Work

A Low‐Potential Terminal Oxidase Associated with the Iron‐Only Nitrogenase from the Nitrogen‐Fixing Bacterium Azotobacter vinelandii

Year: 2021

Type: article

Abstract: Modern agriculture depends on nitrogenous fertilizer produced by the Haber‐Bosch process, but fertilizer use is polluting and its production needs large amounts of methane and emits carbon dioxide. To minimize nitrogen pollution and to reduce the need for fertilizer, there is interest in expressing functional nitrogenase in crop plants, either directly or in prokaryotic symbiotes. The iron‐only alternative nitrogenase is a good candidate for expression, because it requires fewer genes than the more common MoFe nitrogenase. Nitrogenases are inactivated by oxygen, so understanding and overcoming this inhibition will be important for heterologous nitrogenase expression. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic ( i.e. nitrogen‐fixing) growth with the iron‐only nitrogenase, but its enzymatic activity and function are unknown. We have biochemically and structurally characterized Anf3 from the model diazotrophic bacterium Azotobacter vinelandii . Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the FAD cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of ‐420 ± 10 mV and ‐330 ± 10 mV for the two FAD potentials and ‐340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. The combination of the biochemical, electrochemical, and structural results show that Anf3 catalyzes the reduction of oxygen to water. We expect the catalytic mechanism to be similar to that of other heme‐containing oxidases because four electrons can be accumulated: two on the FAD and two on the heme, and then rapidly donated to the bound oxygen for its four‐electron reduction to water. We predict that Anf3 protects the iron‐only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors. Anf3 is therefore a promising candidate to enhance functional nitrogenase expression in heterologous systems.

Source: The FASEB Journal

DOI: 10.1096/fasebj.2021.35.s1.00046