Ecosystem scale evidence for the contribution of vanadium-based nitrogenase to biological nitrogen fixation.

<p>Nitrogen is the primary limiting nutrient in high latitude ecosystems. Biological nitrogen fixation (BNF) by microorganisms associated with cryptogamic covers, such as cyanolichens and bryophytes, is an important source of new reactive nitrogen in pristine, high-latitude ecosystems. BNF is catalyzed by the enzyme nitrogenase, for which three isoforms have been described; the canonical molybdenum (Mo) nitrogenase which requires Mo in its active site and two alternative nitrogenases, the vanadium and iron-only nitrogenases. The low availability of Mo on land has been shown to limit BNF in many ecosystems from the tropical forest to the arctic tundra. Alternative nitrogenases have been suggested as viable alternatives to cope with Mo limitation of BNF, however, field data supporting this long-standing hypothesis have been lacking.</p><p>Here, we elucidated the contribution of the vanadium nitrogenase to BNF by cyanolichens across a 600 km latitudinal transect in eastern Canadian boreal forests. We report a widespread activity of the vanadium nitrogenase which contributed between 15 to 50% of total BNF rates on all sites. Vanadium nitrogenase contribution to BNF was more robust in the northern part of the transect. Vanadium nitrogenase contribution to BNF also changed during the growing season, with a three-fold increase between the early (May) and late (September) growing season. By including the contribution of the vanadium nitrogenase to BNF, estimates of new N input by cyanolichens increase by up to 30%, a significant change in these low N input ecosystems. Finally, we found that Mo availability was the primary driver for the contribution of the vanadium nitrogenase to BNF with a Mo threshold of ~ 250 ng.g<sub>lichen</sub><sup>-1</sup> for the onset of vanadium based BNF.</p><p>This study on N<sub>2</sub>-fixing cyanolichens provides extensive field evidence, at an ecosystem scale, that vanadium-based nitrogenase greatly contributes to BNF when Mo availability is limited. The results showcase the resilience of BNF to micronutrient limitation and reveal a strong link between the biogeochemical cycle of macro- and micronutrients in terrestrial ecosystems. Given widespread findings of Mo limitation of BNF in terrestrial ecosystems, additional consideration of vanadium-based BNF is required in experimental and modeling studies of terrestrial biogeochemistry.</p>

Iron-Only and Vanadium Nitrogenases: Fail-Safe Enzymes or Something More?

The enzyme molybdenum nitrogenase converts atmospheric nitrogen gas to ammonia and is of critical importance for the cycling of nitrogen in the biosphere and for the sustainability of life. Alternative vanadium and iron-only nitrogenases that are homologous to molybdenum nitrogenases are also found in archaea and bacteria, but they have a different transition metal, either vanadium or iron, at their active sites. So far alternative nitrogenases have only been found in microbes that also have molybdenum nitrogenase. They are less widespread than molybdenum nitrogenase in bacteria and archaea, and they are less efficient. The presumption has been that alternative nitrogenases are fail-safe enzymes that are used in situations where molybdenum is limiting. Recent work indicates that vanadium nitrogenase may play a role in the global biological nitrogen cycle and iron-only nitrogenase may contribute products that shape microbial community interactions in nature.

Does the crystal structure of vanadium nitrogenase contain a reaction intermediate? Evidence from quantum refinement

Recently, a crystal structure of V-nitrogenase was presented, showing that one of the µ2 sulphide ions in the active site (S2B) is replaced by a lighter atom, suggested to be NH or NH2, i.e. representing a reaction intermediate. Moreover, a sulphur atom is found 7 Å from the S2B site, suggested to represent a storage site for this ion when it is displaced. We have re-evaluated this structure with quantum refinement, i.e. standard crystallographic refinement in which the empirical restraints (employed to ensure that the final structure makes chemical sense) are replaced by more accurate quantum–mechanical calculations. This allows us to test various interpretations of the structure, employing quantum–mechanical calculations to predict the ideal structure and to use crystallographic measures like the real-space Z-score and electron-density difference maps to decide which structure fits the crystallographic raw data best. We show that the structure contains an OH−-bound state, rather than an N2-derived reaction intermediate. Moreover, the structure shows dual conformations in the active site with ~ 14% undissociated S2B ligand, but the storage site seems to be fully occupied, weakening the suggestion that it represents a storage site for the dissociated ligand. Graphic abstract

