The path of electron transfer to nitrogenase in a phototrophic alpha‐proteobacterium

Fixen, Kathryn R.; Chowdhury, Nilanjan Pal; Martinez‐Perez, Marta; Poudel, Saroj; Boyd, Eric S.; Harwood, Caroline S.

doi:10.1111/1462-2920.14262

Cited by 27 publications

(30 citation statements)

References 44 publications

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“…During growth on succinate, our data are compatible with the classical view that excess H 2 production by the V‐Nase strain leads to slower growth compared with the Mo‐Nase utilizing strains. For example, it has a lower biomass N:C ratio despite a higher abundance of the primary electron donor to Nase, the ferredoxin protein Fer1 (log2 abundance difference of 0.53 ± 2.56 relative to the wild type strain, p < 0.01; Fixen et al ., ). These data indicate that, on succinate, nitrogen acquisition is more limiting to growth than electron balance.…”

Section: Resultsmentioning

confidence: 97%

Carbon substrate re‐orders relative growth of a bacterium using Mo‐, V‐, or Fe‐nitrogenase for nitrogen fixation

Luxem

Kraepiel

Zhang

et al. 2020

Environmental Microbiology

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Summary 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 N2 more slowly and produce more byproduct H2 than the Mo‐nitrogenase, leading to an assumption that their usage results in slower growth. Here we show that, in the metabolically versatile photoheterotroph Rhodopseudomonas palustris, the type of carbon substrate influences the relative rates of diazotrophic growth based on different nitrogenase isoforms. The V‐nitrogenase supports growth as fast as the Mo‐nitrogenase on acetate but not on the more oxidized substrate succinate. Our data suggest that this is due to insufficient electron flux to the V‐nitrogenase isoform on succinate compared with acetate. Despite slightly faster growth based on the V‐nitrogenase on acetate, the wild‐type strain uses exclusively the Mo‐nitrogenase on both carbon substrates. Notably, the differences in H2:N2 stoichiometry by alternative nitrogenases (~1.5 for V‐nitrogenase, ~4–7 for Fe‐nitrogenase) and Mo‐nitrogenase (~1) measured here are lower than prior in vitro estimates. These results indicate that the metabolic costs of V‐based nitrogen fixation could be less significant for growth than previously assumed, helping explain why alternative nitrogenase genes persist in diverse diazotroph lineages and are broadly distributed in the environment.

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Section: Resultsmentioning

confidence: 97%

Carbon substrate re‐orders relative growth of a bacterium using Mo‐, V‐, or Fe‐nitrogenase for nitrogen fixation

Luxem

Kraepiel

Zhang

et al. 2020

Environmental Microbiology

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“…1). The NADH generated can be used by R. palustris cells to reduce flavodoxin or ferredoxin, the direct electron donor of nitrogenase via the FixABCX enzyme complex (22,23). Therefore, organic acid-alcohol pairs are good candidate substrates to study how electron availability affects CH 4 and H 2 production.…”

Section: Discussionmentioning

confidence: 99%

Influence of Energy and Electron Availability on In Vivo Methane and Hydrogen Production by a Variant Molybdenum Nitrogenase

