Patrícia Raleiras

Isolation and Characterization of the Small Subunit of the Uptake Hydrogenase from the Cyanobacterium Nostoc punctiforme

Background: Cyanobacterial uptake hydrogenases perform hydrogen oxidation in nitrogen-fixing cyanobacteria, but their biophysical properties are unknown. Results: The small subunit, HupS, from the Nostoc punctiforme uptake hydrogenase was heterologously expressed and spectroscopically characterized in different redox conditions. Conclusion: Recombinant HupS incorporates three iron-sulfur clusters with unusual iron coordination. Significance: We provide the foundation for engineering of cyanobacterial uptake hydrogenases. 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.

Turning around the electron flow in an uptake hydrogenase. EPR spectroscopy and in vivo activity of a designed mutant in HupSL from Nostoc punctiforme

The filamentous cyanobacterium Nostoc punctiforme ATCC 29133 produces hydrogen via nitrogenase in heterocysts upon onset of nitrogen-fixing conditions. N. punctiforme expresses concomitantly the uptake hydrogenase HupSL, which oxidizes hydrogen in an effort to recover some of the reducing power used up by nitrogenase. Eliminating uptake activity has been employed as a strategy for net hydrogen production in N. punctiforme (Lindberg et al., Int. J. Hydrogen Energy, 2002, 27, 1291–1296). However, nitrogenase activity wanes within a few days. In the present work, we modify the proximal iron-sulfur cluster in the hydrogenase small subunit HupS by introducing the designed mutation C12P in the fusion protein f-HupS for expression in E. coli (Raleiras et al., J. Biol. Chem., 2013, 288, 18345–18352), and in the full HupSL enzyme for expression in N. punctiforme. C12P f-HupS was investigated by EPR spectroscopy and found to form a new paramagnetic species at the proximal cluster site consistent with a [4Fe–4S] to [3Fe–4S] cluster conversion. The new cluster has the features of an unprecedented mixed-coordination [3Fe–4S] metal center. The mutation was found to produce stable protein in vitro, in silico and in vivo. When C12P HupSL was expressed in N. punctiforme, the strain had a consistently higher hydrogen production than the background ΔhupSL mutant. We conclude that the increase in hydrogen production is due to the modification of the proximal iron-sulfur cluster in HupS, leading to a turn of the electron flow in the enzyme.

Comparative electrochemical study of superoxide reductases

Superoxide reductases are involved in relevant biological electron transfer reactions related to protection against oxidative stress caused by reactive oxygen species. The electrochemical features of metalloproteins belonging to the three different classes of enzymes were studied by potentio-dynamic techniques (cyclic and square wave voltammetry): desulfoferrodoxin from Desulfovibrio vulgaris Hildenborough, class I superoxide reductases and neelaredoxin from Desulfovibrio gigas and Treponema pallidum, namely class II and III superoxide reductases, respectively. In addition, a small protein, designated desulforedoxin from D. gigas, which has high homology with the N-terminal domain of class I superoxide reductases, was also investigated. A comparison of the redox potentials and redox behavior of all the proteins is presented, and the results show that SOR center II is thermodynamically more stable than similar centers in different proteins, which may be related to an intramolecular electron transfer function.

Fundamentals and Recent Advances in Hydrogen Production and Nitrogen Fixation in Cyanobacteria

There is an urgent need to develop sustainable solutions to convert solar energy into energy carriers used in the society. In addition to solar cells generating electricity, there are several options to generate solar fuels. Native and engineered cyanobacteria have been as model systems to examine, demonstrate, and develop photobiological hydrogen production. In the present contribution the knowledge and understanding of the native systems in cyanobacteria to generate hydrogen, as well as metabolic modulations and genetic engineering to enhance hydrogen production is presented and summarized. Specifically, the recent insight around ferredoxin/flavodoxin as the likely electron donor to the bidirectional Hox-hydrogenase instead of the generally accepted NAD(P)H is highlighted and discussed. In addition, engineering approaches of [NiFe] hydrogenases for optimal catalytic efficiencies and attempts to express high turnover [FeFe] hydrogenase in cyanobacteria that may facilitate the development of strains to reach target levels of hydrogen production in cyanobacteria are detailed. The fundamental advancements achieved in these fields are summarized in this review.

Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme

The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis.