Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase |
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Authors: | Anders Thapper D R Boer Carlos D Brondino José J G Moura Maria J Romão |
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Institution: | (1) REQUIMTE-CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal;(2) Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina;(3) Present address: Molecular Biomimetics, Department of Photochemistry and Molecular Science, Uppsala University, Box 523, 751 20 Uppsala, Sweden;(4) Present address: Institute of Molecular Biology, C/Josep Samitier 1–5, 08028 Barcelona, Spain |
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Abstract: | 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.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. |
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Keywords: | Molybdenum-containing enzymes Aldehyde oxidoreductase Xanthine oxidase family Electron paramagnetic resonance X-ray |
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