An NMR structural study of nickel-substituted rubredoxin

  • Brian J. Goodfellow
  • Iven C. N. Duarte
  • Anjos L. Macedo
  • Brian F. Volkman
  • Sofia G. Nunes
  • I. Moura
  • John L. Markley
  • José J. G. Moura
Original Paper


The Ni(II) and Zn(II) derivatives of Desulfovibrio vulgaris rubredoxin (DvRd) have been studied by NMR spectroscopy to probe the structure at the metal centre. The βCH2 proton pairs from the cysteines that bind the Ni(II) atom have been identified using 1D nuclear Overhauser enhancement (NOE) difference spectra and sequence specifically assigned via NOE correlations to neighbouring protons and by comparison with the published X-ray crystal structure of a Ni(II) derivative of Clostridium pasteurianum rubredoxin. The solution structures of DvRd(Zn) and DvRd(Ni) have been determined and the paramagnetic form refined using pseudocontact shifts. The determination of the magnetic susceptibility anisotropy tensor allowed the contact and pseudocontact contributions to the observed chemical shifts to be obtained. Analysis of the pseudocontact and contact chemical shifts of the cysteine Hβ protons and backbone protons close to the metal centre allowed conclusions to be drawn as to the geometry and hydrogen-bonding pattern at the metal binding site. The importance of NH–S hydrogen bonds at the metal centre for the delocalization of electron spin density is confirmed for rubredoxins and can be extrapolated to metal centres in Cu proteins: amicyanin, plastocyanin, stellacyanin, azurin and pseudoazurin.


NMR Rubredoxin [Fe–4S] centre Paramagnetic protein Nickel 



Clostridium pasteurianum rubredoxin


Desulfovibrio vulgaris rubredoxin


Heteronuclear single quantum coherence


Magnetic susceptibility anisotropy tensor


Nuclear Overhauser enhancement


Nuclear Overhauser enhancement spectroscopy


Pseudocontact shift


Protein Data Bank


Pyrococcus furiosus rubredoxin




Root-mean-square deviation


Total correlation spectroscopy

Supplementary material

775_2009_613_MOESM1_ESM.doc (1.1 mb)
Supplementary material (DOC 1133 kb)


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Copyright information

© SBIC 2009

Authors and Affiliations

  • Brian J. Goodfellow
    • 1
  • Iven C. N. Duarte
    • 1
  • Anjos L. Macedo
    • 2
  • Brian F. Volkman
    • 3
  • Sofia G. Nunes
    • 4
  • I. Moura
    • 2
  • John L. Markley
    • 5
  • José J. G. Moura
    • 2
  1. 1.CICECO, Departamento de QuímicaUniversidade AveiroAveiroPortugal
  2. 2.REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e TecnologiaUniversidade Nova de LisboaCaparicaPortugal
  3. 3.Department of BiochemistryMedical College of WisconsinMilwaukeeUSA
  4. 4.Valencia Infertility Institute (IVI)ValenciaSpain
  5. 5.Department of Biochemistry, 171A Biochemistry AdditionUniversity of WisconsinMadisonUSA

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