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Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site

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Abstract

Thiol dioxygenases are a subset of non-heme mononuclear iron oxygenases that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield sulfinic acid products. Cysteine dioxygenase (CDO) and 3-mercaptopropionic acid (3MPA) dioxygenase (MDO) are the most extensively characterized members of this enzyme family. As with many non-heme mononuclear iron oxidase/oxygenases, CDO and MDO exhibit an obligate-ordered addition of organic substrate before dioxygen. As this substrate-gated O2-reactivity extends to the oxygen-surrogate, nitric oxide (NO), EPR spectroscopy has long been used to interrogate the [substrate:NO:enzyme] ternary complex. In principle, these studies can be extrapolated to provide information about transient iron-oxo intermediates produced during catalytic turnover with dioxygen. In this work, we demonstrate that cyanide mimics the native thiol-substrate in ordered-addition experiments with MDO cloned from Azotobacter vinelandii (AvMDO). Following treatment of the catalytically active Fe(II)-AvMDO with excess cyanide, addition of NO yields a low-spin (S = 1/2) (CN/NO)-Fe-complex. Continuous wave and pulsed X-band EPR characterization of this complex produced in wild-type and H157N variant AvMDO reveal multiple nuclear hyperfine features diagnostic of interactions within the first- and outer-coordination sphere of the enzymatic Fe-site. Spectroscopically validated computational models indicate simultaneous coordination of two cyanide ligands replaces the bidentate (thiol and carboxylate) coordination of 3MPA allowing for NO-binding at the catalytically relevant O2-binding site. This promiscuous substrate-gated reactivity of AvMDO with NO provides an instructive counterpoint to the high substrate-specificity exhibited by mammalian CDO for l-cysteine.

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Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

CDO:

Cysteine dioxygenase

MDO:

3-Mercaptopropionic acid dioxygenase

3MPA :

3-Mercaptopropionic acid

CYS :

L-cysteine

CA :

Cysteamine (2-aminoethanethiol)

ET :

Ethanethiol

β-Ala:

β-Alanine

3BPA :

3-Bromopropionic acid

3IPA :

3-Iodopropionic acid

CSA :

Cysteine sulfinic acid

HT :

Hypotaurine

3SPA :

3-Sulfinopropionic acid

Av :

Azotobacter vinelandii

CW:

Continuous wave

FT:

Fourier transform (or pulsed): EPR: electron paramagnetic resonance

HYSCORE:

Hyperfine sublevel correlation spectroscopy

DFT:

Density functional theory

References

  1. Dominy JE, Simmons CR, Hirschberger LL, Hwang J, Coloso RM, Stipanuk MH (2007) J Biol Chem 282:25189–25198

    Article  CAS  PubMed  Google Scholar 

  2. Stipanuk M, Simmons C, Andrew Karplus A, Dominy J (2010) Amino Acids, 1–12

  3. Ewetz L, Sorbo B (1966) Biochim Biophys Acta 128:296–305

    Article  CAS  PubMed  Google Scholar 

  4. Sorbo B, Ewetz L (1965) Biochem Biophys Res Commun 18:359–363

    Article  CAS  Google Scholar 

  5. Lombardini JB, Singer TP, Boyer PD (1969) J Biol Chem 244:1172–1175

    Article  CAS  PubMed  Google Scholar 

  6. Gordon C, Emery P, Bradley H, Waring H (1992) Lancett 229:25–26

    Article  Google Scholar 

  7. Dominy JE Jr, Simmons CR, Karplus PA, Gehring AM, Stipanuk MH (2006) J Bacteriol 188:5561–5569

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Heafield MT, Fearn S, Steventon GB, Waring RH, Williams AC, Sturman SG (1990) Neurosci Lett 110:216–220

