Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: The production of nitrogen fertilizer through the chemical Haber-Bosch process requires a very high input energy (temperature & pressure) and therefore necessitates a high consumption of fossil fuels. The chemical production of nitrogen fertilizers also presents significant economic and social costs related to transportation, security, and ecological pollution. In contrast to the chemical approach for producing nitrogen fertilizer nitrogen-fixing organisms can perform the same process but at ambient temperature and pressure. Efforts to lower the energy input by more closely duplicating the biological process, or through direct manipulation of the biological process at an industrial scale, could result in a less hazardous route for delivery that is cost effective with fewer attendant ecological consequences. Achieving this goal can only be accomplished through full understanding of the chemical and regulatory mechanisms associated with the biological process of nitrogen fixation. Our effort in this area is to understand how an inert molecule, such as dinitrogen, can be activated in a living organism such that the strong triple bond can be cleaved at ambient temperature and pressure. Towards this end we have made significant progress in understanding the coordination chemistry that is involved with the binding of dinitrogen to the active site of the biological catalyst for nitrogen fixation. Our strategy is to develop a model that describes each step that occurs in biological nitrogen fixation so that the sequential steps involved in the delivery of electrons and protons during the reduction process can be understood. The biological production of fertilizer, and many other important biological processes, requires the assembly of inorganic entities called iron-sulfur clusters. Iron-sulfur clusters serve as electron carriers during many biological processes. The second major effort in this laboratory is aimed at understanding the biological mechanism for the assembly of iron-sulfur clusters. The importance of this work is that all life sustaining processes, for example, nitrogen fixation, photosynthesis, and respiration, all require iron-sulfur clusters for their activities. Consequently the effective assembly of iron-sulfur clusters is important in efforts to increase plant productivity and disease resistance in agronomically important crops. Defects in iron-sulfur cluster assembly are also associated with a number of debilitating human pathologies such as Friedreich's ataxia. Work in this laboratory has led to the discovery of proteins that are involved in the assembly of iron-sulfur clusters. The action of these proteins appears to provide a near universal mechanism for the activation of iron and sulfur necessary for iron-sulfur cluster formation. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
A comprehensive mechanistic model for nitrogenase has been developed. Results of our experiments have been interpreted to indicate a model where the inert dinitrogen molecule becomes bound to the active site after the enzyme has accumulated four reducing equivalents stored in the form of two metal-bridging hydrides. Displacement of these hydrides by dinitrogen activates the substrate for subsequent reduction by alternating hdrogenation of the bound species. A defecit spending model has also been developed to explain how nucleotide hydrolysis is linked to electron transfer reactions necessary for biological nitrogen fixation to proceed. In separate experiments genetic methods have been used to remodel nitrogenase so that it gains the capacity to reduce carbon monoxide to yield short chain hydrocarbon products or to reduce carbon dioxide, a greenhouse gas, to yield methane or high value short chain hydrocarbons. Although these systems, in the present form, do not operate at a high efficiency the work establishes that the nitrogenase mechanism can be considered in the development of synthetic catalysis aimed at sequestering carbon dioxide with parallel production of high value olefins. Related work on the mechanism for biological formation of simple inorganic metal-sulfur species to support a variety of biological transformations, including, electron transfer, have been completed. The biological process has now been duplicated in vitro and intermediates in the assembly process have been captured in sufficient quantities for analysis by X-ray crystallographic methods. These experiments have revealed the formation of a complex that contains the precursor for iron-sulfur cluster formation. Finally, analysis of gene transcription profiles have revealed all of the biological components involved in iron-sulf cluster assembly. Thus, the overall impact of the project involves a path forward for synthetic catalysts for carbon dioxide sequestration and a starting point for developing therapeutic approaches for disease pathologies related to defects in iron-sulfur cluster formation.
Publications
- Temperature Invariance of the Nitrogenase Electron Transfer Mechanism. (2012) Mayweather D, Danyal K, Dean DR, Seefeldt LC, Hoffman BM. Biochemistry. 51: 8391-8398.
- EXAFS and NRVS reveal a conformational distortion of the FeMo-cofactor in the MoFe nitrogenase propargyl alcohol complex. (2012). George SJ, Barney BM, Mitra D, Igarashi RY, Guo Y, Dean DR, Cramer SP, Seefeldt LC. J Inorg Biochem. 112: 85-92.
- (IscS-IscU)2 complex structures provide insights into Fe2S2 biogenesis and transfer. (2012) Marinoni EN, de Oliveira JS, Nicolet Y, Raulfs EC, Amara P, Dean DR, Fontecilla-Camps JC. Angew Chem 51:5439-42.
- Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase. (2012) Yang ZY, Moure VR, Dean DR, Seefeldt LC. Proc Natl Acad Sci U S A. 109: 19644-19648.
