Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

  • Anders Thapper
  • D. R. Boer
  • Carlos D. BrondinoEmail author
  • José J. G. Moura
  • Maria J. RomãoEmail author
Original Paper


Two arsenite-inhibited forms of each of the aldehyde oxidoreductases from Desulfovibrio gigas and Desulfovibrio desulfuricans have been studied by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy. The molybdenum site of these enzymes shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. Arsenite addition to active as-prepared enzyme or to a reduced desulfo form yields two different species called A and B, respectively, which show different Mo(V) EPR signals. Both EPR signals show strong hyperfine and quadrupolar couplings with an arsenic nucleus, which suggests that arsenic interacts with molybdenum through an equatorial ligand. X-ray data of single crystals prepared from EPR-active samples show in both inhibited forms that the arsenic atom interacts with the molybdenum ion through an oxygen atom at the catalytic labile site and that the sulfido ligand is no longer present. EPR and X-ray data indicate that the main difference between both species is an equatorial ligand to molybdenum which was determined to be an oxo ligand in species A and a hydroxyl/water ligand in species B. The conclusion that the sulfido ligand is not essential to determine the EPR properties in both Mo–As complexes is achieved through EPR measurements on a substantial number of randomly oriented chemically reduced crystals immediately followed by X-ray studies on one of those crystals. EPR saturation studies show that the electron transfer pathway, which is essential for catalysis, is not modified upon inhibition.


Molybdenum-containing enzymes Aldehyde oxidoreductase Xanthine oxidase family Electron paramagnetic resonance X-ray 



Aldehyde oxidoreductase


Aldehyde oxidoreductase from Desulfovibrio desulfuricans ATCC 27774


Aldehyde oxidoreductase from Desulfovibrio gigas


Electronic paramagnetic resonance


Extended X-ray absorption fine structure


N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid


Protein Data Bank


Poly(ethylene glycol) 4000


Quinoline 2-oxidoreductase




Xanthine oxidase



A.T. and D.R.B. thank the Fundação para a Ciência e Tecnologia, Portugal, for funding (grants SFRP/BPD/5689/2001 and SFRH/BPD/20358/2004/E031, respectively, supported by the European Social Funding within the III Communitarian Support Board). D.R.B. and M.J.R. thank the EU (project number HRRN-CT-1999-00084) and the beamline scientists of the ID14-4 and BM14 beamlines of the ESRF in Grenoble for their assistance. C.D.B. and J.J.G.M. thank SECYT (Argentina) and GRICES (Portugal) for a bilateral collaborative grant. This work was supported by projects EC HPRN-CT-1999-00084, POCTI/1999/BME/35078, POCTI/1999/BME/36152 and POCTI/QUI/57641/2004 in Portugal, and by SEPCyT:PICT 2003-06-13872, CONICET PIP 5370/2005 and CAI + D-UNL in Argentina. C.D.B is a member of CONICET (Argentina).

Supplementary material


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Copyright information

© SBIC 2006

Authors and Affiliations

  1. 1.REQUIMTE-CQFB, Departamento de Química, Faculdade de Ciências e TecnologiaUniversidade Nova de LisboaCaparicaPortugal
  2. 2.Departamento de Física, Facultad de Bioquímica y Ciencias BiológicasUniversidad Nacional del LitoralSanta FeArgentina
  3. 3.Molecular Biomimetics, Department of Photochemistry and Molecular ScienceUppsala UniversityUppsalaSweden
  4. 4.Institute of Molecular BiologyBarcelonaSpain

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