| Summary: | Nitrogenase, a bacteria-based enzyme, is the sole enzyme that is able to generate ammonia by atmospheric nitrogen fixation. Thus, improved understanding of its utilization and developing methods to artificially activate it may contribute to basic research, as well as to the design of future artificial systems. Here, we present methods to artificially activate nitrogenase using photoinduced reactions. Two nitrogenase variants originating from <i>Azotobacter vinelandii</i> were examined using photoactivated CdS nanoparticles (NPs) capped with thioglycolic acid (TGA) or 2-mercaptoethanol (ME) ligands. The effect of methyl viologen (MV) as a redox mediator of hydrogen and ammonia generation was tested and analyzed. We further determined the NPs conductive band edges and their effect on the nitrogenase photoactivation. The nano-biohybrid systems comprising CdS NPs and nitrogenase were further imaged by transmission electron microscopy, visualizing their formation for the first time. Our results show that the ME-capped CdS NPs–nitrogenase enzyme biohybrid system with added MV as a redox mediator leads to a five-fold increase in the production of ammonia compared with the non-mediated biohybrid system; nevertheless, it stills lag behind the natural process rate. On the contrary, a maximal hydrogen generation amount was achieved by the αL158C MoFe-P and the ME-capped CdS NPs.
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