- Unification of reaction pathway and kinetic scheme for N2 reduction catalyzed by nitrogenase. (2012) Lukoyanov D, Yang ZY, Barney BM, Dean DR, Seefeldt LC, Hoffman BM. Proc Natl Acad Sci U S A. 109: 5583-5587.
- Electron transfer in nitrogenase catalysis. (2012) Seefeldt LC, Hoffman BM, Dean DR. Curr Opin Chem Biol. 16:19-25.
- Catalytic mechanism of Sep-tRNA:Cys-tRNA synthase: sulfur transfer is mediated by disulfide and persulfide. (2012) Liu Y, Dos Santos PC, Zhu X, Orlando R, Dean DR, Soll D, Yuan J. J Biol Chem. 287: 5426-5433.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: This project involves elucidation of the mechanism for the assembly of simple and complex iron-sulfur clusters in bacteria. The value of the work with respect to output and agronomic and economic impact is that iron-sulfur cluster are simple inorganic prosthetic groups that are essential to sustain biological processes such as nitrogen fixation, photosynthesis and respiration. They are also import cofactors that are necessary for many types of metabolic engineering for economic benefit, for example, the production of biofuels. The output over the present reporting period has involved determination of the transcriptional profile of the model organism Azotobacter vinelandii when grown under a variety of different conditions. Examples of these analyses include cells grown under standard laboratory conditions, cells grown under nitrogen fixing conditions and in the presence of the availability of different metals, and using various mutant strains that are deficient in the regulation of cellular components necessary to control formation of iron-sulfur clusters. Other experiments were aimed at understanding the in vitro assembly of iron-sulfur clusters. This has involved using purified biosynthetic components to direct the in vitro activation of enzymes or proteins that contain iron-sulfur clusters. PARTICIPANTS: Valerie Cash - research technician; Weiya Xu - post-doctoral associate;Katie Dougherty undergraduate researcher TARGET AUDIENCES: The target audience includes researchers and industrial scientists working in the arena of biofuels, and scientists working on approaches for enhancing food production through augmentation of biological nitrogen and carbon fixation. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The main effort during the reporting period was aimed at developing techniques for obtaining transcriptional profiles of Azotobacter vinelandii when grown under different conditions. The requisite technology, which is new to this laboratory, was successfully developed. The major technical hurdle involved the preparation of mRNA that was enriched so that it contains a minimal amount of rRNA contamination. Such procedures subsequently permitted a total genome transcriptome analysis. The results indicated a significant level of metabolic cross-talk between systems for the generalized formation of iron-sulfur clusters and those that are specifically used for activation of biological nitrogen fixation. It was also possible to identify those genes whose expression is specifically linked to the capacity for generalized iron-sulfur cluster formation. The outcome of this work is that it is valuable for the development of strategies to overcome metabolic bottlenecks related to elevated production of enzymes that require iron-sulfur clusters for their activities. Possible applications that are anticipated include augmentation of nitrogen fixation, increased capacity for photosynthesis (enhanced food production) and metabolic engineering strategies related to bioenergy (production of high energy alkane alcohols and hydrogen).
Publications
- Dos Santos, P. C. and Dean, D. R. (2011) Coordination and fine-tuning of nitrogen fixation in Azotobacter vinelandii. Molec Microbiol 79: 1132-1135.
- Hamilton, T. L., Jacobson, M., Ludwig, M., Boyd, E. S., Bryant, D. A., Dean, D. R. and Peters, J. W. (2011) Differential accumulation of nif structural gene mRNA in Azotobacter vinelandii J. Bacteriology 193: 4534-4536.