    Article  CAS  PubMed  Google Scholar 

  9. Sarkar B, Kulharia M, Mantha AK (2017) Int J Exp Pathol 98:52–66

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Brown CD, Neidig ML, Neibergall MB, Lipscomb JD, Solomon EI (2007) J Am Chem Soc 129:7427–7438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yamaguchi K, Sakakibara S, Koga K, Ueda I (1971) Biochim Biophys Acta Gen Subj 237:502–512

    Article  CAS  Google Scholar 

  12. Perry TL, Norman MG, Yong VW, Whiting S, Crichton JU, Hansen S, Kish SJ (1985) Ann Neurol 18:482–489

    Article  CAS  PubMed  Google Scholar 

  13. Bradley H, Gough A, Sokhi RS, Hassell A, Waring R, Emery P (1994) J Rheumatol 21:1192–1196

    CAS  PubMed  Google Scholar 

  14. Deth R, Muratore C, Benzecry J, Power-Charnitsky V-A, Waly M (2008) Neurotoxicology 29:190–201

    Article  CAS  PubMed  Google Scholar 

  15. James SJ, Cutler P, Melnyk S, Jernigan S, Janak L, Gaylor DW, Neubrander JA (2004) Am J Clin Nutr 80:1611–1617

    Article  CAS  PubMed  Google Scholar 

  16. Reddie KG, Carroll KS (2008) Curr Opin Chem Biol 12:746–754

    Article  CAS  PubMed  Google Scholar 

  17. Winyard PG, Moody CJ, Jacob C (2005) Trends Biochem Sci 30:453–461

    Article  CAS  PubMed  Google Scholar 

  18. Trachootham D, Alexandre J, Huang P (2009) Nat Rev Drug Discov 8:579–591

    Article  CAS  PubMed  Google Scholar 

  19. Behave DP, Muse WB, Carroll KS (2007) Infect Disord Drug Targets 7:140–158

    Article  Google Scholar 

  20. Gunawardana DM, Heathcote KC, Flashman E (2021) FEBS J

  21. Weits DA, Giuntoli B, Kosmacz M, Parlanti S, Hubberten H-M, Riegler H, Hoefgen R, Perata P, van Dongen JT, Licausi F (2014) Nat Commun 5:3425

    Article  PubMed  Google Scholar 

  22. White MD, Klecker M, Hopkinson RJ, Weits DA, Mueller C, Naumann C, O’Neill R, Wickens J, Yang J, Brooks-Bartlett JC, Garman EF, Grossmann TN, Dissmeyer N, Flashman E (2017) Nat Commun 8:14690

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Masson N, Keeley TP, Giuntoli B, White MD, Puerta ML, Perata P, Hopkinson RJ, Flashman E, Licausi F, Ratcliffe PJ (2019) Science 365:65–69

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Fernandez RL, Elmendorf LD, Smith RW, Bingman CA, Fox BG, Brunold TC (2021) Biochemistry 60:3728–3737

    Article  CAS  PubMed  Google Scholar 

  25. Wang Y, Shin I, Li J, Liu A (2021) J Biol Chem 297:101176

    Article  CAS  PubMed  Google Scholar 

  26. Tchesnokov EP, Fellner M, Siakkou E, Kleffmann T, Martin LW, Aloi S, Lamont IL, Wilbanks SM, Jameson GNL (2015). J Biol Chem. https://doi.org/10.1074/jbc.M114.635672

    Article  PubMed  PubMed Central  Google Scholar 

  27. Tchesnokov EP, Faponle AS, Davies CG, Quesne MG, Turner R, Fellner M, Souness RJ, Wilbanks SM, de Visser SP, Jameson GNL (2016) Chem Commun (Camb) 52:8814–8817

    Article  CAS  PubMed  Google Scholar 

  28. Crowell JK, Sardar S, Hossain MS, Foss FW, Pierce BS (2016) Arch Biochem Biophys 604:86–94

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pierce BS, Subedi BP, Sardar S, Crowell JK (2015) Biochemistry 54:7477–7490

