NDLI: Purification and Characterization of the [NiFe]-Hydrogenase of Shewanella oneidensis MR-1 ▿

Content Provider	PubMed Central
Author	Shi, Liang Belchik, Sara M. Plymale, Andrew E. Heald, Steve Dohnalkova, Alice C. Sybirna, Kateryna Bottin, Hervé Squier, Thomas C. Zachara, John M. Fredrickson, James K.
Copyright Year	2011
Abstract	Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H2ase) that has been implicated in H2 production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H2ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H2ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H2ase in trans restored the mutant's ability to produce H2 at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H2ase coupled H2 oxidation to reduction of Tc(VII)O4 − and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H2ase-mediated reduction of Tc(VII)O4 − but not methyl viologen. Under the conditions tested, all Tc(VII)O4 − used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O4 − 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)O2·nH2O, which was also the product of Tc(VII)O4 − reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H2ase catalyzes Tc(VII)O4 − reduction directly by coupling to H2 oxidation.
Related Links	http://dx.doi.org/10.1128/aem.00260-11
Ending Page	5590
Page Count	7
Starting Page	5584
File Format	PDF
ISSN	00992240
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	16
Volume Number	77
Language	English
Publisher	American Society for Microbiology
Publisher Date	2011-08-15
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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