An alternative path for the evolution of biological nitrogen fixation.

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.

Introduction

Biological nitrogen fixation, the reduction of dinitrogen (N2) to ammonia, accounts for roughly two-thirds of the fixed nitrogen (N) produced on Earth today (Rubio and Ludden, 2008). The emergence of biological N2 fixation therefore enabled life to access the vast reserves of N present as N2 gas in our atmosphere (Rees, 1993), a feature that would have profoundly impacted the history of life on earth and the biogeochemical cycles that it modulates (Falkowski et al., 2008; Canfield et al., 2010). Today, biological N2 fixation is catalyzed by at least three genetically distinct but evolutionarily related nitrogenases. The majority of present-day biological nitrogen fixation is catalyzed by the molybdenum-nitrogenase (encoded by nifHDK), an oxygen-sensitive, metalloenzyme complex composed of the Fe protein (product of nifH) and the MoFe heterotetramer (products of nifDK; Rubio and Ludden, 2008). The Fe protein is a homodimer bridged by an intersubunit [4Fe–4S] cluster that serves as the obligate electron donor to the MoFe protein (Georgiadis et al., 1992). The MoFe protein is a α2β2 heterotetramer that houses the P-cluster, an [8Fe–7S] cluster that shuttles electrons to the FeMo-cofactor, a [Mo–7Fe–9S-homocitrate] cluster that provides the substrate reduction site (Kim and Rees, 1992). Two “alternative” forms of nitrogenase have also been identified in the genomes of organisms that encode for Nif (Joerger and Bishop, 1988; Raymond et al., 2004; Soboh et al., 2010; Boyd et al., 2011). The nitrogenase encoded by the vnfHDK genes is believed to contain vanadium in place of molybdenum in the active site cofactor, whereas the nitrogenase encoded by the anfHDK genes appears to contain only Fe as the metal constituent of its active site cofactor (Hales et al., 1986; Chisnell et al., 1988). When fixed nitrogen is limiting, the expression and activity of the alternative forms is regulated by the availability of Mo or V (Joerger and Bishop, 1988; Kessler et al., 1997; Hamilton et al., 2011).

Chemical stratigraphic measurements indicate that ancient oceans were limited in soluble Mo prior to the rise of oxygen ∼2.5 Ga (Anbar et al., 2007) due to the insolubility of Mo-sulfides under anoxic conditions (Helz et al., 1996). This prompted the proposal that Anf and Vnf represent primitive forms of nitrogenase that predate Nif (Anbar and Knoll, 2002; Raymond et al., 2004). Past phylogenetic analyses of the nitrogenase structural gene products have failed to provide convincing evidence for the trajectory of specific metal incorporation into the active site cofactor of nitrogenase during its evolution (Raymond et al., 2004; Soboh et al., 2010; Boyd et al., 2011). Here, we examine concatenations of protein homologs of the structural components shared between all known nitrogenases (H, D, and K; Raymond et al., 2004; Mehta and Baross, 2006; Dekas et al., 2009; Boyd et al., 2011). The results of our phylogenetic- and structure-based examination indicate an evolutionary path whereby Mo-dependent nitrogenase gave rise to the alternative forms suggesting that they arose later, perhaps in response to local Mo limitation. These results, when coupled with considerations of the physiology and the biochemistry of nitrogen fixation, lead to a new model for the stepwise evolution of nitrogenase and other related complex metalloproteins.

Materials and Methods

Phylogenetic analysis

Representative homologs of Anf/Vnf/NifHDK and uncharacterized HDK (Table A1 in Appendix) were compiled as previously described (Soboh et al., 2010; Boyd et al., 2011). Individual H, D, and K homologs were aligned using CLUSTALX (version 2.0.8) specifying the Gonnet 250 protein substitution matrix and default gap extension and opening penalties (Larkin et al., 2007) as previously described (Boyd et al., 2011) with ChlLNB/BchLNB from Anabaena variabilis ATCC 29413 and Chlorobium limicola DSM 245 serving as outgroups. The individual alignment blocks were concatenated, subjected to evolutionary model prediction, and the phylogeny of each concatenated protein sequence evaluated using MrBayes (version 3.1.2; Huelsenbeck and Ronquist, 2001) and PhyML (version 3.0; Guindon and Gascuel, 2003) employing the WAG + I + G evolutionary model (Appendix) as identified by ProtTest (version 2.0; Abascal et al., 2005). In phylogenetic reconstructions using MrBayes, tree topologies were sampled every 500 generations over 450,000 generations (after a burnin of 50,000) at likelihood stationarity and after convergence of two separate Markov chain Monte Carlo runs (average SD of split frequencies <0.05). A consensus phylogenetic tree was projected from 1,800 trees using FigTree (version 1.2.2). One hundred bootstrap replicates were performed in phylogenetic reconstructions using PhyML. Matrices describing the Rao phylogenetic dissimilarity of concatenated HDK homologs, inferred using both MrBayes and PhyML, were generated using Phylocom (version 4.0.1; Webb et al., 2008).

