Progress 01/01/06 to 06/30/10
Outputs
OUTPUTS: OUTPUTS: Our main avenue of information dissemination, in addition to scientific publications, is through organizing and running international congresses most often in overseas locales. In 2009, even though held in Montana, USA, participants gathered from all over the world to discuss and critique our efforts and those of others to assist subsistence-level farmers in overcoming poverty by exploiting the benefits and low costs associated with biological nitrogen fixation (BNF) for increasing agriculture productivity without negatively impacting the environment. Crop yield depends on many factors, but the availability of fertilizer-nitrogen is a primary concern. BNF is nature's way of making the enormous reservoir of nitrogen gas in our atmosphere directly usable by the biosphere. Through associations with nitrogen-fixing microorganisms, many plants (and consequently animals) acquire significant amounts of the required fixed nitrogen from BNF naturally, highlighting why BNF is a key metabolic process for food production and the maintenance of life on Earth. Although we emphasize the benefits gained from using nitrogen-fixing plants in local small holdings as well as the appropriate use of soil, water and indigenous soil microbes, we recognize that sustainable agriculture also requires the use of plant cultivars that respond under environmental constraints. We are already organizing future congresses in Australia (2011) and Japan (2013). PARTICIPANTS: PARTICIPANTS: Newton, W.E. PI. Designed experiments, analyzed data, and wrote reports. Dapper, C. Lab. Tech. Conducted experiments and analyzed data. Bothe, H. Collaborator. Designed experiments, analyzed data, and wrote reports. Schmitz, O. Collaborator. Conducted experiments and analyzed data. Yates, M.G. Collaborator. Analyzed data and wrote reports. Cramer, S.P. Collaborator. Provided instrumentation, designed experiments, analyzed data, and wrote reports. George, S. Collaborator. Modified instrumentation and experiments, and analyze data. Yan, L. Collaborator. Conducted experiments and analyzed data. Wang, H. Collaborator. Modified instrumentation, conducted experiments, and analyzed data. TARGET AUDIENCES: TARGET AUDIENCES: Target audiences range from: nitrogen-fertilizer manufacturers through inoculant producers to subsistence-level farmers and cooperatives. In addition, because nitrogen-fixation research is multidisciplinary and the potential for exploiting its benefits for agriculture and environmental protection is great, it attracts the interest of a wide range of scientists, from biochemists through plant physiologists and ecologists to agricultural scientists and extension agents. These are the target audiences for this project. PROJECT MODIFICATIONS: PROJECT MODIFICATIONS: No significant modification to report during this period.
Impacts
OUTCOMES/IMPACT: 2008/10 TO 2009/09 Fundamental scientific understanding is sought so that the process of biological nitrogen fixation may be spread to crops that presently cannot perform it. Because this process depends on only readily renewable resources (sunlight, atmospheric nitrogen gas, and water), it should become a significant component of sustainable agriculture. Currently, fixed nitrogen, as usually provided through urea, ammonia, or nitrate in commercial fertilizers, is not only the most-often limiting factor, it is also certainly the most-energy intensive component in US agriculture. In Virginia alone, annual total fertilizer costs (more than $100 million) account for about 50 percent of farm expenses to produce food and feed with nitrogen fertilizers accounting for about two-thirds of these costs. Thus, alternatives to commercial nitrogen fertilizers could produce considerable financial as well as energy savings, while also decreasing the negative environmental impacts, such as ground and surface water contamination, of fertilizer use. We have focused on how the natural catalyst for making ammonia, a microbial enzyme called nitrogenase, works. Real prospects exist for improving microbial nitrogen fixation, but lack of some fundamental knowledge of how nitrogenase works still limits our efforts. We are attempting to find out how the nitrogen molecule (N2) and related molecules, like carbon monoxide (CO), interact with the enzyme. Recent progress suggests likely locations for their interactions and our on-going research is to describe these sites in detail. Although the detailed mechanism of N2 reduction is now closer to being solved, many details remain to be elucidated. We are certain that our efforts will generate targets for how this enzyme might be manipulated for future agricultural benefit.
