Use of translational fusions to express functional Klebsiella oxytoca dinitrogenase reductase in plant mitochondria

Engineering crop plants with the biological nitrogen fixation pathway is a longstanding goal of modern agriculture. Dinitrogenase reductase (NifH) is a critical component of the biological nitrogen fixation pathway, with multiple roles in metal cofactor assembly and catalysis. This enzyme must be folded correctly as a soluble homodimer and loaded with the [4Fe-4S] metallocluster for function. Previous studies have found that Klebsiella oxytoca (Ko) and Azotobacter vinelandii (Av) NifHs were mostly insoluble when targeted to plant mitochondria. Here we found that a translational fusion of two KoNifH or AvNifH monomers, forming KoNifHH or AvNifHH synthetic dimers, produced a soluble protein when targeted to plant mitochondria and co-expressed with the putative peptidyl-prolyl cis-trans isomerase NifM. KoNifHH isolated after expression in leaf mitochondria at ambient oxygen showed some acetylene reduction activity, which did not require co-expression of the nitrogenase-specific metallocluster machinery NifS and NifU. This activity increased after iron-sulfur cluster reconstitution in vitro with recombinant NifU. In a parallel study, we tested a translational fusion of a variant iron-only dinitrogenase reductase (AvAnfHv6) monomer that was soluble but not active as-isolated from plant mitochondria (AvAnfHHv6) (Gregg et al. 2025a). AvAnfHHv6 was abundant and fully soluble when isolated from plant mitochondria, like its monomer. AvAnfHHv6 was not active as-isolated but could be largely activated by iron-sulfur cluster reconstitution in vitro. This study demonstrates how translational fusions help improve solubility and have the potential to generate an active NifH enzyme within plant mitochondria.