Progress 12/15/14 to 12/14/16
Outputs
Target Audience:I communicated my research output to the scientific community through 3 publications in peer reviewed scientific journals, through a poster presentation at the 2016 spring annual meeting of the American Chemical Society, and an oral presentation to the biology department of California State University, East Bay in January 2016. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided me the opportunity to study the structure of a novel nitrogenase in an agriculturally relevant organism. The results of my work led to the discovery of conserved structural properties of nitrogenases, which has advanced the understanding of the enzymes's electron transfer mechanism. This work has aided my professional development since my coworkers and I were able to publish our results in a high-impact journal. More importantly, the outcome of my research has raised numerous additional questions regarding electron transfer in nitrogenase, questions I wish to answer in future research as an independent PI. Furthermore, the fellowship has contributed to my professional development since it enabled me to build contacts with other NIFA fellows, thereby greatly expanding my professional network. How have the results been disseminated to communities of interest?This work has been disseminated through publications in peer-reviewed scientific journals, a talk at California State University, East Bay, and a conference presentation at the Spring 2016 American Chemical Society national meeting. Furthermore, parts of this work were presented by an undergraduate student who was working under my supervision at the UC San Diego undergraduate research conference. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
To address questions asked in specific aim 1, I determined the binding mechanism of the nitrogenase component proteins FeP and MoFeP in the organism Azotobacter vinelandii (Av). This step in enzymatic turnover is critical to understanding controlled, conformationally gated electron transfer. The FeP binding mechanism has not been firmly established, and transition between the binding conformation (DG1) and the active conformation (DG2) may activate electron transfer. I demonstrated that disrupting FeP-MoFeP interactions in DG1 weakens the FeP binding affinity and lowers nitrogenase activity. However, ATP hydrolysis rates and the ATP hydrolyzed to electron transfer ratio are unaffected, demonstrating that disrupting FeP binding in DG1 does not affect subsequent electron transfer processes. These results are the first to provide direct evidence that FeP binding in DG1 is critical for nitrogenase activity. I propose that DG1 represents an encounter complex, which enables rapid FeP association to MoFeP, and increases the likelihood that collisions between FeP and MoFeP lead to productive electron transfer to substrate. Furthermore, the discovery that DG1 is functionally relevant suggests that the proposed electron transfer activation mechanism during transition from DG1 to DG2 is plausible. To advance specific aim 2, I adapted methods to culture Gluconacetobacter diazotrophicus (Gd), and to express and purify Gd-nitrogenase in high yield. Using purified Gd-MoFeP, I solved its crystal structure and measured its electron paramagnetic resonance (EPR) spectrum in both the reduced and oxidized state, addressing a main goal of the proposal. The structures revealed that the P-cluster of Gd-MoFeP undergoes redox dependent structural changes. Upon oxidation, the P-cluster rearranges and is reversibly coordinated by a Tyr residue. This stands in contrast to the structure of Av-MoFeP and that of other nitrogenases, which feature redox dependent coordination by a Ser ligand. The results of this investigation suggest that having a hard O-based redox active ligand is critical to nitrogenase function. The protein structures also suggest that redox dependent P-cluster rearrangement is conserved between nitrogensaes from different organisms. Furthermore, I started biochemical characterization of Gd-nitrogenase. The FeP-MoFeP binding affinity is weaker than in Av-nitrogenase, and the specific activity is lower. I also started developing cloning methods for generating site-specific mutations in the G. diazotrophicus chromosome by creating a G. diazotrophicus nitrogenase knockout strain in which the gene for the nitrogenase β- subunit is replaced by a kanamycin resistance cassette. This strain demonstrates that it is feasible to generate site specific G. diazotrophicus mutants and it will serve as a platform for making further β- subunit mutants.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Katz, F. E. H., Owens, C. P., Tezcan, F. A. (2016). Electron transfer reactions in biological nitrogen fixation. Isr. J. Chem. 56, 682-692
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Owens, C. P., Katz, F. E. H., Carter, C. H., Oswald, V. F., Tezcan, F. A. (2016) Tyrosine-coordinated P-cluster in G. diazotrophicus nitrogenase: Evidence for the importance of O-based ligands in conformationally gated electron transfer. J. Am. Chem. Soc. 138, 10124-10127
- Type:
Journal Articles
Status:
Accepted
Year Published:
2017
Citation:
Katz, F.E.H., Shi, X., Owens, C.P., Joseph, S., Tezcan, F.A., Determination of NTPase activities in the presence of labile phosphate compounds, Analytical Biochemistry (accepted)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Owens, C. P., Katz, F. E. H., Carter, C. H., Tezcan, F. A. Protein-protein interactions in biological nitrogen fixation, Poster presented at the 251st ACS National Meeting, March 13-17, San Diego, CA