Publications
- PUBLICATIONS (not previously reported): Delfino, I., Cerullo, G., Cannistraro, S., Manzoni, C., Polli, D., Dapper, C., Newton, W.E., Guo, Y., and Cramer, S.P. (2010). Observation of terahertz vibrations in the nitrogenase FeMo cofactor by femtosecond pump-probe spectroscopy. Angew. Chem. Int. Ed., 49: 1-5. DOI: 10.1002/anie.200906787. Bothe, H., Schmitz, O., Yates, M.G., and Newton, W.E. (2010). Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria. Microbiol. Mol. Biol. Rev., in press.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: As reported last time, I have continued to be involved in organizing and running international congresses most often in overseas locales. In 2007, it was held in Cape Town (RSA). This year, although the congress was held in Montana, USA, it attracted participants worldwide in our efforts to assist subsistence farmers overcome poverty by exploiting the benefits and low costs of biological nitrogen fixation (BNF) for increasing agriculture productivity and environmental protection. Crop yield depends on many factors, but the availability of fixed-nitrogen, either mineral or organic, is a primary concern. BNF is nature's way of making the enormous reservoir of nitrogen (N2) gas in our atmosphere directly usable by the biosphere. Through associations with nitrogen-fixing microorganisms, many plants (and consequently animals) acquire significant amounts of fixed nitrogen from BNF, so highlighting why BNF is a key metabolic process for food production and the maintenance of life on Earth. Although we emphasize the benefits gained from using nitrogen-fixing plants in local small holdings as well as the appropriate use of soil, water and indigenous soil microbes, we recognize that sustainable agriculture also requires the use of plant cultivars that respond under environmental constraints. We are already organizing subsequent congresses in Australia (2011) and Japan (2013). PARTICIPANTS: Newton, W.E. PI. Designed experiments, analyzed data, and wrote reports. Dapper, C. Lab. Tech. Conducted experiments and analyzed data. Bothe, H. Collaborator. Designed experiments, analyzed data, and wrote reports. Cramer, S.P. Collaborator. Provided instrumentation, designed experiments, analyzed data, and wrote reports. George, S. Collaborator. Modified instrumentation and experiments, and helped analyze data. Yan, L. Collaborator. Conducted experiments and analyzed data. Wang, H. Collaborator. Modified instrumentation, conducted experiments, and analyze data. TARGET AUDIENCES: Target audiences range from: nitrogen-fertilizer manufacturers through inoculant producers to subsistence-level farmers. In addition, because nitrogen-fixation research is multidisciplinary and the potential for exploiting its benefits for agriculture and environmental protection is great, it attracts the interest of a wide range of scientists, from biochemists through plant physiologists and ecologists to agricultural scientists and extension agents. These are the target audiences for this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Fundamental scientific understanding is sought so that the process of biological nitrogen fixation may be spread to crops that presently cannot perform it. Because this process depends on only readily renewable resources (sunlight, nitrogen gas, and water), it should become a significant component of sustainable agriculture. Currently, fixed nitrogen, as usually provided through urea, ammonia, or nitrate in commercial fertilizers, is most often the limiting factor and certainly the most-energy intensive component in US agriculture. In Virginia alone, annual total fertilizer costs (more than $100 million) account for about 50 percent of farm expenses to produce food and feed with nitrogen fertilizers accounting for about two-thirds of these costs. Thus, alternatives to commercial nitrogen fertilizers could produce considerable financial as well as energy savings, while also decreasing the negative environmental impacts, such as ground and surface water contamination, of fertilizer use. We have focussed on how the natural catalyst, a microbial enzyme called nitrogenase, for making ammonia works. Real prospects exist for improving microbial nitrogen fixation, but improvements are dependent upon acquiring the fundamental knowledge of how nitrogenase works. We are attempting to find out how the N2 and related molecules, like carbon monoxide (CO), interact with the enzyme. Recent studies have suggested locations for these interactions and on-going efforts target these sites in detail. Although the detailed mechanism of N2 reduction is close to being solved, many details remain. We are certain that our efforts will generate targets for how this enzyme might be manipulated for future agricultural benefit.
Publications
- Newton, W.E. (2009). Ammonia synthesis (nitrogen fixation) by biological catalysts, in Encylopedia of Catalysis, John Wiley & Sons, New York, NY, (in press).
