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 = ½) [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 = ½) [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.

Keywords: Cyanobacteria; Electron Paramagnetic Resonance (EPR); Hydrogenase; Iron-Sulfur Protein; Redox.

FIGURE 1.

A, schematic representation of the spatial distribution of metal centers in a NiFe hydrogenase. The large subunit contains the NiFe active site where H₂ is either formed or oxidized. The small subunit contains typically three FeS clusters: a proximal [4Fe-4S] (p) nearer the active site on the large subunit, a medial [3Fe-4S] (m), and a distal [4Fe-4S] (d), which participate in electron transfer. B, comparison between FeS cluster binding motifs of HupS from N. punctiforme ATCC 29133 and: Cyanothece sp. ATCC 51142 HupS; Acidithiobacillus (Ac.) ferrooxidans ATCC 23270 HoxK; D. gigas HydA; Allochromatium (Al.) vinosum DSM 180 HydA. Letters in boldface type indicate amino acids involved in coordination of the FeS clusters, marked as a proximal (p), medial (m), or distal (d) cluster, respectively. The proximal cluster in N. punctiforme HupS, and in other cyanobacterial-like small subunits, contains an asparagine instead of one of the cysteines, and the distal cluster presents a glutamine replacing histidine.

FIGURE 2.

Overview of cloning process of hupS in the pET43.1 plasmid. N. punctiforme hupS was amplified from N. punctiforme. A Strep(II)-tag was subsequently added, and after a cloning step in pSUN119, hupS was cloned downstream of the Nus·Tag coding region as a fusion construct. The construct is under the control of a T7lac promoter, and uses a T7 transcription terminator. The fusion construct, including the Strep(II)-tag and stop codon, is 2667 bp long, giving rise to a 888-aa-long polypeptide, f-HupS. Gray arrows show primer annealing location.

FIGURE 3.

Analysis of the purified f-HupS. A, lanes a–e: Western blot of purification fractions, using chemiluminiscent detection of the Strep(II)-tag. Lane a, protein markers (size shown on left side in kDa). Lane b, crude cell extract, after cell disruption (30 μg total protein). Lane c, supernatant after centrifugation (30 μg of total protein). Lane d, aerobically purified f-HupS (3 μg). Lane e, anaerobically purified f-HupS (2 μg). Lanes f–h, SDS-PAGE of crude extract and anaerobically purified f-HupS. Lane f, protein markers. Lane g, crude cell extract (30 μg total protein). Lane h, anaerobically purified f-HupS (5 μg). Asterisk denotes polypeptide corresponding to full-length f-HupS. Vertical lines denote non-contiguous lanes. Aerobically purified f-HupS presented multiple bands due to protein degradation (lane d), which were absent from anaerobically purified f-HupS (lanes e and h). B, UV-visible absorption spectrum of the anaerobically purified f-HupS (1.9 mg protein/ml). The spectrum was taken in a sealed cuvette, previously purged with nitrogen gas. The arrows mark absorption bands with maxima at ∼340 and 420 nm, which are typically seen in FeS proteins. a.u.: arbitrary units.

FIGURE 4.

X-band EPR spectra of f-HupS. A, top spectrum, f-HupS as purified; bottom spectrum, after oxidation of the sample with ferricyanide. EPR conditions were as follows: 8 microwatts of applied microwave power; temperature = 7 K. B, top spectrum, f-HupS as purified; second spectrum, after reduction with dithionite. EPR conditions: 2 milliwatts of applied microwave power; temperature = 7 K. Third and fourth spectrum (Sim 1, Sim 2), simulated spectra of the two components of the reduced sample spectrum (see main text for details). Bottom spectrum (Sim1+sim2), mathematical addition of sim1 and sim2 simulations, weighed 50% each. C, dithionite-reduced f-HupS measured at different temperatures, at the same microwave power (2 milliwatts). Top spectrum, 15 K. Bottom spectrum, 7 K. The arrows point at spectral features belonging to the two spectral components that change differently with temperature. D, variation in EPR signal amplitudes with different applied microwave power (P) in dithionite-reduced f-HupS, measured at temperature = 15 K. The amplitudes were measured from the resonances indicated with arrows in C. Gray circles, the g = 1.91 resonance; Black squares, the g = 1.88 resonance. The modulation amplitude in all measurements was 10 G. Protein concentration was 9 mg/ml.