JBIC Journal of Biological Inorganic Chemistry

, Volume 21, Issue 8, pp 1021–1035 | Cite as

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity

  • Judy P. Hines
  • Aaron T. Smith
  • Jose P. Jacob
  • Gudrun S. Lukat-Rodgers
  • Ian Barr
  • Kenton R. Rodgers
  • Feng Guo
  • Judith N. Burstyn
Original Paper


The RNA-binding heme protein DiGeorge critical region 8 (DGCR8) and its ribonuclease partner Drosha cleave primary transcripts of microRNA (pri-miRNA) as part of the canonical microRNA (miRNA) processing pathway. Previous studies show that bis-cysteine thiolate-coordinated Fe(III) DGCR8 supports pri-miRNA processing activity, while Fe(II) DGCR8 does not. In this study, we further characterized Fe(II) DGCR8 and tested whether CO or NO might bind and restore pri-miRNA processing activity to the reduced protein. Fe(II) DGCR8 RNA-binding heme domain (Rhed) undergoes a pH-dependent transition from 6-coordinate to 5-coordinate, due to protonation and loss of a lysine ligand; the ligand bound throughout the pH change is a histidine. Fe(II) Rhed binds CO and NO from 6- and 5-coordinate states, forming common CO and NO adducts at all pHs. Fe(II)–CO Rhed is 6-coordinate, low-spin, and pH insensitive with the histidine ligand retained, suggesting that the protonatable lysine ligand has been replaced by CO. Fe(II)–NO Rhed is 5-coordinate and pH insensitive. Fe(II)–NO also forms slowly upon reaction of Fe(III) Rhed with excess NO via a stepwise process. Heme reduction by NO is rate-limiting, and the rate would be negligible at physiological NO concentrations. Importantly, in vitro pri-miRNA processing assays show that both CO- and NO-bound DGCR8 species are inactive. Fe(II), Fe(II)–CO, and Fe(II)–NO Rhed do not bear either of the cysteine ligands found in the Fe(III) state. These data support a model in which the bis-cysteine thiolate ligand environment of Fe(III) DGCR8 is necessary for establishing proper pri-miRNA binding and enabling processing activity.


Heme microRNA RNA processing Carbon monoxide Nitric oxide 



5-Coordinate high spin


6-Coordinate low spin


N-cyclohexyl-3-aminopropanesulfonic acid


N-cyclohexyl-2-aminoethanesulfonic acid


C-terminal tail


Dechloromonas aromatica chlorite dismutase




DiGeorge critical region 8


Dimerization subdomains


Double-stranded RNA-binding domain 1


Double-stranded RNA-binding domain 2




4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid


4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid


Heme-binding domain


Magnetic circular dichroism


2-(N-morpholino)ethanesulfonic acid




3-(N-morpholino)propanesulfonic acid


Nuclear localization signal


Nostdoc sp.


Precursor microRNA


Primary microRNA


Protoporphyrin IX


RNA-binding heme domain


Root-mean-square deviation


Resonance Raman


Size exclusion chromatography





This work was supported by the National Institutes of Health Grants GM094039 (to G.S.L.-R.), AI072719 (to K.R.R.) and GM080563 (to F.G.), and by the National Science Foundation grant CHE-1213739 (to J.N.B.). We would like to thank Prof. Thomas Brunold at the University of Wisconsin-Madison for sharing his MCD and Raman spectrophotometers, and Prof. Joan Valentine at UCLA for allowing us to use her anaerobic chamber.

Supplementary material

775_2016_1398_MOESM1_ESM.pdf (1.7 mb)
Supplementary material 1 (PDF 1716 kb)