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: In addition to the usual on-going dissemination of information through professional journals, we continue to look for other ways to make our findings public so that others can build on our findings and we on theirs. To this end, I have been deeply involved in organizing and running international congresses overseas, for example, in Cape Town (RSA) in January, 2007. Poverty is a severe problem in Africa, Asia, South America and even in pockets of the developed world. Many aspects of these congresses are designed in attempts to help subsistence farmers overcome poverty by increasing the use of biological nitrogen fixation (BNF) in agriculture. Exploiting the benefits and low costs of BNF for agriculture and environmental protection attracts interest from a wide range of people, including chemists, biochemists, plant physiologists, evolutionary biologists, ecologists, agricultural scientists, extension agents, inoculant producers and policy makers from all over the world. Crop yields depend on many factors, but a primary consideration is the availability of fixed-nitrogen, either mineral or organic. Biological nitrogen fixation (BNF) is Nature's way of making the enormous reservoir of nitrogen (N2) gas in our atmosphere directly usable by the biosphere. Then through associations with nitrogen-fixing microorganisms, plants (and consequently animals) can derive a significant proportion of their fixed-nitrogen requirement for growth from BNF. These interactions clearly illustrate why BNF is a key metabolic process for food production and the maintenance of life on Earth. We are deep iinto torganization for the next congrees (in 2009) and are planning subsequent congresses in Australia and Japan for 2011 an 2013, respectively. We try to indicate the benefits from introducting nitrogen-fixing legumes into local small holdings and the appropriate use of soil, water and indigenous soil microbes to provide N and P. However, sustainable agriculture also requires the use of plant cultivars that respond to environmental constraints; these are being considered (by others) through both classical plant-breeding technology and the modern-day engineering of high-performance crops and symbiotic associations. PARTICIPANTS: Newton, W.E. PI. Designed experiments, analyzed data, and wrote reports. Dapper, C. Lab. Tech. Conducted experiments and analyzed data. Fisher, K. Collaborator. Conducted experiments and analyzed data. Cramer, S.P. Collaborator at UC-Davis. Provided instrumentation, designed experiments and helped analyze data. TARGET AUDIENCES: Target audiences range from nitrogen-fertilizer manufacturers through inoculant producers to subsistence-level farmers. In addition, because nitrogen-fixation research is multidisciplinary and the potential for exploiting its benefits for agriculture and environmental protection is great, it attracts the interest of a wide range of scientists, from biochemists through plant physiologists and ecologists to agricultural scientists and extension agents. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This project seeks the fundamental scientific understanding that will allow a general enhancement of the biological nitrogen-fixation process and its spread to crops that presently cannot perform it. This biological approach is dependent on renewable resources (sunlight, nitrogen gas, and water) only and could become a significant component of sustainable agriculture. Currently, fixed nitrogen, as occurs as urea, ammonia, or nitrate in commercial fertilizers, is most often the limiting factor and certainly the most-energy intensive component in US agriculture. In Virginia, annual total fertilizer costs are about $100 million and account for about 50 percent of farm expenses to produce food and feed with fixed-nitrogen fertilizers accounting for about two-thirds of these costs. Thus, alternatives to commercial nitrogen fertilizers could produce considerable financial and energy savings, while also decreasing the negative environmental impacts, such as ground and surface water contamination, of fertilizer use. This project focuses on the workings of the natural microbial catalyst for making ammonia, an enzyme called nitrogenase. The agricultural significance of the microbes, which deliver fixed-nitrogen to certain crops, is well established. Real prospects exist for improving microbial nitrogen fixation, but improvements are dependent upon acquiring a fundamental understanding of how nitrogenase works. This project attempts to understand how the components that make up nitrogenase interact during nitrogen fixation and how their function is controlled by their environment. Recent approaches have concentrated on what happens in the first 1 second after the reaction starts; how are the components poised How is the reaction initiated We found that the various components can respond differently depending on whether or not sufficient energy is available to reduce nitrogen. Although the detailed mechanism of N2 reduction remains unsolved, these findings have generated targets for continued investigation and have indicated how this enzyme might be manipulated for potential agricultural benefit in the future.
