google Scholar 

  14. Lee YC, Martin E, Murad F (2000) Proc Natl Acad Sci USA 97:10763–10768

    Article  PubMed  CAS  Google Scholar 

  15. Karow DS, Pan DH, Davis JH, Behrends S, Mathies RA, Marletta MA (2005) Biochemistry (Mosc) 44:16266–16274

    Article  CAS  Google Scholar 

  16. Burstyn JN, Yu AE, Dierks EA, Hawkins BK, Dawson JH (1995) Biochemistry (Mosc) 34:5896–5903

    Article  CAS  Google Scholar 

  17. Friebe A, Schultz G, Koesling D (1996) EMBO J 15:6863–6868

    PubMed  CAS  Google Scholar 

  18. Denninger JW, Schelvis JPM, Brandish PE, Zhao Y, Babcock GT, Marletta MA (2000) Biochemistry (Mosc) 39:4191–4198

    Article  CAS  Google Scholar 

  19. Ibrahim M, Derbyshire ER, Marletta MA, Spiro TG (2010) Biochemistry (Mosc) 49:3815–3823

    Article  CAS  Google Scholar 

  20. Koglin M, Behrends S (2003) J Biol Chem 278:12590–12597

    Article  PubMed  CAS  Google Scholar 

  21. Li Z, Pal B, Takenaka S, Tsuyama S, Kitagawa T (2005) Biochemistry (Mosc) 44:939–946

    Article  CAS  Google Scholar 

  22. Stone JR, Marletta MA (1998) Chem Biol 5:255–261

    Article  CAS  Google Scholar 

  23. Winger JA, Derbyshire ER, Marletta MA (2007) J Biol Chem 282:897–907

    Article  PubMed  CAS  Google Scholar 

  24. Berry EA, Trumpower BL (1987) Anal Biochem 161:1–15

    Article  PubMed  CAS  Google Scholar 

  25. Chivian D, Kim DE, Malmstrom L, Bradley P, Robertson T, Murphy P, Strauss CE, Bonneau R, Rohl CA, Baker D (2003) Proteins 53(Suppl 6):524–533

    Article  PubMed  CAS  Google Scholar 

  26. Poulos TL (2006) Curr Opin Struct Biol 16:736–743

    Article  PubMed  CAS  Google Scholar 

  27. Ma X, Sayed N, Beuve A, van den Akker F (2007) EMBO J 26:578–588

    Article  PubMed  CAS  Google Scholar 

  28. Autenrieth F, Tajkhorshid E, Baudry J, Luthey-Schulten Z (2004) J Comput Chem 25:1613–1622

    Article  PubMed  CAS  Google Scholar 

  29. Laidig KE, Daggett V (1996) Fold Des 1:335–346

    Article  PubMed  CAS  Google Scholar 

  30. Mackerell AD Jr, Feig M, Brooks CL 3rd (2004) J Comput Chem 25:1400–1415

    Article  PubMed  CAS  Google Scholar 

  31. Guryev OL, Gilep AA, Usanov SA, Estabrook RW (2001) Biochemistry (Mosc) 40:5018–5031

    Article  CAS  Google Scholar 

  32. Hargrove MS, Barrick D, Olson JS (1996) Biochemistry (Mosc) 35:11293–11299

    Article  CAS  Google Scholar 

  33. Hargrove MS, Singleton EW, Quillin ML, Ortiz LA, Phillips GN Jr, Olson JS, Mathews AJ (1994) J Biol Chem 269:4207–4214

    PubMed  CAS  Google Scholar 

  34. Lamar GN, Toi H, Krishnamoorthi R (1984) J Am Chem Soc 106:6395–6401

    Article  CAS  Google Scholar 

  35. Kharitonov VG, Sharma VS, Magde D, Koesling D (1997) Biochemistry (Mosc) 36:6814–6818

    Article  CAS  Google Scholar 

  36. Walker FA (2003) Inorg Chem 42:4526–4544

    Article  PubMed  CAS  Google Scholar 

  37. Iyer LM, Anantharaman V, Aravind L (2003) BMC Genomics 4:5

    Article  PubMed  Google Scholar 

  38. Makino R, Matsuda H, Obayashi E, Shiro Y, Iizuka T, Hori H (1999) J Biol Chem 274:7714–7723

    Article  PubMed  CAS  Google Scholar 

  39. Nioche P, Berka V, Vipond J, Minton N, Tsai AL, Raman CS (2004) Science 306:1550–1553

    Article  PubMed  CAS  Google Scholar 

  40. Stone JR, Sands RH, Dunham WR, Marletta MA (1995) Biochem Biophys Res Commun 207:572–577

    Article  PubMed  CAS  Google Scholar 

  41. Yazawa S, Tsuchiya H, Hori H, Makino R (2006) J Biol Chem 281:21763–21770

    Article  PubMed  CAS  Google Scholar 

  42. Stone JR, Marletta MA (1995) Biochemistry (Mosc) 34:16397–16403

    Article  CAS  Google Scholar 

  43. Tsai AL, Berka V, Martin F, Ma X, van den Akker F, Fabian M, Olson JS (2010) Biochemistry (Mosc) 49:6587–6599

    Article  CAS  Google Scholar 

  44. Kharitonov VG, Russwurm M, Magde D, Sharma VS, Koesling D (1997) Biochem Biophys Res Commun 239:284–286

    Article  PubMed  CAS  Google Scholar 

  45. Zabel U, Hausler C, Weeger M, Schmidt HH (1999) J Biol Chem 274:18149–18152

    Article  PubMed  CAS  Google Scholar 

  46. Kharitonov VG, Sharma VS, Magde D, Koesling D (1999) Biochemistry (Mosc) 38:10699–10706

    Article  CAS  Google Scholar 

  47. Sharma VS, Magde D, Kharitonov VG, Koesling D (1999) Biochem Biophys Res Commun 254:188–191

    Article  PubMed  CAS  Google Scholar 

  48. Ma X, Sayed N, Baskaran P, Beuve A, van den Akker F (2008) J Biol Chem 283:1167–1178

    Article  PubMed  CAS  Google Scholar 

  49. Wagner C, Russwurm M, Jager R, Friebe A, Koesling D (2005) J Biol Chem 280:17687–17693

    Article  PubMed  CAS  Google Scholar 

  50. Zhou Z, Gross S, Roussos C, Meurer S, Muller-Esterl W, Papapetropoulos A (2004) J Biol Chem 279:24935–24943

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors would thank Jiangfeng Du for his support of EPR measurements. This work was financially supported partly by Shanghai Pujiang Talent Project (08PJ14017), National Natural Science Foundation of China (no. 20771029, no. 91013001, no. 31070211), Shanghai Leading Academic Discipline Project (B108), and the PhD program of the Education Ministry of China (20100071110011), to X.T.

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Authors and Affiliations

  1. Department of Chemistry, Fudan University, Shanghai, 200433, China

    Fangfang Zhong, Jie Pan, Xiaoxiao Liu, Tianlei Ying, Zhong-Xian Huang & Xiangshi Tan

  2. Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China

    Hongyan Wang & Xiangshi Tan

  3. Department of Modern Physics, University of Science and Technology of China, Hefei, 230026, China

    Jihu Su

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  1. Fangfang Zhong
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Correspondence to Xiangshi Tan.

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Zhong, F., Pan, J., Liu, X. et al. A novel insight into the heme and NO/CO binding mechanism of the alpha subunit of human soluble guanylate cyclase. J Biol Inorg Chem 16, 1227–1239 (2011). https://doi.org/10.1007/s00775-011-0811-x

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