Research Projects
Nitrogenase and Nitrogen Reduction
The broad, long term goal of the project is to gain structural and mechanistic insights into the role of MgATP in nitrogenase catalysis. Nitrogenase is a complex metal-containing enzyme that catalyzes the conversion of nitrogen gas to ammonia. During nitrogenase catalysis, the iron protein and molybdenum-iron protein associate and dissociate in a manner resulting in the hydrolysis of two molecules of MgATP and the transfer of at least one electron to the MoFe protein. Multiple cycles of iron protein association and dissociation, MgATP hydrolysis, and electron transfer are required for the complete reduction of a single molecule of nitrogen to ammonia. There are a number of aspects of nitrogenase structure/function that are interesting areas of fundamental research. Nitrogenase can be considered an ideal model system for the study of the complex metal cluster mediated catalysis, electron transfer, complex metal cluster assembly, protein-protein interactions, and nucleotide dependent signal transduction. In addition, the involvement of MgATP in nitrogenase catalysis is similar to the role of nucleotides in a large class of nucleotide binding proteins that couple nucleotide binding and hydrolysis to protein conformational changes transduced within a macromolecular assembly. Members of the class include G proteins, Ras p21, RecA, elongation factor Tu, myosin, and transducin, making the role of MgATP binding and hydrolysis one of the most fascinating aspects of nitrogenase research. We have recently been able to determine the structure of a single deletion mutant of the nitrogenase Fe protein that provides a structural mimic of the MgATP bound state. The structural insights described in the preliminary results section of the proposal provide the firmest foundation described to date for generating hypotheses concerning MgATP dependent conformational change in the Fe protein and the initial component protein interactions that trigger MgATP hydrolysis. The proposed studies apply a combined approach involving structure determination by x-ray diffraction methods and site-specific amino acid substitution experiments to gain insights into nucleotide dependent conformational change, macromolecular complex formation, and the specific protein-protein interactions occurring upon complex formation that initiate MgATP hydrolysis in nitrogenase.
Publications
R. K. Szilagyi and J. W. Peters
, "Exploring new frontiers in nitrogenase structure and mechanism
." Curr. Opin. Chem. Biol. 2005
A.-S. Steunou, D. Bhaya, M. Bateson, M. Melendrez, D. M. Ward, E. Brecht, J. W. Peters, M. Kuhl, and A. Grossman
, "In situ metabolic switching in a hot spring cyanobacterial mat of Yellowstone National Park
." Submitted to PNAS (2005)
S. Sen, A. M. Krishnakumar J. McClead, L. C. Seefeldt and J. W. Peters
, "Insights into the role of nucleotide dependent conformational change in nitrogenase catalysis: Structural characterization of the nitrogenase Fe protein Leu127 deletion variant with bound MgATP
." Submitted to the J. Inorg. Biochem. (2005)
S. Sen, and J. W. Peters
, "The thermal adaptation of the nitrogenase Fe protein from thermophilic Methanobacter thermoautotrophicus
." Proteins. 2006 Feb 1; 62(2):450-460
S. Sen, and J. W. Peters
, "The role of MgATP in nitrogenase catalysis.
." In the Proceedings of the 14th International Nitrogen Fixation Congress (2005)
S. B. Jang, M. S. Jeong, L. C. Seefeldt, and J. W. Peters
, "Structural and biochemical implication of single amino acid substitutions in the nucleotide-dependent switch regions of the nitrogenase Fe protein from Azotobacter vinelandii
." J. Biol. Inorg. Chem. 8, 1028-1033 (2004).
P. M. C. Benton and J. W. Peters
, "Nitrogenase Structures
." Nitrogen Fixation (ed. B. E. Smith and R. Richards) Kluwer. pp. 77-96 (2004)
P. M. C. Benton, S. Sen, and J. W. Peters
, "Nitrogenase Structures
."
M. Sørlie, J. Christiansen, B. J. Lemon, J. W. Peters, D. R. Dean, and B. J. Hales
, "Structural and mechanistic interpretation of the EPR signals observed during acetylene reduction by the α-H195Q mutant of nitrogenase
." Biochemistry 40, 1540-1549 (2001)
S. B. Jang, L. C. Seefeldt, and J. W. Peters
, "Insights into Nucleotide Signal Transduction in Nitrogenase: Structure of an Iron Protein with MgADP Bound
." Biochemistry 39, 14745-14752 (2000)
S. B. Jang, L. C. Seefeldt, and J. W. Peters
, "Modulating the Midpoint Potential of the [4Fe-4S] Cluster of the Nitrogenase Fe Protein
." Biochemistry 39, 641-648 (2000)
J.W. Peters, M.H.B. Stowell, M.S. Soltis, M.G. Finnagan, M.K. Johnson, and D.C. Rees
, "Redox Dependent Structural Changes in the Nitrogenase P Cluster
." Biochemistry, 36, 1181-1187 (1997)
J.W. Peters
, "A Brief Overview of Nitrogenase Structure and Function
." Life Support and Biosphere Science, 3, 175-181 (1996).
A. M. Krishnakumar, B. P. Nocek, D. D. Clark, S. A. Ensign and J. W. Peters
, "Identification of a Nitrogenase Protein-Protein Interaction Site Defined by Residues 59 through 67 with the Azotobacter vinelandii Fe Protein
." In preparation (2005)
J.W. Peters, K, Fisher, W.E. Newton, and D.R. Dean
, "Involvement of the P Cluster in Intramolecular Electron Transfer with the Nitrogenase MoFe Protein
." J. Biol. Chem. 270, 27007-27013 (1995)
J.W. Peters, K. Fisher, and D.R. Dean
, "Nitrogenase Structure and Function: A Biochemical Genetic Perspective
." Annu. Rev. Microbiol. 49, 335-366 (1994)
H. J. Chui, J. W. Peters, W. N. Lanzilotta, M. J. Ryle, L. C. Seefeldt, J. B. Howard, and D. C. Rees
, "MgATP-bound and nucleotide-free structures of of a nitrogenase protein complex between Leu 127delta Fe protein and MoFe-protein
." Biochemistry 40, 641-650 (2001)
H. J. Chui, J. W. Peters, W. N. Lanzilotta, M. J. Ryle, L. C. Seefeldt, J. B. Howard, and D. C. Rees
, "MgATP-bound and nucleotide-free structures of of a nitrogenase protein complex between Leu 127delta Fe protein and MoFe-protein
." Biochemistry 40, 641-650 (2001)
S. Sen, R. Igarashi, A. Smith, M. K. Johnson, L. C. Seefeldt, and J. W. Peters
, "A Conformational Mimic of the MgATP bound "On State" of the Nitrogenase Fe Protein
." Biochemistry 43, 1787-1797 (2004)
Personnel:
John Peters
Keywords:
Biochemistry
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