Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N 2 ) to ammonia (NH 3 ). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN’s protection mechanism involves decreasing the binding affinity of CO to nitrogenase’s active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase.

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Sunflower meal, a byproduct of sunflower oil pressing, is not commonly used in alkaline baking applications. This is because chlorogenic acid, the main phenolic antioxidant in sunflower seeds, reacts with protein, giving the baked product a green discoloration. Our group previously demonstrated that a chlorogenic acid esterase from Lactobacillus helveticus hydrolyzes chlorogenic acid in sunflower dough cookie formulations, resulting in cookies that were brown instead of green. This study presents a sensory analysis to determine the acceptability of enzymatically upcycled sunflower meal as an alternative protein source for those allergic to meals from legumes or tree nuts. We hypothesized that the mechanism of esterase‐catalyzed chlorogenic acid breakdown does not influence the cookies’ sensory properties other than color and that consumers would prefer treated, brown cookies over non‐treated cookies. Cookies made from sunflower meal were presented under green lights to mask color and tested by 153 panelists. As expected, the sensory properties (flavor, smell, texture, and overall acceptability) of the treated and non‐treated cookies were not statistically different. These results corroborate proximate analysis, which demonstrated that there was no difference between enzymatically treated and non‐treated cookies other than color and chlorogenic acid content. After the cookie color was revealed, panelists strongly preferred the treated cookies with 58% indicating that they “probably” or “definitely” would purchase the brown cookies, whereas only 5.9% would buy green, non‐treated cookies. These data suggest that esterase‐catalyzed breakdown of chlorogenic acid represents an effective strategy to upcycle sunflower meal for baking applications. Practical Application Sunflower meal is currently used as animal fodder or discarded. A major factor preventing sunflower meal use is its high chlorogenic acid content, which causes a green discoloration of baked goods made from sunflower meals under alkaline conditions. This study presents a sensory analysis in which panelists evaluate cookies made with sunflower flour that was treated with an esterase that breaks down chlorogenic acid. The results show that enzymatic treatment prevents greening and that panelists strongly prefer esterase‐treated, non‐green cookies, thus demonstrating the feasibility of utilizing sunflower flour in baking applications.

Cu,Zn superoxide dismutases (Cu,ZnSOD) are metalloenzymes that catalyze the dismutation of the superoxide anion into oxygen and hydrogen peroxide. These enzymes, for a long time considered peculiar of eukaryotic organisms have been found to be present also in bacteria. From an analysis of their protein sequences we can observe that, with few exceptions, the ligands of metal sites are conserved. Among the bacterial proteins the only one which does not conserve two of the residues able to bind copper is the protein from Bacillus subtilis.The BsSOD protein may be thought as a step of the evolution line from a no-Cu,ZnSOD world to the fully active Cu,ZnSODs. With this in mind we have tried to reconstitute SOD's activity through an artificial evolution obtained by introducing the copper ligands with site-directed mutagenesis. We have cloned the wild type, the two mutants P104H and Y88H-P104H which reintroduce one or both of the copper binding histidines respectively, reestablishing in the first case the ability to bind copper and in the second case the standard copper site of Cu,ZnSOD. We report the structural and biochemical characterization of the three proteins showing the restoration in the double mutant of a partially active Cu,ZnSOD and the resulting mechanistic and physiological implications.