  1. 1.
    Krol J, Loedige I, Filipowicz W (2010) Nat Rev Genet 11:597–610PubMedGoogle Scholar
  2. 2.
    Rana TM (2007) Nat Rev Mol Cell Biol 8:23–36CrossRefPubMedGoogle Scholar
  3. 3.
    Ha M, Kim VN (2014) Nat Rev Mol Cell Biol 15:509–524CrossRefPubMedGoogle Scholar
  4. 4.
    Kim VN, Han J, Siomi MC (2009) Nat Rev Mol Cell Biol 10:126–139CrossRefPubMedGoogle Scholar
  5. 5.
    Denli AM, Tops BBJ, Plasterk RHA, Ketting RF, Hannon GJ (2004) Nature 432:231–235CrossRefPubMedGoogle Scholar
  6. 6.
    Gregory RI, Yan K-P, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, Shiekhattar R (2004) Nature 432:235–240CrossRefPubMedGoogle Scholar
  7. 7.
    Faller M, Matsunaga M, Yin S, Loo JA, Guo F (2007) Nat Struct Mol Biol 14:23–29CrossRefPubMedGoogle Scholar
  8. 8.
    Faller M, Toso D, Matsunaga M, Atanasov I, Senturia R, Chen Y, Zhou ZH, Guo F (2010) RNA 8:1570–1583CrossRefGoogle Scholar
  9. 9.
    Han J, Lee Y, Yeom K-H, Nam J-W, Heo I, Rhee J-K, Sohn SY, Cho Y, Zhang B-T, Kim VN (2006) Cell 125:887–901CrossRefPubMedGoogle Scholar
  10. 10.
    Sohn SY, Bae WJ, Kim JJ, Yeom K-H, Kim VN, Cho Y (2007) Nat Struct Mol Biol 14:847–853CrossRefPubMedGoogle Scholar
  11. 11.
    Quick-Cleveland J, Jacob JP, Weitz SH, Shoffner G, Senturia R, Guo F (2014) Cell Rep 7:1994–2005CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Herbert KM, Sarkar SK, Mills M, Delgado De la Herran HC, Neuman KC, Steitz JA (2015) RNA 22:1–9Google Scholar
  13. 13.
    Kwon SC, Nguyen TA, Choi Y-G, Jo MH, Hohng S, Kim VN, Woo J-S (2016) Cell 164:1–10CrossRefGoogle Scholar
  14. 14.
    Nguyen TA, Jo MH, Choi Y-G, Park J, Kwon SC, Hohng S, Kim VN, Woo J-S (2015) Cell 161:1374–1387CrossRefPubMedGoogle Scholar
  15. 15.
    Weitz SH, Gong M, Barr I, Weiss S, Guo F (2014) Proc Natl Acad Sci USA 111:1861–1866CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Barr I, Smith AT, Senturia R, Chen Y, Scheidemantle BD, Burstyn JN, Guo F (2011) J Biol Chem 286:16716–16725CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Senturia R, Faller M, Yin S, Loo JA, Cascio D, Sawaya MR, Hwang D, Clubb RT, Guo F (2010) Protein Sci 19:1354–1365CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Barr I, Smith AT, Chen Y, Senturia R, Burstyn JN, Guo F (2012) Proc Natl Acad Sci USA 109:1919–1924CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Dioum EM, Rutter J, Tuckerman JR, Gonzalez G, Gilles-Gonzalez M-A, McKnight SL (2002) Science 298:2385–2387CrossRefPubMedGoogle Scholar
  20. 20.
    Shelver D, Thorsteinsson MV, Kerby RL, Chung S-Y, Roberts GP, Reynolds MF, Parks RB, Burstyn JN (1999) Biochemistry 38:2669–2678CrossRefPubMedGoogle Scholar
  21. 21.
    Vogel KM, Spiro TG (1999) Biochemistry 38:2679–2687CrossRefPubMedGoogle Scholar
  22. 22.
    Marvin KA, Kerby RL, Youn H, Roberts GP, Burstyn JN (2008) Biochemistry 47:9016–9028CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Gilles-Gonzalez M-A, Gonzalez G (2004) J Appl Physiol 96:774–783CrossRefPubMedGoogle Scholar
  24. 24.
    Boon EM, Marletta MA (2005) J Inorg Biochem 99:892–902CrossRefPubMedGoogle Scholar
  25. 25.
    Plate L, Marletta MA (2013) Trends Biochem Sci 38:566–575CrossRefPubMedGoogle Scholar
  26. 26.
    Giardina G, Castiglione N, Caruso M, Cutruzzolà F, Rinaldo S (2011) Biochem Soc Trans 39:294–298CrossRefPubMedGoogle Scholar
  27. 27.
    Herzik MA, Jonnalagadda R, Kuriyan J, Marletta MA (2014) Proc Natl Acad Sci USA 111:E4156–E4164CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Tsai A-L, Berka V, Martin F, Ma X, van den Akker F, Fabian M, Olson JS (2010) Biochemistry 49:6587–6599CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Boon EM, Davis JH, Tran R, Karow DS, Huang SH, Pan D, Miazgowicz MM, Mathies RA, Marletta MA (2006) J Biol Chem 281:21892–21902CrossRefPubMedGoogle Scholar