Ecosystem scale evidence for the contribution of vanadium-based nitrogenase to biological nitrogen fixation

<p>Nitrogen is the primary limiting nutrient in high latitude ecosystems. Biological nitrogen fixation (BNF) by microorganisms associated with cryptogamic covers, such as cyanolichens and bryophytes, is an important source of new reactive nitrogen in pristine, high-latitude ecosystems. BNF is catalyzed by the enzyme nitrogenase, for which three isoforms have been described; the canonical molybdenum (Mo) nitrogenase which requires Mo in its active site and two alternative nitrogenases, the vanadium and iron-only nitrogenases. The low availability of Mo on land has been shown to limit BNF in many ecosystems from the tropical forest to the arctic tundra. Alternative nitrogenases have been suggested as viable alternatives to cope with Mo limitation of BNF, however, field data supporting this long-standing hypothesis have been lacking.</p><p>Here, we elucidated the contribution of the vanadium nitrogenase to BNF by cyanolichens across a 600 km latitudinal transect in eastern Canadian boreal forests. We report a widespread activity of the vanadium nitrogenase which contributed between 15 to 50% of total BNF rates on all sites. Vanadium nitrogenase contribution to BNF was more robust in the northern part of the transect. Vanadium nitrogenase contribution to BNF also changed during the growing season, with a three-fold increase between the early (May) and late (September) growing season. By including the contribution of the vanadium nitrogenase to BNF, estimates of new N input by cyanolichens increase by up to 30%, a significant change in these low N input ecosystems. Finally, we found that Mo availability was the primary driver for the contribution of the vanadium nitrogenase to BNF with a Mo threshold of ~ 250 ng.g<sub>lichen</sub><sup>-1</sup> for the onset of vanadium based BNF.</p><p>This study on N<sub>2</sub>-fixing cyanolichens provides extensive field evidence, at an ecosystem scale, that vanadium-based nitrogenase greatly contributes to BNF when Mo availability is limited. The results showcase the resilience of BNF to micronutrient limitation and reveal a strong link between the biogeochemical cycle of macro- and micronutrients in terrestrial ecosystems. Given widespread findings of Mo limitation of BNF in terrestrial ecosystems, additional consideration of vanadium-based BNF is required in experimental and modeling studies of terrestrial biogeochemistry.</p>

Two-Stage Continuous Conversion of Carbon Monoxide to Ethylene by Whole Cells of Azotobacter vinelandii

ABSTRACT Azotobacter vinelandii is an obligate aerobic diazotroph with a verified transient ability to reduce carbon monoxide to ethylene by its vanadium nitrogenase. In this study, we implemented an industrially relevant continuous two-stage stirred-tank system for in vivo biotransformation of a controlled supply of air enriched with 5% carbon monoxide to 302 μg ethylene g−1 glucose consumed. To attain this value, the process required overcoming critical oxygen limitations during cell proliferation while simultaneously avoiding the A. vinelandii respiratory protection mechanism that negatively impacts in vivo nitrogenase activity. Additionally, process conditions allowed the demonstration of carbon monoxide’s solubility as a reaction-limiting factor and a competitor with dinitrogen for the vanadium nitrogenase active site, implying that excess intracellular carbon monoxide could lead to a cessation of cell proliferation and ethylene formation as shown genetically using a new strain of A. vinelandii deficient in carbon monoxide dehydrogenase. IMPORTANCE Ethylene is an essential commodity feedstock used for the generation of a variety of consumer products, but its generation demands energy-intensive processes and is dependent on nonrenewable substrates. This work describes a continuous biological method for investigating the nitrogenase-mediated carbon monoxide reductive coupling involved in ethylene production using whole cells of Azotobacter vinelandii. If eventually adopted by industry, this technology has the potential to significantly reduce the total energy input required and the ethylene recovery costs, as well as decreasing greenhouse gas emissions associated with current production strategies.