title: Isolation and characterization of the small subunit of the uptake hydrogenase from the cyanobacterium Nostoc punctiforme.
abstract: In nitrogen-fixing cyanobacteria, hydrogen evolution is associated with hydrogenases and nitrogenase, making these enzymes interesting targets for genetic engineering aimed at increased hydrogen production. Nostoc punctiforme ATCC 29133 is a filamentous cyanobacterium that expresses the uptake hydrogenase HupSL in heterocysts under nitrogen-fixing conditions. Little is known about the structural and biophysical properties of HupSL. The small subunit, HupS, has been postulated to contain three iron-sulfur clusters, but the details regarding their nature have been unclear due to unusual cluster binding motifs in the amino acid sequence. We now report the cloning and heterologous expression of Nostoc punctiforme HupS as a fusion protein, f-HupS. We have characterized the anaerobically purified protein by UV-visible and EPR spectroscopies. Our results show that f-HupS contains three iron-sulfur clusters. UV-visible absorption of f-HupS has bands ∼340 and 420 nm, typical for iron-sulfur clusters. The EPR spectrum of the oxidized f-HupS shows a narrow g = 2.023 resonance, characteristic of a low-spin (S = &frac12;) [3Fe-4S] cluster. The reduced f-HupS presents complex EPR spectra with overlapping resonances centered on g = 1.94, g = 1.91, and g = 1.88, typical of low-spin (S = &frac12;) [4Fe-4S] clusters. Analysis of the spectroscopic data allowed us to distinguish between two species attributable to two distinct [4Fe-4S] clusters, in addition to the [3Fe-4S] cluster. This indicates that f-HupS binds [4Fe-4S] clusters despite the presence of unusual coordinating amino acids. Furthermore, our expression and purification of what seems to be an intact HupS protein allows future studies on the significance of ligand nature on redox properties of the iron-sulfur clusters of HupS.
authors: Department of Chemistry, Ångström Laboratory, Uppsala University, SE-75120 Uppsala, Sweden.
authors: Patrícia
authors: P
authors: Raleiras
authors: Petra
authors: P
authors: Kellers
authors: Peter
authors: P
authors: Lindblad
authors: Stenbjörn
authors: S
authors: Styring
authors: Ann
authors: A
authors: Magnuson
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D046937:Nostoc / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C043296:nickel-iron hydrogenase; D006864:Hydrogenase
keywords: Cyanobacteria; Electron Paramagnetic Resonance (EPR); Hydrogenase; Iron-Sulfur Protein; Redox
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