Table A1

Accession numbers of representative sequences used in the present study.

ANF/VNF/NIF/UNCHARACTERIZED NITROGENASE HDK HOMOLOGS
Taxon	NifH homologs	NifD homologs	NifK homologs
Acidithiobacillus ferrooxidans ATCC 23270	YP_002219685	YP_002219684	YP_002219683
Alkaliphilusmetalliredigens QYMF	YP_001321310	YP_001321307	YP_001321306
Anabaenavariabilis ATCC 29413 vnf	YP_324416	YP_324526	YP_324527
Anabaenavariabilis ATCC 29413 nif	YP_324741	YP_324742	YP_324743
Azotobactervinelandii AvOP nif	YP_002797378	YP_002797379	YP_002797380
Azotobactervinelandii AvOP anf	YP_002801975	YP_002801974	YP_002801972
Azotobactervinelandii AvOP vnf	YP_002797502	YP_002797497	YP_002797495
Beijerinckiaindica subsp. indica ATCC 9039	YP_001831615	YP_001831616	YP_001831617
Caldicellulosiruptorsaccharolyticus DSM 8903	YP_001181234	YP_001181231	YP_001181230
Candidatus Azobacteroides pseudotrichonymphae CFP2	YP_002309219	YP_002309222	YP_002309223
Candidatus Desulforudis audaxviator MP104C	YP_001716343	YP_001716346	YP_001716347
Candidatus Methanosphaerula palustris E1-9c	YP_002465657	YP_002465654	YP_002465653
Chlorobiumtepidum TLS000	NP_662417	NP_662420	NP_662421
Chloroherpetonthalassium ATCC 35110	YP_001996732	YP_001996735	YP_001996737
Chloroherpetonthalassium ATCC 35110	YP_001995946	YP_001995943	YP_001995942
Clostridiumacetobutylicum ATCC 824	NP_346894	NP_346897	NP_346898
Clostridiumbeijerinckii NCIMB 8052	ABR34169	ABR34172	ABR34173
Clostridiumkluyveri NBRC 12016	YP_001395138	YP_001395137	YP_001395135
Dehalococcoidesethenogenes 195	YP_181872	YP_181869	YP_181868
Desulfitobacteriumhafniense Y51	YP_520504	YP_520503	YP_520502
Desulfotomaculumreducens MI-1	YP_001114150	YP_001114147	YP_001114146
Desulfovibriovulgaris subsp. vulgaris DP4	YP_009055	YP_961292	YP_009051
Geobacterlovleyi SZ	YP_001950896	YP_001950895	YP_001950894
Geobacter sp. M21	YP_003021955	YP_003021954	YP_003021953
Heliobacteriummodesticaldum Ice1	YP_001679706	YP_001679707	YP_001679708
Klebsiellapneumoniae 342	YP_002237565	YP_002237564	YP_002237563
Lyngbya sp. PCC 8106	ZP_01620768	ZP_01620767	ZP_01620766
Magnetospirillummagnetotacticum MS-1	YP_420937	ZP_00054386	ZP_00054385
Methanobacteriumthermoautotrophicum str. Delta H	NP_276673	NP_276676	NP_276677
Methanococcusaeolicus Nankai-3	YP_001325622	YP_001325619	YP_001325618
Methanococcusmaripaludis strain S2	NP_987973	NP_987976	NP_987977
Methanococcusvannielii SB	YP_001322591	YP_001322588	YP_001322587
Methanosarcinaacetivorans str. C2A	NP_618766	NP_618769	NP_618770
Methanosarcinaacetivorans str. C2A	NP_616152	NP_616155	NP_616157
Methanosarcinaacetivorans str. C2A	NP_616144	NP_616149	NP_616147
Methylacidiphiluminfernorum V4	YP_001940528	YP_001940526	YP_001940525
Methylocellasilvestris BL2	YP_002363879	YP_002363878	YP_002363877
Nostoc sp. PCC 7120	NP_485497	NP_485484	NP_485483
Opitutaceae bacterium TAV2	ZP_03723745	ZP_03723746	ZP_03723747
Pelobactercarbinolicus DSM 2380	YP_357508	YP_357509	YP_357510
Rhodobactersphaeroides ATCC 17025	YP_001167452	YP_001167453	YP_001167454
Rhodopseudomonaspalustris BisA53	YP_782790	YP_782789	YP_782787
Rhodopseudomonaspalustris HaA2	YP_484590	YP_484591	YP_484592
Rhodospirillumcentenum SW	YP_002299844	YP_484591	YP_002299842
Rhodospirillumrubrum ATCC 11170	YP_426483	YP_426482	YP_426480
