Abstract
The N2 fixing bacterium Azotobacter vinelandii carries a molybdenum storage protein, referred to as MoSto, able to bind 25-fold more Mo than needed for maximum activity of its Mo nitrogenase. Here we have investigated a plausible role of MoSto as obligate intermediate in the pathway that provides Mo for the biosynthesis of nitrogenase iron–molybdenum cofactor (FeMo-co). The in vitro FeMo-co synthesis and insertion assay demonstrated that purified MoSto functions as Mo donor and that direct interaction with FeMo-co biosynthetic proteins stimulated Mo donation. The phenotype of an A. vinelandii strain lacking the MoSto subunit genes (ΔmosAB) was analyzed. Consistent with its role as storage protein, the ΔmosAB strain showed severe impairment to accumulate intracellular Mo and lower resilience than wild type to Mo starvation as demonstrated by decreased in vivo nitrogenase activity and competitive growth index. In addition, it was more sensitive than the wild type to diazotrophic growth inhibition by W. The ΔmosAB strain was found to readily derepress vnfDGK upon Mo step down, in contrast to the wild type that derepressed Vnf proteins only after prolonged Mo starvation. The ΔmosAB mutation was then introduced in a strain lacking V and Fe-only nitrogenase structural genes (Δvnf Δanf) to investigate possible compensations from these alternative systems. When grown in Mo-depleted medium, the ΔmosAB and mosAB+ strains showed low but similar nitrogenase activities regardless of the presence of Vnf proteins. This study highlights the selective advantage that MoSto confers to A. vinelandii in situations of metal limitation as those found in many soil ecosystems. Such a favorable trait should be included in the gene complement of future nitrogen fixing plants.
Highlights
Nitrogenase, the enzyme complex that catalyzes the fixation of N2 into NH3, is one of the most relevant enzymes in the nitrogen cycle since it converts inert N into a biologically usable form
molybdenumstorage protein (MoSto) Serves as Mo Donor for in vitro FeMo-co Biosynthesis
Two versions of MoSto were purified to test their capacity to serve as Mo donor in the in vitro FeMo-co synthesis assay
Summary
Nitrogenase, the enzyme complex that catalyzes the fixation of N2 into NH3, is one of the most relevant enzymes in the nitrogen cycle since it converts inert N into a biologically usable form. The Mo-nitrogenase consists of a dinitrogenase component, a NifDK heterotetramer containing two pairs of metalloclusters named. P-cluster (8Fe-7S) and FeMo-co (7Fe-9S-C-Mo-R-homocitrate) (Kim and Rees, 1992; Einsle et al, 2002; Rubio and Ludden, 2008; Spatzal et al, 2011), and a dinitrogenase reductase component formed by two NifH homodimers each one carrying a [4Fe4S] cluster (Georgiadis et al, 1992). The dinitrogenase components of the alternative nitrogenases contain additional subunits (VnfG or AnfG) essential for N2 reduction (Chatterjee et al, 1997; Krahn et al, 2002) and present subtle differences in cofactor structure (Sippel and Einsle, 2017). Amino acid sequence comparisons of NifD/VnfD/AnfD and NifK/VnfK/AnfK indicate that residues that serve as ligands to the metal cofactors are conserved in all three nitrogenases (Joerger et al, 1990)
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