  30. 30.
    Boon EM, Marletta MA (2006) J Am Chem Soc 128:10022–10023CrossRefPubMedGoogle Scholar
  31. 31.
    Ding XD, Weichsel A, Andersen JF, Shokhireva TK, Balfour C, Pierik AJ, Averill BA, Montfort WR, Walker FA (1999) J Am Chem Soc 121:128–138CrossRefGoogle Scholar
  32. 32.
    Walker FA (2005) J Inorg Biochem 99:216–236CrossRefPubMedGoogle Scholar
  33. 33.
    Wasser IM, de Vries S, Moënne-Loccoz P, Schröder I, Karlin KD (2002) Chem Rev 102:1201–1234CrossRefPubMedGoogle Scholar
  34. 34.
    Averill BA (1996) Chem Rev 96:2951–2964CrossRefPubMedGoogle Scholar
  35. 35.
    Daiber A, Shoun H, Ullrich V (2005) J Inorg Biochem 99:185–193CrossRefPubMedGoogle Scholar
  36. 36.
    Shiro Y, Fujii M, Iizuka T, Adachi S-I, Tsukamoto K, Nakahara K, Shoun H (1995) J Biol Chem 270:1617–1623CrossRefPubMedGoogle Scholar
  37. 37.
    Obayashi E, Tsukamoto K, Adachi S-I, Takahashi S, Nomura M, Iizuka T, Shoun H, Shiro Y (1997) J Am Chem Soc 119:7807–7816CrossRefGoogle Scholar
  38. 38.
    Praneeth VKK, Paulat F, Berto TC, George SD, Näther C, Sulok CD, Lehnert N (2008) J Am Chem Soc 130:15288–15303CrossRefPubMedGoogle Scholar
  39. 39.
    Barr I, Guo F (2014) In: Arenz C (ed) miRNA maturation. Humana Press, New York, pp 73–86CrossRefGoogle Scholar
  40. 40.
    Spiro TG, Strekas TC (1974) J Am Chem Soc 96:338–345CrossRefPubMedGoogle Scholar
  41. 41.
    Kitagawa T, Kyogoku Y, Iizuka T, Ikeda-Saito M, Yamanaka T (1975) J Biochem 78:719–728PubMedGoogle Scholar
  42. 42.
    Hu S, Smith KM, Spiro TG (1996) J Am Chem Soc 118:12638–12646CrossRefGoogle Scholar
  43. 43.
    Tomita T, Gonzalez G, Chang AL, Ikeda-Saito M, Gilles-Gonzalez M-A (2002) Biochemistry 41:4819–4826CrossRefPubMedGoogle Scholar
  44. 44.
    Spiro TG (1975) Biochim Biophys Acta 416:169–189CrossRefPubMedGoogle Scholar
  45. 45.
    Dawson JH, Andersson LA, Sono M (1983) J Biol Chem 258:13637–13645PubMedGoogle Scholar
  46. 46.
    Dawson RMC, Elliot DC, Elliot WH, Jones KM (1986) Data for biochemical research. Oxford Science Publications, OxfordGoogle Scholar
  47. 47.
    Khangulov SV, Sossong TM Jr, Ash DE, Dismukes GC (1998) Biochemistry 37:8539–8550CrossRefPubMedGoogle Scholar
  48. 48.
    Ferraroni M, Tilli S, Briganti F, Chegwidden WR, Supuran CT, Wiebauer KE, Tashian RE, Scozzafava A (2002) Biochemistry 41:6237–6244CrossRefPubMedGoogle Scholar
  49. 49.
    Blanc B, Mayfield JA, McDonald CA, Lukat-Rodgers GS, Rodgers KR, DuBois JL (2012) Biochemistry 51:1895–1910CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Streit BR, Blanc B, Lukat-Rodgers GS, Rodgers KR, DuBois JL (2010) J Am Chem Soc 132:5711–5724CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Dhawan IK, Shelver D, Thorsteinsson MV, Roberts GP, Johnson MK (1999) Biochemistry 38:12805–12813CrossRefPubMedGoogle Scholar
  52. 52.
    Marvin KA, Reinking JL, Lee AJ, Pardee K, Krause HM, Burstyn JN (2009) Biochemistry 48:7056–7071CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Senturia R, Laganowsky A, Barr I, Scheidemantle BD, Guo F (2012) PLoS One:7Google Scholar
  54. 54.
    Stein P, Mitchell M, Spiro TG (1980) J Am Chem Soc 102:7795–7797CrossRefGoogle Scholar
  55. 55.
    Bangcharoenpaurpong O, Schomacker KT, Champion PM (1984) J Am Chem Soc 106:5688–5698CrossRefGoogle Scholar
  56. 56.
    Desbois A, Lutz M (1981) Biochim Biophys Acta Protein Struct 671:168–176CrossRefGoogle Scholar
  57. 57.
    Makinen MW, Churg AK (1983) In: Lever ABP, Gray HB (eds) Iron porphyrins, part i. Addison-Wesley Publishing Company, Reading, pp 141–236Google Scholar
  58. 58.