    Article  CAS  PubMed  Google Scholar 

  30. York NJ, Lockart MM, Sardar S, Khadka N, Shi W, Stenkamp RE, Zhang J, Kiser PD, Pierce BS (2021) J Biol Chem 296:100496

    Article  Google Scholar 

  31. York NJ, Lockart MM, Pierce BS (2021) Inorg Chem 60:18639–18651

    Article  CAS  PubMed  Google Scholar 

  32. Sardar S, Weitz A, Hendrich MP, Pierce BS (2019) Biochemistry 58:5135–5150

    Article  CAS  PubMed  Google Scholar 

  33. Li W, Blaesi EJ, Pecore MD, Crowell JK, Pierce BS (2013) Biochemistry 52:9104–9119

    Article  CAS  PubMed  Google Scholar 

  34. Fernandez RL, Juntunen ND, Brunold TC (2022) Acc Chem Res 55:2480–2490

    Article  CAS  PubMed  Google Scholar 

  35. Weitz AC, Giri N, Caranto JD, Kurtz DM, Bominaar EL, Hendrich MP (2017) J Am Chem Soc 139:12009–12019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Tierney DL, Rocklin AM, Lipscomb JD, Que L, Hoffman BM (2005) J Am Chem Soc 127:7005–7013

    Article  CAS  PubMed  Google Scholar 

  37. Casey TM, Grzyska PK, Hausinger RP, McCracken J (2013) J Phys Chem B 117:10384–10394

    Article  CAS  PubMed  Google Scholar 

  38. Li J, Koto T, Davis I, Liu A (2019) Biochemistry 58:2218–2227

    Article  CAS  PubMed  Google Scholar 

  39. McCracken J, Eser BE, Mannikko D, Krzyaniak MD, Fitzpatrick PF (2015) Biochemistry 54:3759–3771

    Article  CAS  PubMed  Google Scholar 

  40. Pierce BS, Gardner JD, Bailey LJ, Brunold TC, Fox BG (2007) Biochemistry 46:8569–8578

    Article  CAS  PubMed  Google Scholar 

  41. McQuilken AC, Ha Y, Sutherlin KD, Siegler MA, Hodgson KO, Hedman B, Solomon EI, Jameson GNL, Goldberg DP (2013) J Am Chem Soc 135:14024–14027