Zheng

Harwood

2019

Appl Environ Microbiol

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The anoxygenic phototrophic bacterium Rhodopseudomonas palustris produces methane (CH4) from carbon dioxide (CO2) and hydrogen (H2) from protons (H+) when it expresses a variant form of molybdenum (Mo) nitrogenase that has two amino acid substitutions near its active site. We examined the influence of light energy and electron availability on in vivo production of these biofuels. Nitrogenase activity requires large amounts of ATP, and cells exposed to increasing light intensities produced increasing amounts of CH4 and H2. As expected for a phototroph, intracellular ATP increased with increasing light intensity, but there was only a loose correlation between ATP content and CH4 and H2 production. There was a much stronger correlation between decreased intracellular ADP and increased gas production with increased light intensity, suggesting that the rate-limiting step for CH4 and H2 production by R. palustris is inhibition of nitrogenase by ADP. Increasing the amounts of electrons available to nitrogenase by providing cells with organic alcohols, using nongrowing cells, blocking electrons from entering the Calvin cycle, or blocking H2 uptake resulted in higher yields of H2 and, in some cases, CH4. Our results provide a more complete understanding of the constraints on nitrogenase-based production of biofuels. IMPORTANCE A variant form of Mo nitrogenase catalyzes the conversion of CO2 and protons to the biofuels CH4 and H2. A constant supply of electrons and ATP is needed to drive these reduction reactions. The bacterium R. palustris generates ATP from light and has a versatile metabolism that makes it ideal for manipulating electron availability intracellularly. We therefore explored its potential as a biocatalyst for CH4 and H2 production. We found that intracellular ADP had a major effect on biofuel production, more pronounced than the effect caused by ATP. This is probably due to inhibition of nitrogenase activity by ADP. In general, the amount of CH4 produced by the variant nitrogenase in vivo was affected by electron availability much less than was the amount of H2 produced. This study shows the nature of constraints on in vivo biofuel production by variant Mo nitrogenase.

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“…This may point to the use of Flds as an adaptive strategy to fix N 2 in oxic environments. Moreover, under iron-deficient conditions that characterize most circumneutral oxic environments, diazotrophs tend to synthesize Flds preferentially as primary electron donors to Nif [92]. Studies have also shown that electron delivery by Fd or Fld can be complemented by other Fds or Flds that are encoded in the genomes of diazotrophs [70], [93], [94], [73], [95].…”

Section: Sources Of Reducing Equivalents During Aerobic and Anaerobicmentioning

confidence: 99%

Geobiological feedbacks, oxygen, and the evolution of nitrogenase

Mus¹,

Colman²,

Peters³

et al. 2019

Free Radical Biology and Medicine

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Biological nitrogen fixation via the activity of nitrogenase is arguably one of the most important biological innovations, allowing for an increase in global productivity that eventually permitted the emergence of complex forms of life. The complex metalloenzyme termed nitrogenase contains complex iron-sulfur cofactors. Three versions of nitrogenase exist that differ mainly by the presence or absence of a heterometal at the active site metal cluster (either Mo or V). Mo-dependent nitrogenase is the most common while V-dependent or heterometal independent (Fe-only) are often termed alternative nitrogenases since they have lower activities and are expressed in the absence of Mo. Phylogenetic evidence indicates that biological nitrogen fixation emerged in an anaerobic, thermophilic ancestor of hydrogenotrophic methanogens and later diversified via lateral gene transfer into anaerobic bacteria, and eventually aerobic bacteria. Isotopic evidence indicates that nitrogenase activity existed at 3.2 Ga prior to the advent of oxygenic photosynthesis and rise of oxygen in the atmosphere, implying the presence of favorable environmental conditions for oxygen-sensitive nitrogenase to evolve. Following the proliferation of oxygenic phototrophs, diazotrophic organisms had to develop strategies to protect nitrogenase from oxygen inactivation and generate the right balance of low potential reducing equivalents and cellular energy for growth and nitrogen fixation activity. Here we review the fundamental advances in our understanding of biological nitrogen fixation in the context of the emergence, evolution, and distribution of nitrogenase, with an emphasis placed on key events associated with its emergence and diversification from anoxic into oxic environmental conditions.

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The path of electron transfer to nitrogenase in a phototrophic alpha‐proteobacterium

Cited by 27 publications

References 44 publications

Carbon substrate re‐orders relative growth of a bacterium using Mo‐, V‐, or Fe‐nitrogenase for nitrogen fixation

Carbon substrate re‐orders relative growth of a bacterium using Mo‐, V‐, or Fe‐nitrogenase for nitrogen fixation

Influence of Energy and Electron Availability on In Vivo Methane and Hydrogen Production by a Variant Molybdenum Nitrogenase

Geobiological feedbacks, oxygen, and the evolution of nitrogenase

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