Document Detail


Purification and characterization of the [NiFe]-hydrogenase of Shewanella oneidensis MR-1.
MedLine Citation:
PMID:  21724888     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Liang Shi; Sara M Belchik; Andrew E Plymale; Steve Heald; Alice C Dohnalkova; Kateryna Sybirna; Hervé Bottin; Thomas C Squier; John M Zachara; James K Fredrickson
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2011-07-01
Journal Detail:
Title:  Applied and environmental microbiology     Volume:  77     ISSN:  1098-5336     ISO Abbreviation:  Appl. Environ. Microbiol.     Publication Date:  2011 Aug 
Date Detail:
Created Date:  2011-08-05     Completed Date:  2011-12-15     Revised Date:  2013-06-28    
Medline Journal Info:
Nlm Unique ID:  7605801     Medline TA:  Appl Environ Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5584-90     Citation Subset:  IM    
Affiliation:
Microbiology Group, Pacific Northwest National Laboratory, Richland, WA 99352, USA. liang.shi@pnnl.gov
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MeSH Terms
Descriptor/Qualifier:
Blotting, Western
Buffers
Cloning, Molecular
Genes, Bacterial*
Genetic Complementation Test
Hydrogen / metabolism*
Hydrogenase / genetics,  isolation & purification*,  metabolism
Microscopy, Electron, Transmission
Organotechnetium Compounds / metabolism
Oxidation-Reduction
Paraquat / metabolism
Shewanella / enzymology*,  genetics
Spectrometry, X-Ray Emission / methods
Technetium / metabolism*
Chemical
Reg. No./Substance:
0/Buffers; 0/Organotechnetium Compounds; 1333-74-0/Hydrogen; 4685-14-7/Paraquat; 7440-26-8/Technetium; EC 1.12.-/nickel-iron hydrogenase; EC 1.12.7.2/Hydrogenase
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