Roseiflexuscastenholzii DSM 13941	YP_001434094	YP_001434092	YP_001434091
Roseiflexus sp. RS-1	YP_001275558	YP_001275556	YP_001275555
Sinorhizobiummedicae WSM419	YP_001314762	YP_001314761	YP_001314760
Synechococcus sp. JA-2-3B′a	YP_475238	YP_476681	YP_476682
Syntrophobacterfumaroxidans MPOB	YP_845148	YP_845145	YP_845144
Thermodesulfovibrioyellowstonii DSM 11347	YP_002249508	YP_002249507	YP_002249506
Zymomonasmobilis subsp. mobilis ZM4	ZP_04760608	YP_163559	YP_163560
Methanocaldococcusinfernus ME	YP_003615677	YP_003615674	YP_003615673
Syntrophothermuslipocalidus DSM 12680	YP_003703438	YP_003703435	YP_003703434
Methanocaldococcusvulcanius M7	YP_003247421	YP_003247424	YP_003247425
Methanosarcinamazei strain Goe1	NP_632743	NP_632746	NP_632747
Methanocaldococcus sp. FS406-22	YP_003457468	YP_003457471	YP_003457472
Methanothermococcusokinawensis IH1	ZP_07330063	ZP_07330060	ZP_07330059
Oscillochloristrichoides	ZP_07684114	ZP_07684112	ZP_07684111
Anabaenavariabilis ATCC 29413	YP_322845	YP_322843	YP_323968
Chlorobiumlimicola DSM 245	YP_001944195	YP_001944197	YP_001944196
NFL HOMOLOGS
Taxon	NflH homologs	NflD homologs
Methanobacteriumthermoautotrophicum	AAB85148	AAB85997
Methanobrevibactersmithii	YP_001274280	YP_001273733
Methanocaldococcusjannaschii	NP_247874	NP_248427
Methanocaldococcus sp. FS406-22	YP_003458692	YP_003458054
Methanococcoidesburtonii	YP_565723	YP_565722
Methanococcusaeolicus	YP_001325412	YP_001325501
Methanococcusmaripaludis C5	YP_001098039	YP_001097721
Methanococcusmaripaludis C6	YP_001548852	YP_001548533
Methanococcusmaripaludis C7	YP_001330364	YP_001330639
Methanococcusmaripaludis S2	CAF29703	CAF29984
Methanococcusvannielii	YP_001323663	YP_001323921
Methanocorpusculumlabreanum	YP_001029608	YP_001029961
Methanopyruskandleri	AAM02629	AAM02598
Methanosaetathermophila	YP_843600	YP_842548
Methanosarcinaacetivorans	AAM05043	AAM06983
Methanosarcinabarkeri	YP_303736	YP_303910
Methanosarcinamazei	AAM30210	AAM30211
Methanosphaerastadtmanae	YP_448145	YP_448474
CHL/BCH HOMOLOGS
Taxon	ChlL/BchL homologs	ChlN/BchN homologs	ChlB/BchB
Bradyrhizobium sp. BTAi1	YP_001242230	YP_001242233	YP_001242232
Methylobacteriumpopuli BJ001	YP_001927985	YP_001927988	YP_001927987
Erythrobacter sp. NAP1	ZP_01041680	ZP_01041677	ZP_01041678
Hoefleaphototrophica DFL-43	ZP_02167537	ZP_02167540	ZP_02167539
Halorhodospirahalophila SL1	YP_001003200	YP_001003203	YP_001003202
Prochlorococcus sp. CC9311	YP_731178	YP_731176	YP_731177
Synechococcus sp. RCC307	YP_001227822	YP_001227820	YP_001227821
Prochlorococcusmarinus MIT 9515	YP_001010923	YP_001010925	YP_001010924
Roseobacter sp. AzwK-3b	ZP_01902759	ZP_01902756	YP_002464757
Chloroflexusaggregans DSM 9485	YP_002464754	YP_002464757	YP_002464756
Roseiflexuscastenholzii HLO8, DSM 13941	YP_001431649	YP_001431647	YP_001431648
Chlorobiumphaeobacteroides DSM 266	YP_912798	YP_912800	YP_912799
Cyanothece sp. CCY 0110	ZP_01730952	ZP_01730955	ZP_01728917
Synechococcus sp. JA-2-3B	YP_477257	YP_477255	YP_478401
Gloeobacterviolaceus PCC 7421	NP_925316	NP_925315	NP_923161
Heliobacteriummodesticaldum Ice1	YP_001679876	YP_001679877	YP_001679878
Anabaenavariabilis ATCC 29413	YP_322845	YP_322843	YP_323968
Chlorobiumlimicola DSM 245	YP_001944195	YP_001944197	YP_001944196