- Hamilton, T. L., Ludwig, M., Dixon, R., Boyd, E. S., Dos Santos, P. C., Setubal, J., Bryant, D. A., Dean, D. R. and Peters, J. W. (2011) Transcriptional profiling of nitrogen fixation in Azotobacter vinelandii J. Bacteriology. 193: 4477-4486
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Small inorganic structures composed of iron and sulfur are constructed by cells to perform a variety of essential catalytic, regulatory and oxidation/reduction roles that support metabolism. Among the metabolic processes that require the participation of iron-sulfur clusters is included biological nitrogen fixation. Biological formation of iron-sulfur clusters required to support nitrogen fixation involves the participation of two protein partners. One of these is a cysteine desulfurase (NifS) and an iron-binding scaffold protein (NifU) upon which the nascent cluster is formed. It has now been possible through genetic manipulation and biochemical purification procedures to capture NifS and NifU at various stages of the assembly process. These include the separately isolated proteins, proteins that have formed a complex but have not initiated sulfur transfer, a complex that contains a completed iron-sulfur cluster and the active form of NifU that contains an iron-sulfur cluster but is not complexed with NifS. Genetic and biochemical constructs developed during the course of this project have been widely disseminated to the research community and have enabled numerous advances in the field beyond the scope of this specific project. Experiments are now in progress that are aimed towards understanding the consequences at the gene expression level that result as a consequence of impairment of iron-sulfur cluster formation or during the transition from conditions where nitrogen fixation is unnecessary to conditions that demand nitrogen fixation for growth. PARTICIPANTS: Dennis R. Dean (Principal Investigator), Jaim De Oliviera (Post-Doctoral Associate), Timothy J. Larson (collaborator), Robert H. White (Collaborator), Valerie L. Cash (Collaborator), Lance C. Seefeldt (Collaborator, Utah State University) Jeverson Frazzon (Post-Doctoral Associate) TARGET AUDIENCES: Agricultural and biomedical research scientists. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
A knowledge of the biological mechanism for assembly of iron-sulfur clusters is related to a more fundamental understanding of how iron and sulfur are trafficked and sequestered in forms that do not damage cells. Identification and genetic manipulation of this system continues to contribute to three primary outcomes. First, genetic transfer of a well-characterized system has now been used by many other investigators for the augmentation of the production of certain enzymes in active forms that was not previously possible. Second, defects in iron-sulfur cluster assembly and delivery components have now been linked to a variety of genetic disorders resulting in the possibility for the development of approaches to diminish or eliminate the physiological consequences of such defects. Third, it is reasonable to expect that augmentation in plant productivity can be augmented by genetic improvement in a capacity for iron-sulfur cluster formation. A fourth outcome that has emerged is the availability of a comprehensive "transcriptome" analysis of gene expression in the model organism, Azotobacter vinelandii, under different growth conditions. This work has provided new targets for genetic modifications to improve the capacity for nitrogen fixation and acquisition of trace metals needed for effective metabolism.
Publications
- Sarma, R. B. M. Barney, S. Keable, D. R. Dean and J. W. Peters (2010) Insights into substrate binding at FeMo-cofactor in nitrogenase from the structure of an alpha 70 Ile MoFe protein. J. Inorg. Biochem. 104: 385-389.
- Danyal, K., D. Mayweather, D. R. Dean, L. C. Seefeldt and B. M. Hoffman (2010) Conformational gating of electron transfer from the nitrogenase Fe protein to MoFe protein. J. Am. Chem. Soc. 132:6894-6896
- Danyal, K., Inglet, B., Vincent, K. A., Barney, B. M., Hoffman, B. M., Armstrong, F. A., Dean, D. R. and Seefeldt, L. C. (2010) Uncoupling nitrogenase: Catalytic reduction of hydrazine to ammonia by a MoFe protein in the absence of Fe protein-ATP. JACS 132:13197-13199
- Dos Santos, P. C. and D. R. Dean (2010) Electrons in Fe-S protein assembly. Nature Chemical Biology 6:700-701
- Yang, Z., Seefeldt, L. C., Dean, D. R., Cramer, S. P., and S. J. George (2010) Steric control of the Hi-CO MoFe nitrogenase complex revealed by stopped-flow infra-red spectroscopy. Angewandte Chemie in press
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Small inorganic structures composed of iron and sulfur are constructed by cells to perform a variety of essential catalytic, regulatory and oxidation/reduction roles that support metabolism. Among the metabolic processes that require the participation of iron-sulfur clusters is included biological nitrogen fixation. Biological formation of iron-sulfur clusters required to support nitrogen fixation involves the participation of two protein partners. One of these is a cysteine desulfurase (NifS) and an iron-binding scaffold protein (NifU) upon which the nascent cluster is formed. It has now been possible through genetic manipulation and biochemical purification procedures to capture NifS and NifU at various stages of the assembly process. These include the separately isolated proteins, proteins that have formed a complex but have not initiated sulfur transfer, a complex that contains a completed iron-sulfur cluster and the active form of NifU that contains an iron-sulfur cluster but is not complexed with NifS. Genetic and biochemical constructs developed during the course of this project have been widely disseminated to the research community and have enabled numerous advances in the field beyond the scope of this specific project. In addition, this project has led to the organization of a consortium of scientists that have sequenced and annotated the genome of the nitrogen fixing soil bacterium Azotobacter vinelandii. PARTICIPANTS: Dennis R. Dean (Principal Investigator), Timothy Larson (Collaborator), Joao Setubal (Collaborator), Robert White (Collaborator), Valerie Cash (Research Technician), Estella Raulfs (Graduate Student), Ina O'Carroll (Graduate Student,) Kyle Cromer (Undergraduate Student), Jeverson Frazzon (Post-doctoral Associate), Ana Frazzon (Post-doctoral Associate). TARGET AUDIENCES: Agricultural and biomedical researcher scientists. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
A knowledge of the biological mechanism for assembly of iron-sulfur clusters is related to a more fundamental understanding of how iron and sulfur are trafficked and sequestered in forms that do not damage cells. Identification and genetic manipulation of this system continues to contribute to three primary outcomes. First, genetic transfer of a well-characterized system has now been used by many other investigators for the augmentation of the production of certain enzymes in active forms that was not previously possible. Second, defects in iron-sulfur cluster assembly and delivery components have now been linked to a variety of genetic disorders resulting in the possibility for the development of approaches to diminish or eliminate the physiological consequences of such defects. Third, it is reasonable to expect that augmentation in plant productivity can be augmented by genetic improvement in a capacity for iron-sulfur cluster formation. A fourth outcome that has emerged is the availability of the genome sequence of Azotobacter vinelandii. This organism provides a model system for studies aimed at understanding the chemical mechanism of biological nitrogen fixation as well as for the assembly of complex metal-containing iron-sulfur clusters involved in nitrogen, carbon and sulfur metabolism.
