title: Purification and characterization of the [NiFe]-hydrogenase of Shewanella oneidensis MR-1.
abstract: Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H(2)ase) that has been implicated in H(2) production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H(2)ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H(2)ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H(2)ase in trans restored the mutant's ability to produce H(2) at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H(2)ase coupled H(2) oxidation to reduction of Tc(VII)O(4)(-) and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H(2)ase-mediated reduction of Tc(VII)O(4)(-) but not methyl viologen. Under the conditions tested, all Tc(VII)O(4)(-) used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O(4)(-) was reduced. The reduced products were soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc precipitates reduced in HEPES buffer were aggregates of crystallites with diameters of ∼5 nm. Measurements with X-ray absorption near-edge spectroscopy revealed that the reduction products were a mixture of Tc(IV) and Tc(V) in Tris buffer but only Tc(IV) in HEPES buffer. Measurements with extended X-ray adsorption fine structure showed that while the Tc bonding environment in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O(2)·nH(2)O, which was also the product of Tc(VII)O(4)(-) reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H(2)ase catalyzes Tc(VII)O(4)(-) reduction directly by coupling to H(2) oxidation.
authors: Microbiology Group, Pacific Northwest National Laboratory, Richland, WA 99352, USA. liang.shi@pnnl.gov
authors: Liang
authors: L
authors: Shi
authors: Sara M
authors: SM
authors: Belchik
authors: Andrew E
authors: AE
authors: Plymale
authors: Steve
authors: S
authors: Heald
authors: Alice C
authors: AC
authors: Dohnalkova
authors: Kateryna
authors: K
authors: Sybirna
authors: Hervé
authors: H
authors: Bottin
authors: Thomas C
authors: TC
authors: Squier
authors: John M
authors: JM
authors: Zachara
authors: James K
authors: JK
authors: Fredrickson
mesh_terms: D015153:Blotting, Western; D002021:Buffers; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D015609:Organotechnetium Compounds / Q000378:metabolism; D010084:Oxidation-Reduction; D010269:Paraquat / Q000378:metabolism; D020592:Shewanella / Q000201:enzymology* / Q000235:genetics; D013052:Spectrometry, X-Ray Emission / Q000379:methods; D013667:Technetium / Q000378:metabolism*
chemical_list: D002021:Buffers; D015609:Organotechnetium Compounds; D013667:Technetium; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010269:Paraquat