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

• The small subunit, HupS, from a cyanobacterial uptake hydrogenase was expressed and isolated.
• HupS was subject to flash photolysis using Ru(bpy)3 as sensitizer and ascorbate as electron donor.
• Photoinduced electron injection in the medial FeS cluster of HupS was observed by EPR spectroscopy.

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.

The small subunit, HupS, from the cyanobacterial uptake hydrogenase, has been isolated in the absence of the large subunit. Using Ru(bpy)32 + as photosensitizer and sodium ascorbate as electron donor, electrons were injected into the isolated HupS protein by flash photolysis. Reduction of FeS clusters were observed by EPR spectroscopy.Figure optionsDownload as PowerPoint slide