Enzyme Structure and Mechanism

john.peterschemistry.montana.edu

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.

Selected 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)

Keywords:
Biochemistry

Epoxides and ketones are widely used in industry and have been shown to have potential mutagenic, carcinogenic, and toxic effects. Therefore, there is considerable interest in microbial based bioconversions of these compounds into less environmentally detrimental compounds. Epoxides are utilized in a variety of industrial processes as intermediates in organic synthesis. Due to the strained three-membered ring structure of these compounds, epoxides react readily with a number of nucleophiles {Swaving, 1998 #1315}. Optically pure epoxides are intermediates in the synthesis of a number of bioactive compounds including leukotrienes, pheromones, antibiotics, and an HIV protease inhibitor. Due to the reactive nature of these compounds and their ability to react with cellular components including DNA and proteins, they are of considerable concern as potential human health hazards. The ability of epoxides to form covalent adducts to DNA can result in mutagenic and carcinogenic effects. Acetone is a toxic molecule that is synthesized industrially and formed biologically during bacterial fermentation and mammalian starvation. A number of bacteria are able to grow with acetone as a source of carbon and energy. In addition, acetone is formed as an intermediate in the metabolism of propane and isopropanol by some bacteria. Bacterial pathways of acetone metabolism and the biochemical properties of acetone-metabolizing enzymes are poorly understood. The widespread use of epoxides and ketones has resulted in an increased interest in the mechanisms by which various microorganisms transform these compounds into less environmentally detrimental compounds. From a basic science standpoint the bioconversions we propose to study represent novel microbial based CO2 fixation / carboxylation mechanisms that have only recently been identified. The results obtained in the study will reveal new insights into these novel carboxylation reactions and will provide the basis for the comparison of the mechanism of these interesting enzymes to other more well characterized CO2 fixing and carboxylating enzymes.

Selected Publications

A. M. Krishnakumar, B. P. Nocek, D. D. Clark, S. A. Ensign and J. W. Peters :
Structural basis for stereo selectivity in 2-® hydroxypropylthioethanesulfonate (R-HPC), 2(S) hydroxypropylthioethanesulfonate dehydrogenases in Xanthobacter autotrophicus
In preparation (2005)

A. S. Pandey, B. P. Nocek, D. D.Clark, D., S. A. Ensign, J.W. Peters :
Mechanistic implications of the structure of the mixed-disulfide intermediate of the disulfide oxidoreductase, 2-ketopropyl coenzyme M oxidoreductase / carboxylase
Biochemistry 2006 Jan 10:45 (1) 113-120

A.J. Copik, B. Nocek, S.I. Swierszek, S. Ruebesh, S.B. Jang, L. Meng, V.M. D\'Souza, J.W. Peters, B. Bennett, R.C. Holz :
EPR and X-ray crystallographic characterization of the product bound form of the Mn(II)-loaded methionyl aminopeptidase from Pyrococcus furiosus
Biochemistry 44, 121-129 (2005)

B.P. Nocek, J. Boyd, S.A. Ensign, and J.W. Peters :
Crystallization and preliminary x-ray analysis of an acetone carboxylase from Xanthobacter autotrophicus strain Py2
Acta Crystallography D 60, 385-387

B. P. Nocek, S.B. Jang, M. Jeong, D.D. Clark, S.A. Ensign, and J. W. Peters :
Structural basis for CO2 fixation by a member of the disulfide oxidoreductase family of enzymes: 2-ketopropyl coenzyme M oxidoreductase carboxylase
Biochemistry 41: 12907-12913 (2002)

S. B. Jang, M. S. Jeong, D.D. Clark, S. A. Ensign, J.W. Peters :
Crystallization and preliminary X ray analysis of a NADPH 2-ketopropyl-coenzyme M oxidoreductase/carboxylase
Acta Crystallogr. D57, 445-447 (2001)

B. P. Nocek, D. D. Clark, S. A. Ensign and J. W. Peters :
Crystallization and preliminary X-ray analysis of 2-R-hydroxypropyl-coenzyme M dehydrogenase
Acta Crystallogr. D58, 1470-1473 (2002)