Representative sequences were selected to sample the primary lineages of each homolog, using approaches as outlined in Boyd et al. (.

Structural analyses

The structures of the representative H, D, and K homologs selected for phylogenetic analysis were inferred using the CPH homology server for protein homology modeling (Nielsen et al., 2010) using the NifH (Georgiadis et al., 1992), NifD (Peters et al., 1997; Chiu et al., 2001; Mayer et al., 2002), and NifK (Peters et al., 1997; Chiu et al., 2001; Mayer et al., 2002) crystal structures from Azotobacter vinelandii AvOP. The inferred structures for each H, D, and K homolog were imported into PyMol (version 1.4). The root-mean-square-deviations (RMSD) in the Cαi positions were calculated for each individual inferred H, D, and K homolog structure in relation to the other inferred H, D, or K homolog structures resulting in a pairwise matrix describing the structural RMSD (e.g., structural dissimilarity) for H, D, and K homologs. RMSDs generated for H, D, and K homologs, were normalized to compensate for differing HDK protein lengths, and the normalized H, D, and K matrices were then averaged to produce an HDK RMSD matrix for use in statistical analyses. PyMol was also used to generate images of sequence conservation in the active site cavity of Anf/Vnf/NifDK homologs.

Statistical analyses

Mantel regressions of dissimilarity matrices were performed using XL Stat (version 2009.5.01). Ten thousand permutations employing two-tailed t-tests were used to determine the strength and significance of the relationships between dissimilarity matrices, respectively.

Results and Discussion

Bayesian and maximum-likelihood phylogenetic analyses of concatenated protein homologs of the required structural components (H, D, and K) encoded by characterized and putative anf, vnf, and nif regulons (Raymond et al., 2004; Mehta and Baross, 2006; Dekas et al., 2009; Boyd et al., 2011) yielded congruent topologies (Figure A1 in Appendix) with well-supported lineages that correspond to the nitrogenase active site metal content (Figures 1 and A2 in Appendix). nif-Encoded HDK protein homologs formed two distinct lineages, one of which was comprised of proteins derived solely from hydrogenotrophic methanogens that branched at the base of the tree. The second Nif lineage was comprised of more recently evolved NifHDK homologs from both bacterial and methanogen genomes. These findings are consistent with the results of a recent phylogenetic analysis which indicate that Nif emerged within the hydrogenotrophic methanogen lineage ∼2.2 Ga (Boyd et al., 2011), at a time when Mo concentrations in oceans are thought to have begun to increase (Anbar et al., 2007).

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 1

Bayesian inferred phylogenetic tree of concatenated HDK homologs (see Figure . 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 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.