Publications
- Newton, W.E., Fisher, K., Huynh, B.H., Edmondson, D.E., Tavares, P., Pereira, A.S., and Lowe, D.J. (2007). Azotobacter vinelandii nitrogenase MoFe protein: Pre-steady state spectroscopic studies of the metal cofactors, In F.D. Dakora, S.B.M. Chimpango, A.J. Valentine, C. Elmerich, and W.E. Newton (eds.), Biological nitrogen fixation: Towards poverty alleviation through sustainable agriculture. Springer, Dordrecht, The Netherlands, pp. 331-333.
- Dilworth, M.J., James, E.K., Sprent, J.I., and Newton, W.E. (2008). Nitrogen-fixing leguminous associations. Springer, Dordrecht, The Netherlands, 402 pp.
- Dakora, F.D., Chimpango, S.B.M., Valentine, A.J., Elmerich, C., and Newton, W.E. (2008). Biological nitrogen fixation: Towards poverty alleviation through sustainable agriculture. Springer, Dordrecht, The Netherlands, 392 pp.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: As we continue to gain new and deeper insights into the fundamentals of the catalytic mechanism of the unique microbial enzyme, called nitrogenase, on which life as we know it depends, we ensure that all of our findings are made public through professional journals. In this way, others can build on our findings and we on theirs. In addition, I was deeply involved in organizing an international congress in Cape Town (RSA) in January, 2007. Poverty is a severe problem in Africa, Asia, South America and even in pockets of the developed world. The congress addressed poverty alleviation via the expanded use of biological nitrogen fixation (BNF) in agriculture. Because nitrogen-fixation research is multidisciplinary, exploiting its benefits for agriculture and environmental protection has continued to attract research by diverse groups of scientists, including chemists, biochemists, plant physiologists, evolutionary biologists, ecologists, agricultural scientists, extension
agents, and inoculant producers from all parts of the world. Crop yields depend on many factors, but a primary consideration is the availability of fixed-nitrogen, either mineral or organic. Biological nitrogen fixation (BNF) is Nature's way of making the enormous reservoir of nitrogen (N2) gas in our atmosphere directly usable by the biosphere. Then through associations with nitrogen-fixing microorganisms, plants (and consequently animals) can derive a significant proportion of their fixed-nitrogen requirement for growth from BNF. These interactions clearly illustrate why BNF is a key metabolic process for food production and the maintenance of life on Earth. The Congress theme was the application of BNF to sustainable agriculture, poverty alleviation, and environmental concerns, but also included the effects of stresses, bioremediation, and forestry. Highlights included how to benefit from: the introduction of nitrogen-fixing legumes into local small holdings; the appropriate use of
both soil and water; the use of indigenous soil microbes to provide N and P; and from good agricultural practices generally. However, sustainable agriculture not only depends on appropriate agricultural practices but, to maintain high yields, it requires the use of plant cultivars that respond to environmental constraints. Now, access to, and use of, the incredible insights acquired through plant and bacterial genomics also drives both classical plant-breeding technology and the modern-day engineering of high-performance crops and symbiotic associations. Indeed, refined gene-sequence maps of some simpler legumes have led to exciting genomic work in crop legumes. The lively discussions that followed the formal poster sessions are clear indicators of the interest in and potential of this area.
PARTICIPANTS: Newton, W.E. PI. Designed experiments, analyzed data, and wrote reports. Dapper, C. Lab. Tech. Conducted experiments and analyzed data. Fisher, K. Collaborator. Conducted experiments and analyzed data. Lowe, D.J. Collaborator. Provided instrumentation, modified experiments and helped analyze data. Huynh, B.H. Collaborator. Provided instrumentation, designed experiments and helped analyze data. Edmondson, D.E. Collaborator. Provided instrumentation and collected data. Tavares, P. Collaborator. Conducted experiments and analyzed data. Pereira, A.S. Collaborator. Collected and analyzed data. Cramer, S.P. Collaborator. Provided instrumentation, designed experiments and helped analyze data.
TARGET AUDIENCES: Target audiences range from: nitrogen-fertilizer manufacturers through inoculant producers to subsistence-level farmers. In addition, because nitrogen-fixation research is multidisciplinary and the potential for exploiting its benefits for agriculture and environmental protection is great, it attracts the interest of a wide range of scientists, from biochemists through plant physiologists and ecologists to agricultural scientists and extension agents. these are the target audiences for this project.