    Article  CAS  PubMed  Google Scholar 

  42. Stoll S, Schweiger A (2006) J Magn Reson 178:42–55

    Article  CAS  PubMed  Google Scholar 

  43. Neese F (2017) Wiley Interdiscip Rev Comput Mol Sci 8:e1327

    Google Scholar 

  44. Tao J, Perdew JP, Staroverov VN, Scuseria GE (2003) Phys Rev Lett 91:146401

    Article  PubMed  Google Scholar 

  45. Weigend F, Ahlrichs R (2005) Phys Chem Chem Phys 7:3297–3305

    Article  CAS  PubMed  Google Scholar 

  46. Grimme S, Antony J, Ehrlich S, Krieg H (2010) J Chem Phys 132:154104

    Article  PubMed  Google Scholar 

  47. Vahtras O, Almlöf J, Feyereisen MW (1993) Chem Phys Lett 213:514–518

    Article  CAS  Google Scholar 

  48. Kendall RA, Früchtl HA (1997) Theor Chem Acc 97:158–163

    Article  CAS  Google Scholar 

  49. Cossi M, Rega N, Scalmani G, Barone V (2003) J Comput Chem 24:669–681

    Article  CAS  PubMed  Google Scholar 

  50. Staroverov VN, Scuseria GE, Tao J, Perdew JP (2003) J Chem Phys 119:12129–12137

    Article  CAS  Google Scholar 

  51. Römelt M, Ye S, Neese F (2009) Inorg Chem 48:784–785

    Article  PubMed  Google Scholar 

  52. Rega N, Cossi M, Barone V (1996) J Chem Phys 105:11060–11067

    Article  CAS  Google Scholar 

  53. Timoshin AA, Vanin AF, Orlova TR, Sanina NA, Ruuge EK, Aldoshin SM, Chazov EI (2007) Nitric Oxide 16:286–293

    Article  CAS  PubMed  Google Scholar 

  54. Vanin AF (2018) Cell Biochem Biophys 76:3–17

    Article  CAS  PubMed  Google Scholar 

  55. Vanin AF, Serezhenkov VA, Mikoyan VD, Genkin MV (1998) Nitric Oxide 2:224–234

    Article  CAS  PubMed  Google Scholar 

  56. Ye S, Neese F (2010) J Am Chem Soc 132:3646–3647

    Article  CAS  PubMed  Google Scholar 

  57. Sellmann D, Blum N, Heinemann FW, Hess BA (2001) Chem Eur J 7:1874–1880

    Article  CAS  PubMed  Google Scholar 

  58. Enemark JH, Feltham RD (1974) Coord Chem Rev 13:339–406

    Article  CAS  Google Scholar 

  59. Lewandowska H, Kalinowska M, Brzóska K, Wójciuk K, Wójciuk G, Kruszewski M (2011) Dalton Trans 40:8273–8289

    Article  CAS  PubMed  Google Scholar 

  60. Truzzi DR, Augusto O, Iretskii AV, Ford PC (2019) Inorg Chem 58:13446–13456

    Article  CAS  PubMed  Google Scholar 

  61. Reginato N, McCrory CTC, Pervitsky D, Li L (1999) J Am Chem Soc 121:10217–10218

    Article  CAS  Google Scholar 

  62. Tsai F-T, Kuo T-S, Liaw W-F (2009) J Am Chem Soc 131:3426–3427

    Article  CAS  PubMed  Google Scholar 

  63. Blaesi EJ, Gardner JD, Fox BG, Brunold TC (2013) Biochemistry 52:6040–6051

    Article  CAS  PubMed  Google Scholar 

  64. Li M, Bonnet D, Bill E, Neese F, Weyhermèuller T, Blum N, Sellmann D, Wieghardt K (2002) Inorg Chem 41:3444–3456

    Article  CAS  PubMed  Google Scholar 

  65. Orville AM, Lipscomb JD (1997) Biochemistry 36:14044–14055

    Article  CAS  PubMed  Google Scholar 

  66. Stevens TH, Chan SI (1981) J Biol Chem 256:1069–1071

    Article  CAS  PubMed  Google Scholar 

  67. Yonetani T, Yamamoto H, Erman JE, Leigh JS, Reed GH (1972) J Biol Chem 247:2447–2455

    Article  CAS  PubMed  Google Scholar 

  68. Fischer AA, Miller JR, Jodts RJ, Ekanayake DM, Lindeman SV, Brunold TC, Fiedler AT (2019) Inorg Chem 58:16487–16499

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Abragam A, Bleaney B (1970) Electron paramagnetic resonance of transition ions (International series of monographs on physics)

  70. Dikanov SA, Davydov RM, Gräslund A, Bowman MK (1998) J Am Chem Soc 120:6797–6805

    Article  CAS  Google Scholar 

  71. Forlano PM, Olabe JA, Magallanes JF, Blesa MA (1997) Can J Chem 75:9–13

    Article  CAS  Google Scholar 

  72. Traore ES, Liu A (2022) ACS Catal 12:6191–6208

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institutes of Health (2 R15 GM117511-03) to B.S.P.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Alabama, 250 Hackberry Lane, Tuscaloosa, AL, 35487, USA

    Nicholas J. York, Allison N. Schmittou & Brad S. Pierce

  2. Department of Chemistry and Biochemistry, Samford University, 800 Lakeshore Drive, Homewood, AL, 35229, USA

    Molly M. Lockart

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York, N.J., Lockart, M.M., Schmittou, A.N. et al. Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site. J Biol Inorg Chem 28, 285–299 (2023). https://doi.org/10.1007/s00775-023-01990-7

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