The fundamental biological processes of nitrogen fixation, nitrification and photosynthesis are catalyzed by complexes of electron transfer proteins. X-ray diffraction methods will be used to determine the three-dimensional structures of the component proteins in these systems. The structures will permit an evaluation of the protein - cofactor and cofactor - cofactor interactions that control the redox properties and electron transfer mechanisms of the proteins; the structural interactions that are responsible for the specificity in electron transfer between donor and acceptor proteins; and the mechanisms of energy transduction associated with the coupling of electron transfer to ATP hydrolysis (nitrogenase) and light absorption (photosynthesis). The specific structural objectives in these areas are: 1.Nitrogenase. Biological nitrogen fixation is catalyzed by the nitrogenase complex, which consists of iron (Fe-) protein and molybdenum iron (MoFe-) protein. The three-dimensional structures of Fe-protein from Azotobacter vinelandii and Clostridium pasteurianum will be completed. Structures of Fe-protein - nucleotide complexes and site directed mutants of Fe-protein will be determined to describe the mechanism of electron transfer, and the coupling of ATP hydrolysis to this process. Cocrystallizations and subsequent structure determinations of Fe-protein complexed with the physiological electron transfer partners MoFe-protein, ferredoxin and flavodoxin will be attempted. 2.Photosynthetic Reaction Center (RC). Refinement of the structures of the bacterial photosynthetic RCs from Rhodobacter sphaeroides strains R-26 and 2.4.1 will be completed. Structures of RCs in various oxidation states, from site directed mutants, and under low temperature conditions will be determined to provide a structural basis for understanding the efficiency of electron transfer in this system. The structure of the Rb. sphaeroides cytochrome c2 that serves as the physiological reductant for the oxidized RC will be determined, and co-crystallization of the cytochrome - RC complex will be attempted. As the only class of membrane proteins of known atomic structure, analysis of the RC will also focus on general implications for membrane protein structure. 3.Nitrification. The structure of the tetraheme cytochrome c-554, involved in nitrification reactions of Nitrosomonas europea, will be determined. Crystallization and structure determination of hydroxylamine oxidoreductase from this organism will be attempted.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM045162-01
Application #
3304516
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1991-01-01
Project End
1994-12-31
Budget Start
1991-01-01
Budget End
1991-12-31
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Engineering
DUNS #
078731668
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Arias, Renee J; Kaiser, Jens T; Rees, Douglas C (2018) The ""speed limit"" for macromolecular crystal growth. Protein Sci 27:1837-1841
Segal, Helen M; Spatzal, Thomas; Hill, Michael G et al. (2017) Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II. Protein Sci 26:1984-1993
Morrison, Christine N; Spatzal, Thomas; Rees, Douglas C (2017) Reversible Protonated Resting State of the Nitrogenase Active Site. J Am Chem Soc 139:10856-10862
Spatzal, Thomas; Schlesier, Julia; Burger, Eva-Maria et al. (2016) Nitrogenase FeMoco investigated by spatially resolved anomalous dispersion refinement. Nat Commun 7:10902
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Mattle, Daniel; Zhang, Limei; Sitsel, Oleg et al. (2015) A sulfur-based transport pathway in Cu+-ATPases. EMBO Rep 16:728-40
Zhang, Li Mei; Morrison, Christine N; Kaiser, Jens T et al. (2015) Nitrogenase MoFe protein from Clostridium pasteurianum at 1.08?Å resolution: comparison with the Azotobacter vinelandii MoFe protein. Acta Crystallogr D Biol Crystallogr 71:274-82
Nguyen, Phong T; Li, Qi Wen; Kadaba, Neena S et al. (2015) The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter-substrate binding protein complex. Biol Chem 396:1127-34
Morrison, Christine N; Hoy, Julie A; Zhang, Limei et al. (2015) Substrate pathways in the nitrogenase MoFe protein by experimental identification of small molecule binding sites. Biochemistry 54:2052-60
Tezcan, F Akif; Kaiser, Jens T; Howard, James B et al. (2015) Structural evidence for asymmetrical nucleotide interactions in nitrogenase. J Am Chem Soc 137:146-9

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