Impacts
This project seeks the fundamental scientific understanding that will allow a general enhancement of the biological nitrogen-fixation process and its spread to crops that presently cannot perform it. This biological approach is dependent on renewable resources (sunlight, nitrogen gas, and water) only and could become a significant component of sustainable agriculture. Currently, fixed nitrogen, as occurs as urea, ammonia, or nitrate in commercial fertilizers, is most often the limiting factor and certainly the most-energy intensive component in US agriculture. In Virginia, annual total fertilizer costs are about $100 million and account for about 50 percent of farm expenses to produce food and feed with fixed-nitrogen fertilizers accounting for about two-thirds of these costs. Thus, alternatives to commercial nitrogen fertilizers could produce considerable financial and energy savings, while also decreasing the negative environmental impacts, such as ground and surface
water contamination, of fertilizer use. This project focuses on the workings of the natural microbial catalyst for making ammonia, an enzyme called nitrogenase. The agricultural significance of the microbes, which deliver fixed-nitrogen to certain crops, is well established. Real prospects exist for improving microbial nitrogen fixation, but improvements are dependent upon acquiring a fundamental understanding of how nitrogenase works. This project attempts to understand how the components that make up nitrogenase interact during nitrogen fixation and how their function is controlled by their environment. Recent approaches have concentrated on what happens in the first 1 second after the reaction starts; how are the components poised? How is the reaction initiated? We found that the various components can respond differently depending on whether or not sufficient energy is available to reduce nitrogen. Although the detailed mechanism of N2 reduction reamins unsolved, these findings
have generated targets for continued investigation and have indicated how this enzyme might be manipulated for potential agricultural benefit in the future.
Publications
- Fisher, K., Lowe, D.J., Tavares, P., Pereira, A.S., Huynh, B.H., Edmondson, D.E.,and Newton, W.E. (2007). Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function. J. Inorg. Biochem. 101: 1649-1656.
- Newton, W.E., Fisher, K., Huynh, B.H., Edmondson, D.E., Tavares, P., Pereira, A.S., and Lowe, D.J. (2007). Azotobacter vinelandii nitrogenase MoFe protein: Pre-steady state spectroscopic studies of the metal cofactors, In F.D. Dakora, S.B.M. Chimpango, A.J. Valentine, C. Elmerich, and W.E. Newton (eds.), Biological nitrogen fixation: Towards poverty alleviation through sustainable agriculture. Springer, Dordrecht, The Netherlands, (in press).
- Pawlowski, K., and Newton, W.E. (2007). Nitrogen-fixing actinorhizal associations. Springer, Dordrecht, The Netherlands, 310 pp.
- Bothe, H., Ferguson, S.J., and Newton, W.E. (2007). Bology of the nitrogen cycle. Elsevier, Amsterdam, The Netherlands, 427 pp.
- Dakora,F.D., Chimpango, S.B.M., Valentine, A.J., Elmerich, C., and Newton, W.E. (2007). Biological nitrogen fixation: Towards poverty alleviation through sustainable agriculture. Springer, Dordrecht, The Netherlands, (in press).
