An alternative path for the evolution of biological nitrogen fixation

Abstract

Nitrogenase catalyzed nitrogen fixation is the process by which life converts dinitrogen gas into fixed nitrogen in the form of bioavailable ammonia. The most common form of nitrogenase today requires a complex metal cluster containing molybdenum (Mo), although alternative forms exist which contain vanadium (V) or only iron (Fe). It has been suggested that Mo-independent forms of nitrogenase (V and Fe) were responsible for N(2) fixation on early Earth because oceans were Mo-depleted and Fe-rich. Phylogenetic- and structure-based examinations of multiple nitrogenase proteins suggest that such an evolutionary path is unlikely. Rather, our results indicate an evolutionary path whereby Mo-dependent nitrogenase emerged within the methanogenic archaea and then gave rise to the alternative forms suggesting that they arose later, perhaps in response to local Mo limitation. Structural inferences of nitrogenase proteins and related paralogs suggest that the ancestor of all nitrogenases had an open cavity capable of binding metal clusters which conferred reactivity. The evolution of the nitrogenase ancestor and its associated bound metal cluster was controlled by the availability of fixed nitrogen in combination with local environmental factors that influenced metal availability until a point in Earth's geologic history where the most desirable metal, Mo, became sufficiently bioavailable to bring about and refine the solution (Mo-nitrogenase) we see perpetuated in extant biology.

Keywords: evolution; great oxidation event; iron; metalloenzyme; nitrogenase; oxygen molybdenum; sulfide; vanadium.

Figure 1

Bayesian inferred phylogenetic tree of concatenated HDK homologs (see Figure A1 in Appendix for maximum-likelihood inferred tree). Posterior probabilities are indicated above or below nodes. Branches are colored dark blue (Mo-nitrogenase, Nif), green (V-nitrogenase, Vnf), purple (Fe-nitrogenase, Anf), red (uncharacterized nitrogenase), and light blue (uncharacterized homolog). The hash at the root was introduced to conserve space.

Figure 2

Bayesian inferred phylogenetic reconstruction of Anf/Vnf/NifD, BchN, and NflD proteins. The putative substrates and cofactors for each protein lineage are indicated below each respective clade. Posterior probabilities for each collapsed node are indicated. Nodes have been collapsed and hashes introduced to conserve space.

Figure 3

Model depicting the divergence of nitrogenase (NifD) and protochlorophyllide reductase (ChlN/BchN) from a NflD ancestor. The stepwise evolution of cofactor biosynthesis leading to the acquisition of metal specificity in the covalently bound active site metallocluster, where Mo acquisition and Mo-nitrogenase predates V acquisition and V-nitrogenase, and V acquisition predates Fe-only nitrogenase. ChlN/BchN bind substrates in their active site cavities non-covalently and release these substrates following reduction (Muraki et al., 2010). Abbreviations: Mo, molybdenum; V, vanadium; Nif, Mo-dependent nitrogenase; Vnf, V-dependent nitrogenase; Anf, Fe-only nitrogenase; Bch, BchN protein involved in bacteriochlorophyll biosynthesis; Chl, ChlN protein involved in chlorophyll biosynthesis.

Figure A1

Plot of a Mantel regression of a matrix describing the Rao phylogenetic dissimilarity of concatenated HDK homologs inferred by PhyML as a function of the Rao phylogenetic dissimilarity of concatenated HDK homologs inferred by MrBayes. The strong positively trending correlation suggests that the topologies of the two trees are congruent.

Figure A2

Maximum-likelihood inferred phylogenetic tree of concatenated HDK homologs. Bootstrap values based on 100 replicates are indicated at each node. Branches are colored dark blue (Mo-nitrogenase, Nif), green (V-nitrogenase, Vnf), purple (Fe-nitrogenase, Anf), red (uncharacterized nitrogenase), and light blue (uncharacterized homolog). The hash at the root was introduced to conserve space.

Figure A3

Plot of a Mantel regression of a matrix describing the average RMSD for H, D, and K protein structures inferred using homology modeling as a function of the Rao phylogenetic dissimilarity of concatenated HDK homologs inferred by MrBayes. The strong correlation suggests a relationship between the evolution of sequences and their inferred structures, implying that the HDK structure is evolving. The slope of the line linear regression (∼2) suggests that the evolution of protein structure is constrained to a greater extent than the evolution of the primary sequences.

Figure A4

Structural alignment of the inferred structures of DK homologs indicating conservation in the active site (A) and P-cluster binding cavity (B). Ribbon diagram of the superimposition of NifDK from Azotobacter vinelandii AvOP (D, violet and K, gray), NifDK from Methanococcus maripaludis strain S2 (D, wheat and NifK, blue), UncDK from Methanocaldococcus sp. FS406-22 (D, cyan and K, orange), UncDK from Roseiflexus sp. RS-1 (D, marine, and K, sand), VnfDK from Methanosarcina acetivorans str. C2A (D, raspberry, and K, pale green), and AnfDK from Azotobacter vinelandii AvOP (D, green, and K, salmon), with the FeMo-co (A) and P-cluster (B) depicted as stick representations. Dark red, Fe; yellow, S; gray, C; red, O; teal, Mo; unknown, magenta. Protein Data Bank ID for Azotobacter vinelandii AvOP 1MIN.

Figure A5

Amino acid sequence conservation in selected residues that ligate FeMo-co (Cys-275, red box, and His-442, blue box) and that have been implicated as important in the FeMo-co binding pocket (indicated by a gray box). Representative NifD, VnfD, AnfD, and UncD (uncharacterized nitrogenase). Numbering is based on NifD from Azotobacter vinelandii AvOP. Abbreviations: A.v., Azotobacter vinelandii AvOP; M.a., Methanosarcina acetivorans str. C2A; M.m., Methanococcus maripaludis strain S2; R.c., Roseiflexus sp. RS-1; M.c. Methanocaldococcus sp. FS406-22. The conservation in the active site environment and active site custer ligands between classes of nitrogenase suggest that once the active site cavity evolved, it was maintained through time.