Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase

The Azotobacter vinelandii molybdenum nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires an existing [Fe-S] cluster to form a [Mo-Fe3-S4] group, which acts as a specific molybdenum donor during nitrogenase FeMo-co biosynthesis. Here, we show biochemical evidence supporting the role of NifU as the [Fe-S] cluster donor. Protein-protein interaction studies involving apo-NifQ and as-isolated NifU demonstrated their interaction, which was only effective when NifQ lacked its [Fe-S] cluster. Incubation of apo-NifQ with [Fe4-S4]-loaded NifU increased the iron content of the former, contingent on both proteins being able to interact with one another. As a result of this interaction, a [Fe4-S4] cluster was transferred from NifU to NifQ. In A. vinelandii, NifQ was preferentially metalated by NifU rather than by the [Fe-S] cluster scaffold protein IscU. These results indicate the necessity of co-expressing NifU and NifQ to efficiently provide molybdenum for FeMo-co biosynthesis when engineering nitrogenase in plants.

This work was supported in part by the Bill & Melinda Gates Foundation (INV-005889). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. The work was also supported by grant PID2021-124060OB-100 from the Ministerio de Ciencia, Innovación/Agencia Estatal de Investigación/10.13039/50110001103 and “ERDF A way of making Europe” to MG-G. Y.G. acknowledges the funding supporting from the National Institutes of Health (NIH R01GM125924). EB was funded by the Severo Ochoa Programme for Centres of Excellence in R&D from Agencia Estatal de Investigación of Spain (grant SEV-2016–0672) received by Centro de Biotecnología y Genómica de Plantas (UPM-INIA/CSIC). ER-N and JAC-G were supported by Formación de Personal de Investigación fellowships PRE2018-084895 and PRE2022-101253, respectively. XJ is recipient of a doctoral fellowship from Universidad Politécnica de Madrid.

With funding form the Spanish government through the "Severa Ochoa Centre of Excellence" accreditation (SEV-2016–0672).

Peer reviewed