The oxygen sensitivity of [4Fe-4S] clusters on the nitrogenase scaffold protein NifU

The extreme oxygen sensitivity of nitrogenase enzymes poses a major obstacle to their use in biotechnology, particularly in engineering nitrogen-fixing plants. While the vulnerability of nitrogenase components NifH and NifDK to oxygen is well established, the sensitivity of the metal clusters on scaffold proteins involved in nitrogenase biosynthesis has not been previously reported. In this study, we investigate the oxygen sensitivity of the [4Fe-4S] clusters on the scaffold protein NifU, which supplies these clusters to several downstream targets, including NifH. Using UV/Vis, Mössbauer, and Electron Paramagnetic Resonance (EPR) spectroscopy, we show that exposure to oxygen converts the [4Fe-4S] clusters into [2Fe-2S] clusters. This degradation occurs rapidly, even at low oxygen levels (2 %). However, a fraction of the [4Fe-4S] clusters remain intact under these conditions, allowing NifU to activate apo-NifH by transferring functional clusters. In contrast, at atmospheric oxygen levels (21 %), all [4Fe-4S] clusters are damaged over 10 min, and NifU quickly loses its ability to reactivate apo-NifH.