Artificial, Photoinduced Activation of Nitrogenase Using Directed and Mediated Electron Transfer Processes

Abstract

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 Azotobacter vinelandii 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.

Tailoring Quantum Dot Sizes for Optimal Photoinduced Catalytic Activation of Nitrogenase

“…In a follow‐up work, they investigated the mechanism and the kinetics in which photoexcited electrons are transferred to the MoFe−P during the reduction process of N 2 into ammonia using electron paramagnetic resonance (EPR) spectroscopy [33,34] . Recently, we have examined the catalytic activity of the nitrogenase in the presence of 10 nm CdS NPs modified with charged and uncharged ligands [35] . In parallel, Cha and co‐workers examined different methodologies to covalently link the nitrogenase with CdS NRs to achieve efficient, non‐diffusional electron transfer processes [27] .…”

“…The QD long‐chain ligands were replaced with short ones using 2‐mercaptoethanol (ME) ligands that ensure solubility in aqueous conditions and enable short electron transfer distances required for artificial activation of the proteins (see Figure S2). Azotobacter vinelandii were grown and the nitrogenase enzyme was expressed by ammonium depletion in the growth medium, as have been described before [35,39–41] . The Fe−P and the nitrogenase proteins were isolated and purified under a strict oxygen‐free environment using a home‐built path through an adapter which enabled the integration of a glovebox and a fast protein liquid chromatography (FPLC) setup (see experimental section).…”

“…Azotobacter vinelandii DJ995 and DJ1194 strains were grown and the His‐tagged MoFe−P variants, WT and αL158C, were expressed and purified, respectively as was published before [35,39–41] . Protein purifications were performed in an oxygen‐free environment (O 2 <0.2 ppm) using homemade designed feedthrough connectors to fit the glove box.…”

“…The precipitated cells were resuspended in the same Tris buffer containing NaCl (100 m m ). Cells were lysed by a sonicator installed in a glove box as was published before [35] . The cell lysate was centrifuged (30 min, 10 000 rpm, 4 °C).…”

“…Over 10 CV of gradient elution from 100 to 500 m m NaCl at 4 mL min −1 different fractions were collected. The Fe–P fraction was further purified using size exclusion chromatography (SEC) on a GE Superdex 200 Increase 10/300 GL column with a 50 m m Tris buffer, pH 7.6, and 200 mM NaCl (as described in our previous work) [35] . His‐tagged MoFe−P was purified on a 1 ml GE HisPrepTM FF affinity column using a step gradient.…”