Nested within the two Nif sublineages is a monophyletic lineage comprised of Vnf and Anf nitrogenase, indicating that Vnf and Anf are derived from Nif. It is likely that the ancestor of the Anf/Vnf lineage resulted from gene duplication within the hydrogenotrophic methanogen lineage and was not singly laterally transferred, a finding that is consistent with the observation that anf and vnf have yet to be identified in a genome that does not also encode for nif (Raymond et al., 2004; Boyd et al., 2011). VnfHDK homologs nest AnfHDK homologs with strong statistical support, providing evidence that Anf is derived from Vnf, both of which are derived from Nif. The latter conclusion appears to be supported by the results of a recent transcriptomic profiling of A. vinelandii, which indicated that a number of nif-encoded genes are up-regulated under conditions favoring the expression of Vnf and Anf (Hamilton et al., 2011). The observation that VnfHDK and AnfHDK from Methanosarcinales branch closely, coupled with the fact that these operons are located proximal in the genomes of these organisms, may suggest that anf is the result of a recent duplication of the vnf operon within the Methanosarcinales lineage. The acquisition of vnf within the Methanosarcinales lineage may have been the result of a lateral gene transfer (LGT) event with a firmicute, a finding that is consistent with the close spatial proximity noted between members of the Methanosarcinales and Firmicutes in a variety of anoxic environments (Stams, 1994) and with previous reports of LGT of individual genes and metabolic pathways between these two anaerobic lineages (Beiko et al., 2005; Fournier and Gogarten, 2008; Boyd et al., 2011). Importantly, evidence presented here and elsewhere (Raymond et al., 2004; Boyd et al., 2011) indicates that nif may have been acquired in the Methanosarcinales via LGT; however, it is unclear based on this dataset if that event predates the acquisition of vnf in this lineage and the subsequent duplication of vnf that resulted in anf. Nevertheless, considering that the biosynthesis of Anf and Vnf examined to date require nif-encoded gene products (Joerger et al., 1986; Kennedy and Dean, 1992; Hamilton et al., 2011), the acquisition of vnf and the duplication of vnf that led to anf are most likely to postdate the acquisition of nif within this lineage. Collectively, the evidence suggests that both Nif and Anf evolved in the methanogenic archaea, a guild of organisms which typically inhabit anoxic environments where Mo is in limited supply (Helz et al., 1996). Together with the fact that the expression of Anf and Vnf is tightly regulated by the availability of Mo and V (Joerger and Bishop, 1988; Hamilton et al., 2011), this set of observations suggests that the transient fluctuations in metal availability in anoxic environments may have been the impetus to incorporate new metals into the active site cluster of nitrogenase.

We also examined the evolutionary history of HDK homologs from the genomes of organisms that have been shown to fix N2 (Mehta and Baross, 2006; Dekas et al., 2009), but for which detailed biochemical analysis of the active site cofactor has yet to be performed (denoted as “uncharacterized nitrogenase” in Figures 1 and A2 in Appendix). These proteins formed a monophyletic lineage that branched after Nif derived from hydrogenotrophic methanogens and the Anf/Vnf lineages (Figure 1) indicating they emerged after Nif and Vnf, and possibly Anf. The “uncharacterized nitrogenase” lineage is comprised of proteins derived from strictly anaerobic taxa within the Firmicutes, as well as the methanogenic and methanotrophic archaea (Figure 1). A separate lineage comprised of uncharacterized HDK homologs that have only been identified in the genomes of filamentous anoxygenic phototrophic bacteria, branches after Nif, Vnf, and the uncharacterized nitrogenases indicating that they are the most recently evolved lineage of putative nitrogenase. A physiological or biochemical role for these proteins has yet to be conclusively demonstrated.

We inferred the protein structures of HDK homologs from anf, vnf, nif, and uncharacterized operons using homology modeling based on the structures of NifHDK from A. vinelandii (Georgiadis et al., 1992; Kim and Rees, 1992). Pairwise comparisons of the inferred protein structures enabled the generation of a matrix that describes their structural dissimilarity. A regression of this matrix and a matrix describing the phylogenetic dissimilarity of the concatenated HDK proteins revealed a significant and positive relationship (Mantel R2 = 0.23, p < 0.01; Figure A3 in Appendix). This indicates that the structure of nitrogenase has evolved significantly through time. Conserved residues that line the active site pocket in Anf/Vnf/NifD inferred through homology modeling (Figure A4 in Appendix) suggest that once the active site cavity evolved, the majority of the residues in the cavity and the cluster-coordinating ligands were maintained despite differences in the metal composition of the cofactor (Figure A5 in Appendix).

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.

