Biochemistry & Organic Chemistry

Ancient Nitrogenases Are ATP Dependent

By Keith Cowing
Status Report
June 20, 2024
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Ancient Nitrogenases Are ATP Dependent
Evolution of the ATP/ADP-binding site in nitrogenase NifH. (A) Nitrogenase NifHDK protein phylogeny. Phylogenetic positions of ancestral (AncAK029) and extant A. vinelandii (“Extant A. vin”) nitrogenase variants discussed in the main text are highlighted by colored circles. (B) Protein sequence alignment of NifH ATPase signature motifs (labeled) (54) site index from A. vinelandii NifH. (C) Residue-level intermolecular interactions in the ADP-binding site of A. vinelandii NifH (PDB 1FP6). All displayed residues are conserved between AncAK029 and WT. Curved lines delineate residues with nonspecific interactions that shape the binding site. (D) Estimated age range of nitrogenase ancestors mapped to Earth’s environmental history. Nitrogenases hosted by heterocystous cyanobacteria provide a minimum age constraint based on oldest fossil evidence (60). Atmospheric oxygenation plot and relative marine metal abundances are from Lyons et al. (61). — mBio

Life depends on a conserved set of chemical energy currencies that are relics of early biochemistry. One of these is ATP, a molecule that, when paired with a divalent metal ion such as Mg2+, can be hydrolyzed to support numerous cellular and molecular processes.

Despite its centrality to extant biochemistry, it is unclear whether ATP supported the function of ancient enzymes. We investigate the evolutionary necessity of ATP by experimentally reconstructing an ancestral variant of the N2-reducing enzyme nitrogenase. The Proterozoic ancestor is predicted to be ~540–2,300 million years old, post-dating the Great Oxidation Event. Growth rates under nitrogen-fixing conditions are ~80% of those of wild type in Azotobacter vinelandii.

In the extant enzyme, the hydrolysis of two MgATP is coupled to electron transfer to support substrate reduction. The ancestor has a strict requirement for ATP with no other nucleotide triphosphate analogs (GTP, ITP, and UTP) supporting activity. Alternative divalent metal ions (Fe2+, Co2+, and Mn2+) support activity with ATP but with diminished activities compared to Mg2+, similar to the extant enzyme.

Additionally, it is shown that the ancestor has an identical efficiency in ATP hydrolyzed per electron transferred to the extant of two. Our results provide direct laboratory evidence of ATP usage by an ancient enzyme.

Ancient nitrogenases are ATP dependent, mBio (open access)


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