ABSTRACT Biological nitrogen fixation is catalyzed by the enzyme nitrogenase. Two forms of this metalloenzyme, the vanadium (V)- and iron (Fe)-only nitrogenases, were recently found to reduce small amounts of carbon dioxide (CO 2 ) into the potent greenhouse gas methane (CH 4 ). Here, we report carbon ( 13 C/ 12 C) and hydrogen ( 2 H/ 1 H) stable isotopic compositions and fractionations of methane generated by V- and Fe-only nitrogenases in the metabolically versatile nitrogen fixer Rhodopseudomonas palustris . The stable carbon isotope fractionation imparted by both forms of alternative nitrogenase are within the range observed for hydrogenotrophic methanogenesis ( 13 α CO2/CH4 = 1.051 ± 0.002 for V-nitrogenase and 1.055 ± 0.001 for Fe-only nitrogenase; values are means ± standard errors). In contrast, the hydrogen isotope fractionations ( 2 α H2O/CH4 = 2.071 ± 0.014 for V-nitrogenase and 2.078 ± 0.018 for Fe-only nitrogenase) are the largest of any known biogenic or geogenic pathway. The large 2 α H2O/CH4 shows that the reaction pathway nitrogenases use to form methane strongly discriminates against 2 H, and that 2 α H2O/CH4 distinguishes nitrogenase-derived methane from all other known biotic and abiotic sources. These findings on nitrogenase-derived methane will help constrain carbon and nitrogen flows in microbial communities and the role of the alternative nitrogenases in global biogeochemical cycles. IMPORTANCE All forms of life require nitrogen for growth. Many different kinds of microbes living in diverse environments make inert nitrogen gas from the atmosphere bioavailable using a special enzyme, nitrogenase. Nitrogenase has a wide substrate range, and, in addition to producing bioavailable nitrogen, some forms of nitrogenase also produce small amounts of the greenhouse gas methane. This is different from other microbes that produce methane to generate energy. Until now, there was no good way to determine when microbes with nitrogenases are making methane in nature. Here, we present an isotopic fingerprint that allows scientists to distinguish methane from microbes making it for energy versus those making it as a by-product of nitrogen acquisition. With this new fingerprint, it will be possible to improve our understanding of the relationship between methane production and nitrogen acquisition in nature.
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Carbon substrate re‐orders relative growth of a bacterium using Mo‐, V‐, or Fe‐nitrogenase for nitrogen fixation
Biological nitrogen fixation is catalyzed by the molybdenum (Mo), vanadium (V) and iron (Fe)‐only nitrogenase metalloenzymes. Studies with purified enzymes have found that the ‘alternative’ V‐ and Fe‐nitrogenases generally reduce N2more slowly and produce more byproduct H2than the Mo‐nitrogenase, leading to an assumption that their usage results in slower growth. Here we show that, in the metabolically versatile photoheterotroph
- Award ID(s):
- 1631814
- NSF-PAR ID:
- 10457974
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Environmental Microbiology
- Volume:
- 22
- Issue:
- 4
- ISSN:
- 1462-2912
- Page Range / eLocation ID:
- p. 1397-1408
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Life on Earth depends on N2‐fixing microbes to make ammonia from atmospheric N2gas by the nitrogenase enzyme. Most nitrogenases use Mo as a cofactor; however, V and Fe are also possible. N2fixation was once believed to have evolved during the Archean‐Proterozoic times using Fe as a cofactor. However, δ15N values of paleo‐ocean sediments suggest Mo and V cofactors despite their low concentrations in the paleo‐oceans. This apparent paradox is based on an untested assumption that only soluble metals are bioavailable. In this study, laboratory experiments were performed to test the bioavailability of mineral‐associated trace metals to a model N2‐fixing bacterium
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Atomi, Haruyuki (Ed.)Methanogens and closely related methanotrophs are the only archaea known or predicted to possess nitrogenase. Methanogens play critical roles in both the global biological nitrogen and carbon cycles. Moreover, methanogens are an ancient microbial lineage and nitrogenase likely originated in methanogens. An understanding of the usage and properties of nitrogenases in methanogens can provide new insight into the evolution of nitrogen fixation and aid in the development nitrogenase-based biotechnology. This study provides the first evidence that a methanogen can produce all three forms of nitrogenases, including simultaneously. The results reveal components of Mo-nitrogenase regulate or are needed to produce V-nitrogenase and Fe-nitrogenase in methanogens, a result not seen in bacteria. Overall, this study provides a foundation to understand the assembly, regulation, and activity of the alternative nitrogenases in methanogens.more » « less
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Abstract The nitrogenase metalloenzyme family, essential for supplying fixed nitrogen to the biosphere, is one of life's key biogeochemical innovations. The three forms of nitrogenase differ in their metal dependence, each binding either a FeMo‐, FeV‐, or FeFe‐cofactor where the reduction of dinitrogen takes place. The history of nitrogenase metal dependence has been of particular interest due to the possible implication that ancient marine metal availabilities have significantly constrained nitrogenase evolution over geologic time. Here, we reconstructed the evolutionary history of nitrogenases, and combined phylogenetic reconstruction, ancestral sequence inference, and structural homology modeling to evaluate the potential metal dependence of ancient nitrogenases. We find that active‐site sequence features can reliably distinguish extant Mo‐nitrogenases from V‐ and Fe‐nitrogenases and that inferred ancestral sequences at the deepest nodes of the phylogeny suggest these ancient proteins most resemble modern Mo‐nitrogenases. Taxa representing early‐branching nitrogenase lineages lack one or more biosynthetic
nifE andnifN genes that both contribute to the assembly of the FeMo‐cofactor in studied organisms, suggesting that early Mo‐nitrogenases may have utilized an alternate and/or simplified pathway for cofactor biosynthesis. Our results underscore the profound impacts that protein‐level innovations likely had on shaping global biogeochemical cycles throughout the Precambrian, in contrast to organism‐level innovations that characterize the Phanerozoic Eon. -
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Recent spectroscopic, kinetic, photophysical, and thermodynamic measurements show activation of nitrogenase for N2→ 2NH3reduction involves the reductive elimination (
re ) of H2from two [Fe–H–Fe] bridging hydrides bound to the catalytic [7Fe–9S–Mo–C–homocitrate] FeMo-cofactor (FeMo-co). These studies rationalize the Lowe–Thorneley kinetic scheme’s proposal of mechanistically obligatory formation of one H2for each N2reduced. They also provide an overall framework for understanding the mechanism of nitrogen fixation by nitrogenase. However, they directly pose fundamental questions addressed computationally here. We here report an extensive computational investigation of the structure and energetics of possible nitrogenase intermediates using structural models for the active site with a broad range in complexity, while evaluating a diverse set of density functional theory flavors. (i ) This shows that to prevent spurious disruption of FeMo-co having accumulated 4[e −/H+] it is necessary to include: all residues (and water molecules) interacting directly with FeMo-co via specific H-bond interactions; nonspecific local electrostatic interactions; and steric confinement. (ii ) These calculations indicate an important role of sulfide hemilability in the overall conversion ofE 0to a diazene-level intermediate. (iii ) Perhaps most importantly, they explain (iiia ) how the enzyme mechanistically couples exothermic H2formation to endothermic cleavage of the N≡N triple bond in a nearly thermoneutralre /oxidative-addition equilibrium, (iiib ) while preventing the “futile” generation of two H2without N2reduction: hydridere generates an H2complex, but H2is only lost when displaced by N2, to form an end-on N2complex that proceeds to a diazene-level intermediate.