Keywords:
Biochemistry

The reversible conversion of molecular hydrogen to protons and electrons is a central reaction in the global biological energy cycle. Hydrogenase enzymes catalyze a large percentage of this reaction, and thus a more detailed understanding of these enzymes is of wide interest in biotechnology, biochemistry, and energy sciences. These enzymes are present in various microorganisms and function either in the utilization of Hydrogen as a growth substrate (Hydrogen uptake) or in certain anaerobic bacteria to dispose of excess electrons by combining them with protons to form Hydrogen. The X-ray crystal structure of the iron-only hydrogenase from the anaerobic soil microorganism Clostridium pasteurianum (CpI) was determined to 1.8 angstrom resolution in the group. CpI is a highly complex protein containing twenty iron atoms arranged into five individual metal cluster assemblies. The active site cluster or "H cluster" is structurally unprecedented among previously characterized biological iron-sulfur clusters. Our ongoing hydrogenase research involves probing the mechanism of hydrogenases by biochemical, structural, and physical methods. We are collaborating with Prof. Joan Broderick of the department on the mechanism of the H cluster. In addition, we are collaborating with investigators throughout the world, probing avenues to produce hydrogen efficiently as renewable energy. This work includes a collaboration here at MSU with Trevor Douglas of the department on the design, synthesis, and characterization of hydrogen producing biomimetic materials.

Selected Publications

J.W. Peters, R. K. Szilagyi, A. Naumov, T. Douglas :
A radical solution for the biosynthesis of the H cluster of hydrogenase
FEBS Lett. 2005 Dec 22

B. Bennett, B. J. Lemon, and J. W. Peters :
Reversible Carbon Monoxide Binding and Inhibition at the Active Site of the Fe-Only Hydrogenase (CpI)
Biochemistry 39, 7455-7460 (2000)

B. J. Lemon and J. W. Peters :
Photochemistry at the Active Site of the Carbon Monoxide Inhibited Form of the Iron-Only Hydrogenase (CpI)
J. Am. Chem. Soc. 122, 3793-3794 (2000)

Y. Nicolet, B. J. Lemon, J. C. Fontecilla-Camps, and J. W. Peters :
A Novel FeS Cluster in Fe-only Hydrogenases
Trends in Biochemical Sciences 25, 138-144 (2000)

J.W. Peters :
Structure and Mechanism of Fe-only Hydrogenase
Curr. Opin. Struct. Biol. 6, 670-676 (1999)

J. W. Peters and H. D. Bellamy :
Extension of Fe MAD phases in the structure determination of a multiple [FeS] cluster containing hydrogenase
J. Appl. Cryst. 32, 1180-1182 (1999)

B. J. Lemon, and J. W. Peters :
Binding of Exongenously Added Carbon Monoxide at the Active Site of the Fe-Only Hydrogenase from Clostridium pasteurianum
Biochemistry 38, 12969-12973 (1999)

J.W. Peters, W.N. Lanzilotta, B.J. Lemon, and L.C. Seefeldt :
The X-ray Crystal Structure of the Fe Hydrogenase (CpI) from Clostridium pasteurianum to 1.8 A Resolution
Science 282, 1853 1858 (1998)

Z. Varpness, J. W. Peters, M. Young, and T. Douglas :
Biomimetic synthesis of a H2 catalyst using a protein cage architecture
Nanoletters 5, 2306-2309 (2005)

T. E. Elgren, O. A. Zadvorny, E. Brecht, T. Douglas, N. A. Zorin, M. J. Maroney, and J. W. Peters :
Immobilization of Active Hydrogenases by Encapsulation in Polymeric Porous Gels
Nanoletters 5, 2085-2087 (2005)

L. Girbal, G. von Abendroth, M. Winkler, P. M. C. Benton, I. Meynial-Salles, C. Croux, J. W. Peters, T. Happe, and P. Soucaille :
Homologous and heterologous over-expression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases
Appl. Environ. Microbiol. 71, 2777-2781 (2005)

Z. Chen, B. J. Lemon, S. Huang, D. Swartz, J. W. Peters, and K. A. Bagley :
Infrared Studies of the CO Inhibited form of the Fe-Only Hydrogenase from Clostridium pasteurianum I (CpI): Examination of its Light-Sensitivity at Cryogenic Temperatures
Biochemistry 41, 2036-2043 (2002)

B. J. Lemon and J. W. Peters :
Fe-only Hydrogenases
Handbook of Metalloproteins. (eds. A. Messerschmidt, R. Huber, T. Poulos, & K. Weighardt) pp. 738-750 (2001)

Keywords:
Biochemistry

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