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Progress 12/15/14 to 12/14/15
Outputs
Target Audience:My efforts were communicted to the scientific community through a publication in the Journal of the American Chemical Society. I also reached academic audiences at local universities (CSU San Bernardino, UC Irvine and Chapman University),where I presented my work. Furthermore, I presented my work toother NIFA post- and predoctoral fellows at the NIFA fellows meeting. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided opportunities to pursue novel research characterizingA. vinelandiiandG. diazotrophicusnitrogenase, and disseminate results of my work through publication and presentations. Furthermore, I was given the opportunity to supervise the work of an undergraduate student whose project was based on my research. The student investigated the hypothesis thatweakeningA. vinelandiinitrogenase encounter complex formation also alters downstream steps in nitrogenase turnover. The results of his research suggest that disrupting the encounter complex does not affect later stages of nitrogenase turnover. These resultsindicate that the respectiverolesof the encounter complex and the active complex in turnover are distinct. This work was presented at two undergraduate research conferences at UC San Diego. In addition to gaining mentorship experience,this project has given me the opportunity to develop my teaching skills by attending workshops at the UC San Diego Center for Teaching Excellence. How have the results been disseminated to communities of interest?The work has been disseminated to the scientific community through 1 scientific publication in the Journal of the American Chemical Society. Furthermore, I presented my work to the chemistry/biochemistry departments of local universities (CSU San Bernardino, UC Irvine and Chapman University). What do you plan to do during the next reporting period to accomplish the goals?Plans for objective 1: 1) Investigate activation of electron transfer in A. vinelandii nitrogenase. To do so, I will generate mutations in the A. vinelandii FeP-MoFeP complex interface along the transition pathway between encounter complex and active complex. These mutations may disrupt activation of electron transfer and will determine if the electron transfer activation step occurs during transition from the encounter complex to the active complex. 2) Investigate the possibility that activation of electron transfer occurs exclusively in the active complex. To probe this possibility, I will generate mutations in the interface of the activated FeP-MoFeP complex and study their effects on enzymatic activity and electron transfer. Plans for objective 2: 1) Solve the G. diazotrophicus MoFeP crystal structure. 2) Investigate redox dependent structural changes surrounding the G. diazotrophicus nitrogenase P-cluster as part of a broader effort to characterize electron transfer between metal clusters during G. diazotrophicus nitrogenase turnover. 3) Determine the association mechanism between FeP and MoFeP in G. diazotrophicus nitrogenase, specifically if G. diazotrophicus nitrogenase forms a distinct encounter complex like A. vinelandii nitrogenase.
Impacts
What was accomplished under these goals?
Accomplishments towards objective 1: Objective 1 of this project is to determine how protein-protein interactions between nitrogenase FeP and nitrogenase MoFeP in A. vinelandii enable electron transfer between the respective nitrogenase metal clusters. In this reporting period, I determined how FeP and MoFeP associate in the first step of A. vinelandii nitrogenase turnover. FeP forms an encounter complex with MoFeP that is stabilized by electrostatic interactions. Formation of this encounter complex is required for efficient nitrogenase turnover since disrupting the encounter complex weakens FeP association to MoFeP and lowers nitrogenase activity. The encounter complex is structurally distinct from the catalytically active nitrogenase complex, suggesting that FeP undergoes a large conformation change between encounter complex and active complex. The results of this investigation raise the possibility that electron transfer between the nitrogenase metal clusters is activated as part of the transition between encounter complex and active complex. Accomplishments towards objective 2: Objective 2 of this project is to characterize nitrogenase from the organism G. diazotrophicus. During this reporting period, I optimized protocols to maximize nitrogenase expression in G. diazotrophicus laboratory growths, and I modified G. diazotrophicus nitrogenase purification methods. I have started G. diazotrophicus nitrogenase MoFeP crystallization trials to determine its protein structure. Furthermore, I have begun comparing the mechanism of G. diazotrophicus nitrogenase turnover with that of A. vinelandii nitrogenase using enzymatic assays with G. diazotrophicus nitrogenase.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Owens, C. P., Katz, F. E. H., Carter, C. H., Luca, M. A., Tezcan, F. A. (2015). Evidence for functionally relevant encounter complexes in nitrogenase catalysis. J. Am. Chem. Soc. 137, 12704-12712
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