Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2185272 | Journal of Molecular Biology | 2011 | 17 Pages |
The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe–4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C4mim]Cl−. In addition, UV–Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.
Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (130 K)Download as PowerPoint slideResearch Highlights► The periplasmic nitrate reductase (NapAB) from C. necator is a heterodimeric Mo protein. ► We report the crystal structures of the oxidized form (1.5 Å) and the partially reduced form (1.7 Å). ► In both forms, a sulfur atom (not an oxygen atom) is the sixth molybdenum ligand. ► This has important mechanistic implications for this family of proteins. ► The reduced form was obtained with ionic liquids, and the ligand Cys was displaced.