We examined phylogenetic and structural relationships among proteins that are evolutionarily related to nitrogenase, including those required to biosynthesize bacteriochlorophyll (BchN; Hearst et al., 1985; Burke et al., 1993) and those that have been proposed to catalyze an analogous reaction in Ni porphyrin F430 biosynthesis (NflD; Staples et al., 2007). Phylogenetic reconstruction of Anf/Vnf/NifD, BchN, and NflD revealed three lineages, with NflD proteins forming a lineage that bisects a lineage comprising Anf/Vnf/NifD and a lineage comprising ChlN/BchN (Figure 2). These findings are consistent with a previous phylogenetic analysis of concatenated Anf/Vnf/NifHD, BchLN, and NflHD proteins (Raymond et al., 2004), which together suggest that Nfl is ancestral to Anf/Vnf/Nif and Bch (Staples et al., 2007). Intriguingly, NflD proteins share little sequence conservation with the active site cavity of Anf/Vnf/NifD and BchN. Likewise, the cofactor coordinating ligands in Anf/Vnf/NifD are not conserved in BchN sequences, although the crystal structure of BchN reveals an open cavity for the binding of protochlorophyllide instead of the bound cofactor observed in nitrogenase (Muraki et al., 2010). Homology modeling of NflD from Methanocaldococcus jannaschii DSM 2661 threaded on the structure of BchN (Muraki et al., 2010) revealed an open cavity that is similar to that of BchN that may serve as the substrate binding site. Thus, the two derived states (Anf/Vnf/NifD and BchN) have maintained similar structural architecture to that of the inferred ancestral state (e.g., NflD) but appear to have fine-tuned cavity residues to bind target substrates as the paralogs diversified. This leads to a model for the emergence of nitrogenase (Figure 3) whereby a gene encoding for an ancestral protein complex with a cavity similar to that observed in the inferred structure of NflD duplicated, leading to the evolutionary precursor of BchN and Anf/Vnf/NifD. Serendipitously, metals (e.g., Fe) or metal clusters (e.g., 4Fe–4S) were bound in the cavity of the ancestor in a non-specific manner, resulting in an enzyme complex with altered reactivity, perhaps toward N2 reduction. In response to selective pressure of limited fixed nitrogen on early Earth, genes and associated gene products were presumably recruited to improve the enzyme stepwise through the modification of the metal cofactor (Rubio and Ludden, 2008; Hu and Ribbe, 2011). In parallel, the active site was refined to yield a cavity that binds the active site cofactor FeMo-co thereby fine-tuning the structural determinants for nitrogenase catalysis. In this mechanism, it is not inconceivable that the size and dimension of the nitrogenase cofactor were constrained somewhat by the structure of the ancestor. This might be supported by the observation that the end-to-end dimensions of the FeMo-cofactor of Mo-nitrogenase are not that different from those of bacteriochlorophyll or F430. Given that the as isolated FeMo-cofactor is not reactive toward N2 on its own, the aforementioned stepwise evolution of nitrogenase may be the only mechanism by which the biochemical pathway for cofactor biosynthesis could have evolved in response to the selective pressure of fixed nitrogen limitation.

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.

In summary, the Mo-nitrogenase we see today in extant biology is not likely to be the first nitrogenase associated with early life on Earth, a finding that is in line with the dogma supported by geochemistry (Anbar and Knoll, 2002; Anbar, 2008). However, in contrast with what has been proposed previously (Anbar and Knoll, 2002; Raymond et al., 2004; Anbar, 2008), the results indicate that alternative nitrogenases (V- and Fe-only forms) are not ancestors of the Mo-nitrogenase but rather are derived from Mo-nitrogenase. The common ancestor of Nif/Vnf/Anf, Bch/Chl, and Nfl had a cavity capable of binding certain porphyrins and/or metal cluster fragments. The nature of the ancestral nitrogenase enzyme and its associated bound metal cluster was likely controlled by the selective pressure imposed by fixed nitrogen limitation in combination with local environmental metal availability until a point in Earth history (e.g., the “Great Oxidation Event”) when Mo became sufficiently bioavailable (Anbar and Knoll, 2002; Anbar, 2008) and the most favorable solution for biological nitrogen fixation (Mo-nitrogenase) emerged that is reflective of today’s extant enzyme. These results reveal a new paradigm for the evolution of biological nitrogen fixation and provide key insights into the manner in which early life forms might have exploited the reactivity of their mineral environment prior to evolving the refined complex metalloenzymes observed today.

Author Contributions

Eric S. Boyd designed the study and performed phylogenetic and statistical analyses. Trinity L. Hamilton generated the inferred protein structures. John W. Peters supervised the work. All authors contributed to the writing of the manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.