The role of αPhe-291 residue in phosphate binding by Escherichia coli F1F0-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F1F0 membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.

Single-atom catalysts (SACs) with metal–nitrogen (M–N) sites are one of the most promising electrocatalysts for electrochemical carbon dioxide reduction (ECO₂R). However, challenges in simultaneously enhancing the activity and selectivity greatly limit the efficiency of ECO₂R due to the improper interaction of reactants/intermediates on these catalytic sites. Herein, we report a carbon-based nickel (Ni) cluster catalyst containing both single-atom and cluster sites (NiNx-T, T = 500–800) through a ligandmediated method and realize a highly active and selective electrocatalytic CO₂R process. The catalytic performance can be regulated by the dispersion of Ni–N species via controlling the pyrolysis condition. Benefitting from the synergistic effect of pyrrolicnitrogen coordinated Ni single-atom and cluster sites, NiNx-600 exhibits a satisfying catalytic performance, including a high partial current density of 61.85 mA cm⁻² and a high turnover frequency (TOF) of 7,291 h⁻¹ at −1.2 V vs. RHE, and almost 100% selectivity toward carbon monoxide (CO) production, as well as good stability under 10 h of continuous electrolysis. This work discloses the significant role of regulating the coordination environment of the transition metal sites and the synergistic effect between the isolated single-site and cluster site in enhancing the ECO₂R performance.