    Jung C, Ristau O (1977) Chem Phys Lett 49:103–108CrossRefGoogle Scholar
  59. 59.
    Spiro TG, Wasbotten IH (2005) J Inorg Biochem 99:34–44CrossRefPubMedGoogle Scholar
  60. 60.
    Enemark JH, Feltham RD (1974) Coord Chem Rev 13:339–406CrossRefGoogle Scholar
  61. 61.
    Linder DP, Rodgers KR, Banister J, Wyllie GRA, Ellison MK, Scheidt WR (2004) J Am Chem Soc 126:14136–14148CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Stone JR, Marletta MA (1994) Biochemistry 33:5636–5640CrossRefPubMedGoogle Scholar
  63. 63.
    Taoka S, Banerjee R (2001) J Inorg Biochem 87:245–251CrossRefPubMedGoogle Scholar
  64. 64.
    Decatur SM, Franzen S, DePillis GD, Dyer RB, Woodruff WH, Boxer SG (1996) Biochemistry 35:4939–4944CrossRefPubMedGoogle Scholar
  65. 65.
    Palmer G (1983) In: Lever ABP, Gray HB (eds) Iron porphyrins, part ii. Addison-Wesley Publishing Compnay, Reading, pp 43–88Google Scholar
  66. 66.
    Neese F (1997) Electronic structure and spectroscopy of novel copper chromophores in biology. Atelier für Gestaltung und Verlag, KonstanzGoogle Scholar
  67. 67.
    Reynolds MF, Parks RB, Burstyn JN (2000) Biochemistry 39:388–396CrossRefPubMedGoogle Scholar
  68. 68.
    Stone JR, Sands RH, Dunham WR, Marletta MA (1995) Biochem Biophys Res Commun 207:572–577CrossRefPubMedGoogle Scholar
  69. 69.
    Hille R, Olson JS, Palmer G (1979) J Biol Chem 254:12110–12120PubMedGoogle Scholar
  70. 70.
    Yoshimura T, Suzuki S, Nakahara A, Iwasaki H, Masuko M, Matsubara T (1986) Biochemistry 25:2436–2442CrossRefGoogle Scholar
  71. 71.
    Rodgers KR, Lukat-Rodgers GS, Tang L (2000) J Biol Inorg Chem 5:642–654CrossRefPubMedGoogle Scholar
  72. 72.
    Du J, Perera R, Dawson JH (2011) Inorg Chem 50:1242–1249CrossRefPubMedPubMedCentralGoogle Scholar
  73. 73.
    Martinez SE, Huang D, Szczepaniak A, Cramer WA, Smith JL (1994) Structure 2:95–105CrossRefPubMedGoogle Scholar
  74. 74.
    Varhač R, Antalík M, Bánó M (2004) J Biol Inorg Chem 9:12–22CrossRefPubMedGoogle Scholar
  75. 75.
    Youn H, Kerby RL, Thorsteinsson MV, Clark RW, Burstyn JN, Roberts GP (2002) J Biol Chem 277:33616–33623CrossRefPubMedGoogle Scholar
  76. 76.
    Zeng Y, Yi R, Cullen BR (2005) EMBO J 24:138–148CrossRefPubMedGoogle Scholar
  77. 77.
    Yeom K-H, Lee Y, Han J, Suh MR, Kim VN (2006) Nucleic Acids Res 34:4622–4629CrossRefPubMedPubMedCentralGoogle Scholar
  78. 78.
    Ma H, Wu Y, Choi J-G, Wu H (2013) Proc Natl Acad Sci USA 110:20687–20692CrossRefPubMedPubMedCentralGoogle Scholar
  79. 79.
    Barr I, Weitz SH, Atkin T, Hsu P, Karayiorgou M, Gogos JA, Weiss S, Guo F (2015) Chem Biol 22:793–802CrossRefPubMedPubMedCentralGoogle Scholar
  80. 80.
    Smith AT, Majtan T, Freeman KM, Su Y, Kraus JP, Burstyn JN (2011) Inorg Chem 50:4417–4427CrossRefPubMedPubMedCentralGoogle Scholar
  81. 81.
    Adar F (1978) In: Dolphin D (ed) The porphyrins, vol iii. Academic Press, New York, pp 167–210CrossRefGoogle Scholar

Copyright information

© SBIC 2016

Authors and Affiliations

  • Judy P. Hines
    • 1
  • Aaron T. Smith
    • 2
  • Jose P. Jacob
    • 3
  • Gudrun S. Lukat-Rodgers
    • 4
  • Ian Barr
    • 3
  • Kenton R. Rodgers
    • 4
  • Feng Guo
    • 3
  • Judith N. Burstyn
    • 1
  1. 1.Department of ChemistryUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Department of Chemistry and BiochemistryUniversity of Maryland, Baltimore CountyBaltimoreUSA
  3. 3.Department of Biological ChemistryDavid Geffen School of Medicine, Molecular Biology Institute, University of California Los AngelesLos AngelesUSA
  4. 4.Department of Chemistry and Molecular BiologyNorth Dakota State UniversityFargoUSA

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