Fig. 5.

Evolution of G-subunit functional specialization and Vnf/Anf metal dependence. a) Hypothesized functional specialization of Vnf and Anf G-subunit proteins. Specialized sequence motifs, represented by sequence logos, of ancestral G-subunit proteins (VnfAnf^Anc, Vnf^Anc, and Anf^Anc) are shown beside their respective nodes in the stylized nitrogenase phylogeny. Sequence logos scale with amino acid posterior probabilities at each site. VnfAnf^Anc dependence on FeV-co is inferred based on comparisons of specialized sequence motifs (indicated by a bold asterisk above FeV-co for VnfAnf^Anc). Branch lengths in the phylogeny do not scale with the timeline in b). b) Geological context of G-subunit emergence in VnfAnf^Anc nitrogenase. Plot shows the association between changes in bulk marine concentrations of redox-sensitive metals involved in nitrogen fixation (Mo, V, and Fe) and the progressive oxygenation of the Earth’s surface environment. Ancient vanadium concentrations are not well known, although oxygenation likely impacted the speciation and solubility of V and Mo similarly over geologic time (Emerson and Huested 1991; Moore et al. 2020). Metal concentrations are from Zerkle et al. (2005) and references therein and p_O2 data are from Lyons et al. (2014). Estimated age constraints of nitrogenase origins and VnfAnf^Anc (i.e. G-subunit emergence) are from Stueken et al. (2015) and Parsons et al. (2021), respectively. GOE, great oxidation event; PAL, present atmospheric level. c) Schematic of the experimental strategy to replace A. vinelandii VnfG with the ancestral VnfAnf^Anc G-subunit (G^Anc) or A. vinelandii AnfG and characterization via the acetylene (C₂H₂) reduction assay. d) Genome engineering approach to modify the vnfG gene of A. vinelandii by reciprocal recombination with donor plasmid DNA. All reported strains have Nif inactivated by nifD replacement with a kanamycin resistance cassette (KanR). e) Cellular acetylene reduction activity of engineered A. vinelandii strains (n = 4 biological replicates for WT vnfG and ΔvnfG::anfG; n = 3 for ΔvnfG::G^Anc and ΔvnfG). Individual data points are shown by dark circles and mean values are shown by bar plots. Statistical tests were performed by one-way ANOVA and post hoc Tukey’s Honestly Significant Difference test.