Methane (CH) is a potent greenhouse gas that is released from fossil fuels and is also produced by microbial activity, with at least one billion tonnes of CH being formed and consumed by microorganisms in a single year . Complex methanogenesis pathways used by archaea are the main route for bioconversion of carbon dioxide (CO) to CH in nature. Here, we report that wild-type iron-iron (Fe-only) nitrogenase from the bacterium Rhodopseudomonas palustris reduces CO simultaneously with nitrogen gas (N) and protons to yield CH, ammonia (NH) and hydrogen gas (H) in a single enzymatic step. The amount of CH produced by purified Fe-only nitrogenase was low compared to its other products, but CH production by this enzyme in R. palustris was sufficient to support the growth of an obligate CH-utilizing Methylomonas strain when the two microorganisms were grown in co-culture, with oxygen (O) added at intervals. Other nitrogen-fixing bacteria that we tested also formed CH when expressing Fe-only nitrogenase, suggesting that this is a general property of this enzyme. The genomes of 9% of diverse nitrogen-fixing microorganisms from a range of environments encode Fe-only nitrogenase. Our data suggest that active Fe-only nitrogenase, present in diverse microorganisms, contributes CH that could shape microbial community interactions.

Nitrogenase catalyzes the reduction of dinitrogen (N) using low potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O) sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd/Fld-reducing enzymes in 359 genomes of putative N fixers (diazotrophs). Six distinct lineages of nitrogenase were identified and their distributions largely corresponded to differences in the host cells' ability to integrate O or light into energy metabolism. Predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the level of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation. Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O or light into their energy metabolism. Acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the nitrogen fixation (Rnf) complex.

Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes. Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.

Nitrogenase is an ATP-requiring enzyme capable of carrying out multielectron reductions of inert molecules. A purified remodeled nitrogenase containing two amino acid substitutions near the site of its FeMo cofactor was recently described as having the capacity to reduce carbon dioxide (CO 2 ) to methane (CH 4 ). Here, we developed the anoxygenic phototroph, Rhodopseudomonas palustris, as a biocatalyst capable of light-driven CO 2 reduction to CH 4 in vivo using this remodeled nitrogenase. Conversion of CO 2 to CH 4 by R. palustris required constitutive expression of nitrogenase, which was achieved by using a variant of the transcription factor NifA that is able to activate expression of nitrogenase under all growth conditions. Also, light was required for generation of ATP by cyclic photophosphorylation. CH 4 production by R. palustris could be controlled by manipulating the distribution of electrons and energy available to nitrogenase. This work shows the feasibility of using microbes to generate hydrocarbons from CO 2 in one enzymatic step using light energy.A n essential process for life and an important step in the biogeochemical nitrogen cycle is nitrogen fixation by nitrogenase, in which nitrogen gas (N 2 ) is converted to ammonia (NH 3 ) (1). The difficult reduction of N 2 to two NH 3 occurs at an FeMoS cluster called FeMo cofactor in Mo-dependent nitrogenase in a reaction that requires ATP hydrolysis and dihydrogen production as shown in 1Nitrogenase deprived of access to N 2 but provided with a source of electrons produces H 2 exclusively. Also, the ability of nitrogenase to carry out the multielectron reduction of an inert molecule is not limited to reduction of N 2 . This enzyme can also reduce other molecules with double and triple bonds, including carboncontaining compounds (reviewed in ref.2). Recently, we found that a remodeled nitrogenase with substitutions in two key amino acids near the FeMo cofactor is capable of reducing carbon dioxide (CO 2 ) to methane (CH 4 ) in vitro. This enzyme did not retain its ability to reduce N 2 but was active in H 2 production (3). It was unclear if the remodeled nitrogenase gene could confer to bacteria the ability to reduce CO 2 to CH 4 . Here, we describe a biocatalyst capable of generating the energyrich hydrocarbon CH 4 by reduction of CO 2 using a remodeled nitrogenase. Development of this biocatalyst required selection of an appropriate microbial host, because large amounts of cellular reductant and ATP are used by nitrogenase, and as a consequence, this energetically expensive enzyme is repressed by both transcriptional and posttranslational regulatory mechanisms when an alternative nitrogen source, like ammonium, is available (4). Use of nitrogenase to generate a product not used by the organism would require overcoming these regulatory constraints to achieve expression of active enzyme, while at the same time providing cells with ammonium for growth. We reasoned that the anoxygenic photosynthetic bacterium Rhodopseudomonas palustris would be a good ch...