Publications
- Setubal JC, dos Santos P, Goldman BS, Ertesvag, H, Espin G, Rubio LM, Valla S, Almeida NF, Balasubramanian D, Cromes L, Curatti L, Du Z, Godsy E, Goodner B, Hellner-Burris K, Hernandez JA, Houmiel K, Imperial J, Kennedy C, Larson TJ, Latreille P, Ligon LS, Lu J, Maerk M, Miller NM, Norton S, O'Carroll IP, Paulsen I, Raulfs EC, Roemer R, Rosser J, Segura D, Slater S, Stricklin SL, Studholme DJ, Sun J, Viana CJ, Wallin E, Wang B, Wheeler C, Zhu H, Dean DR, Dixon R, Wood D. Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J Bacteriol. 2009 191:4534-45.
- Seefeldt, L. C. B. M. Hoffman and D. R. Dean. 2009 Mechanism of Mo-dependent nitrogenase. Annu. Rev. Biochem. 78:701-722.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Our goal is to understand the biological mechanisms for understanding how cells construct small inorganic entities called iron-sulfur clusters which are used to support metabolic processes such as nitrogen fixation, photosynthesis and respiration. Towards this goal it was possible to establish using living cells that such clusters are synthesized on a protein scaffold. This scaffold is then used to deliver pre-assembled clusters to other proteins. It was also possible to establish that defects in iron-sulfur cluster formation causes deleterious effects on the capacity for cellular metabolism and and increase in iron-sulfur cluster formation increases the respiratory capacity of cells. Genetic constructs, including modified cells and plasmids used in this project have been made freely available to the scientific community. PARTICIPANTS: Valerie L. Cash research technician Estella C. Raulfs graduate student Ina O Carroll graduate student Patricia Dos Santos post-doctoral associate TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A knowledge of the biological mechanism for assembly of iron-sulfur clusters is related to a more fundamental understanding of how iron and sulfur are trafficked and sequestered in forms that do not damage cells. Identification and genetic manipulation of this system has resulted in three primary outcomes. First, genetic transfer of a well-characterized system has now been used by many other investigators for the augmentation of the production of certain enzymes in active forms that was not previously possible. Second, defects in iron-sulfur cluster assembly and delivery components have now been linked to a variety of genetic disorders resulting in the possibility for the development of approaches to diminish or eliminate the physiological consequences of such defects. Third, it is reasonable to expect that augmentation in plant productivity can be augmented by genetic improvement in a capacity for iron-sulfur cluster formation.
Publications
- Raulfs, E. C., O Carroll, I. P., Dos Santos, P. C., Unciuleac, M-C, and Dean, D. R. (2008) In vivo iron-sulfur cluster formation. Proceedings of the National Academy of Science (USA) 105: 8591-8596.
- Bandyopadhy, S., Naik, S. G., O Carroll, I. P., Huynh, B. H., Dean, D. R., Johnson, M. K. and Dos Santos, P. C. (2008) A proposed role for the Azotobacter vinelandii NfuA protein as an intermediate iron-sulfur cluster carrier J. Biol. Chem. 283: 14092-14099.
- Seefeldt, L. C., Hoffman, B. M, and Dean, D. R. (2008) Nitrogenase Mechanism Annual Reviews of Biochemistry. In Press.
- Dos Santos, P. C. and Dean, D. R. (2008) A newly discovered role for iron-sulfur clusters.. Proceedings of the National Academy of Sciences (USA) 105: 11589-11590.
- Hoffman, B. M., Dean, D. R., and Seefeldt, L. C. (2008) Climbing Nitrogenase: Towards the mechanism of N2 reduction. Accounts of Chemical Research. In Press.
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