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Progress 01/01/06 to 09/30/06
Outputs
Fixed nitrogen is essential for life. However, nitrogen fixation is performed only by certain microbes containing the enzyme nitrogenase. The agricultural significance of symbiotic microbes that deliver fixed-nitrogen to their hosts is well established. Real prospects exist that the economy of nitrogen fixation can be improved through the genetic manipulation of nitrogen-fixing species. Such improvements are dependent upon acquiring a fundamental understanding of the biochemical action and genetic regulation of the nitrogen-fixing enzymes. This project attempts to understand how nitrogenase is organized to catalyze the reduction of N2. In particular, it focuses on the role in this process of the MoFe protein, the larger of the two component proteins that constitute nitrogenase, and specifically on the functions of both of its biologically unique prosthetic groups, the FeMo-cofactor and the P cluster. Understanding their chemical reactivity is fundamental to
understanding how N2 is fixed biologically and how nitrogen fixation might be either utilized or manipulated more effectively in agricultural systems. Our efforts are aimed at determining how the FeMo-cofactor, the acknowledged site of N2 binding, and the P cluster operate and interact during nitrogen fixation and how their function is controlled by their polypeptide environment. One approach (freeze-quench electron paramagnetic resonance; EPR) has shown that a transient signal (1b) appears during wild-type Azotobacter vinelandii nitrogenase turnover at room temperature and at pH7.4. Signal 1b forms at the expense of the FeMo-cofactor-based signal (1a) and is independent of the substrate (N2, C2H2, H+) being reduced. Computer simulations of the kinetics of its formation indicate that it arises from a three-electron reduced state (E3) of the MoFe protein. The variant alpha-H195Q and alpha-H195N MoFe proteins develop signal 1b quicker than wild type, but the alpha-Q191K variant exhibits
no signal 1b. All three MoFe variants reduce H+ and C2H2, but only alpha-Q191K cannot bind N2. Thus, the observation of signal 1b for alpha-H195Q and alpha-H195N correlate with N2 being bound the E3 redox level. Involvement of the P-cluster in enzyme turnover arises from related stopped-flow spectrophotometry, which indicates that P-cluster oxidation occurs within the time course of the freeze-quench EPR observations with wild type, alpha-H195Q and alpha-H195N, but not with alpha-Q191K. A related effort, using a new technique (nuclear resonance vibrational spectroscopy; NRVS), has supplied data that is consistent with the presence of a small ion/atom in the center of the FeMo-cofactor. Current work is designed to determine whether it is C, N or O by growing the bacteria on isotopically labeled media. A third approach attempts to pinpoint the exact N2-binding site on the FeMo-cofactor, with either the molybdenum (Mo) atom or a central iron (Fe) atom being the likley key player.
Substitutions at an amino acid residue (alpha-lysine-426) near the Mo atom selectively perturbs the N2-reduction process without affecting the C2H2-reduction process, indicating the involvement of Mo in N2 reduction.
Impacts
Fixed nitrogen, as occurs in commercial fertilizers, is relatively scarce and is most often the limitng factor and most-energy intensive component in US agriculture. In Virginia, annual total fertilizer costs are about $100 million and account for 50 percent of farm expenses to produce feed and 12 percent of total farm expenses. Nitrogen fertilizers account for about two-thirds of these costs. Thus, alternatives to commercial nitrogen fertilizers could produce considerable savings, while also decreasing the negative environmental impacts, such as ground and surface water contamination, of fertilizer use. This project seeks the scientific understanding that will allow enhancement of the biological nitrogen-fixation process generally and its dispersal to crops that presently cannot perform it. This biological approach is dependent on renewable resources (sunlight, nitrogen gas, and water) only and could become a significant component of sustainable agriculture.
Publications
- Elmerich, C., and Newton, W.E. 2006. Associative and Endophytic Nitrogen-fixing Bacteria and Cyanobacterial Associations. Springer, Dordrecht, The Netherlands, 321pp.
- Newton, W.E. 2006. Biological Nitrogen Fixation and Nitrification, in Biological Inorganic Chemistry: Structure and Reactivity (I. Bertini, H.B. Gray, E.I. Stiefel, and J.S. Valentine, Eds.), University Science Books, Sausalito, CA, pp. 468-493.
- Xiao, Y., Fisher, K., Smith, M.C., Newton, W.E., Case, D.A., George, S.J., Wong, H., Sturhahn, W., Alp, E.E., Zhao, J., Yoda, Y., and Cramer, S.P. 2006. How Nitrogenase Shakes: Initial Information about P-clusters and FeMo-cofactor Normal Modes from Nuclear Resonance Vibrational Spectroscopy (NRVS), J. Am. Chem. Soc., 128: 7608-7612.
- Fisher, K., Dilworth, M.J., and Newton, W.E. 2006. Azotobacter vinelandii Vanadium Nitrogenase: Formaldehyde is a Product of Catalyzed HCN Reduction and Excess Ammonia arises Directly from Catalyzed Azide Reduction, Biochemistry, 45: 4190-4198.
- Durrant, M.C., Francis, A., Lowe, D.J., Newton, W.E., and Fisher, K. 2006. Evidence for a Dynamic Role for Homocitrate during Nitrogen Fixation: The Effect of Substitution at the alpha-Lys-426 Position in the MoFe-Protein of Azotobacter vinelandii, Biochem. J., 397: 261-270.
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