Medina Michael S, Bretzing Kevin O, Aviles Richard A, Chong Kiersten M, Espinoza Alejandro, Garcia Chloe Nicole G, Katz Benjamin B, Kharwa Ruchita N, Hernandez Andrea, Lee Justin L, Lee Terrence M, Lo Verde Christine, Strul Max W, Wong Emily Y, Owens Cedric P
Schmid College of Science and Technology, Chapman University, Orange, California, USA.
Department of Chemistry, University of California, Irvine, Irvine, California, USA.
J Biol Chem. 2021 Jan-Jun;296:100501. doi: 10.1016/j.jbc.2021.100501. Epub 2021 Mar 2.
Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N) to ammonia (NH). 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.
固氮酶是唯一能够催化固氮作用的酶，即将双氮气体（N₂）还原为氨（NH₃）。固氮酶受到环境气体一氧化碳（CO）的强烈抑制。固氮细菌依靠蛋白质CowN在有CO的情况下生长。然而，CowN发挥作用的机制尚不清楚。在这里，我们展示了CowN的生化特性，并研究了CowN如何保护固氮酶免受CO的影响。我们确定CowN直接与固氮酶相互作用，并且CowN的保护作用相对于CowN浓度呈现双曲线动力学。在CO浓度为0.001个大气压时，CowN几乎能恢复固氮酶的全部活性。我们的结果进一步表明，CowN的保护机制包括将CO与固氮酶活性位点的结合亲和力降低约十倍，而不中断底物周转。综上所述，我们的工作表明CowN是固氮作用中一种重要的辅助蛋白，它赋予固氮酶对CO的耐受性。
