Lab Research Descriptions

The NASA Astrobiology Biogeocatalysis Research Center

MSU is privileged to be the lead institution for one of sixteen Nodes of the NASA Astrobiology Institute. The Institute examines the origin and nature of life on earth in the context of the potential and nature for life on other earth based bodies. In addition, the institute is actively engaged in research that explores the nature of matter in the universe and the habitable zones of other solar systems. The Astrobiology teams are highly integrated and catalyze multi-disciplinary research and education. The Astrobiology Biogeocatalysis Research Center (ABRC) at MSU is a group of ten lead principle investigators, post-doctoral associates, graduate students, undergraduates, and education and public outreach staff.

The major research theme of the ABRC is in the area of prebiotic chemistry and specifically the role for iron-sulfur mineral motifs in the transition between the non-living and the living world. The project has three major thrusts including 1) iron-sulfur mineral catalysis, 2) iron-sulfur enzyme catalysis, and 3) biomimetic approaches to bridging iron-sulfur mineral and iron-sulfur enzyme structure and reactivity. These projects are highly integrated and the characterization of the unique iron-sulfur centers of nitrogenase and hydrogenase provide the inspiration to examine the structure determinants for effective nitrogen reduction and reversible hydrogen oxidation catalysis.

In our laboratory we are collaborating closely with the research labs of Professors Joan Broderick and Robert Szilagyi to examine the structural features of the unique iron-sulfur containing prosthetic groups at the active site of nitrogenases and hydrogenases. In addition, a major thrust of the project is to elucidate the biochemical steps involved in the synthesis of these unique cofactors. Although in each case, gene products have been revealed that are involved in the process, there is much yet to be learned about the substrates for the unique ligand sets of these cofactors and sequence of enzymatic steps required to produce intact clusters and

mature active enzymes.

In the context of the Astrobiology Institute project, the results of these studies will be exploited to drive the mineral catalysis and biomimetic thrusts of the project. Detailed knowledge of the structural determinants for iron-sulfur based nitrogen reduction and reversible hydrogen oxidation will improve our understanding of the potential for iron-sulfur based catalysis as an important factor in the origin of a rudimentary metabolism and the origin of life.

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Robust Phototrophic Microorganisms for Biological Hydrogen Production

The laboratory is currently is part of an AirForce Office of Scientific Research (AFOSR) sponsored Multiple University Research Initiatives (MURI) project which is lead by PI Charles Dismukes of Princeton University's Department of Chemistry. The project is aimed at the optimization of light driven or mediated hydrogen production for alternative energy. We are exploring hydrogen production in either algae or Cyanobacteria for optimal hydrogen production and in parallel we are attempting to identify the organisms with the highest hydrogen production potential, thus laying the groundwork for metabolic engineering to create organisms with enhanced hydrogen production capabilities.

Within the aims of this project, we are pursuing structural characterization of the NiFe hydrogenase from the purple sulfur bacterium Thiocapsa rosepersicina. There is a tremendous interest in this enzyme in biotechnology due to its enhanced stability. The enzyme is optimally active at 80º C and is stable for days at room temperature and essentially insensitive to oxygen. The purified enzyme exists as an interesting supermolecular complex made up of six hydrogenase heterodimeric units. The structure will be the first from a phototrophic bacteria and knowledge of the structure will improve our understanding of oxygen tolerance and thermal stability and will provide a template for heterologous expression and metabolic engineering.

In addition to the structural work on novel [NiFe]-hydrogenases as a complement to our work on hydrogenase biosynthesis in the context of prebiotic chemistry supported by NASA, we are examining the metabolism of organisms that harbor [FeFe]-hydrogenases in an attempt to reveal the substrates that are derived from central metabolism that are involved in hydrogenase H cluster biosynthesis. These studies are essential if we are to effectively genetically engineer organisms to express high levels of [FeFe]-hydrogenase enzymes for photobiological hydrogen production.

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The Role of MgATP in Nitrogenase Catalysis

This NIH supported project is aimed at providing the structural basis for understanding the sequence of conformational changes within the nitrogenase Fe protein that effectively gate unidirectional electron transfer and support the reduction of dinitrogen to ammonia which occurs by the activity to the MoFe protein partner. This process is an analog of many signal transduction mechanisms, such as the function of myosin in muscle contraction or transducin in vision. The Fe protein is an interesting member of the class, having a dimeric quaternary structure and a redox active [4Fe-4S] cluster. Arguably the Fe protein is an attractive model system for the study of signal transduction processes by virtue of its [4Fe-4S] cluster, since structural changes induce perturbations in the spectroscopic properties of the cluster, which can be monitored by EPR, CD, and Resonance Raman spectroscopies. In this project, we are attempting to provide a better understanding of the linkage between spectroscopic properties and protein structure using the aforementioned spectroscopic methods to analyze the electronic properties of the [4Fe-4S] cluster in combination with

x-ray diffraction methods and solution small angle x-ray scattering to analyze protein structure. In this approach we are attempting to gain insights into the factors in the protein environment that are key to tuning the physicochemical properties of the [4Fe-4S] cluster. These studies are fundamentally important to a general understanding as to how specific protein environments tune metal-cluster properties, and will provide insights into protein control of redox potentials in FeS cluster-dependent electron transfer reactions.

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Hydrogen Metabolism and Nitrogen Fixation in Thermal Environments

This work, supported in part by AFOSR and in part by NASA as part of the MSU Thermal Biology Institute, is focused on analyzing the occurrence of enzymes involved in hydrogen metabolism (hydrogenases) and nitrogen fixation (nitrogenase) in diverse environments where evolutionarily deeply-rooted microorganisms predominate. Fundamental evolutionary questions of great interest remain to be answered for both hydrogenases and nitrogenases. For example, nitrogen fixation is widely distributed among prokaryotes and occurs in both bacteria and archeae. Among the Archeae, however, nitrogen fixation has been shown to occur only amongst the methanogens. For hydrogen metabolism, the evolutionary questions are even more interesting. The metal-containing hydrogenases that formally catalyze reversible hydrogen oxidation can be divided into two classes, the [FeFe]-hydrogenases and the [NiFe]-hydrogenases, reflecting the respective compositions of their metal-containing active sites. The [FeFe]-hydrogenases are found in many bacteria and lower Eukaryotes, including algae and protozoa, but have not been found to occur amongst any Archaea. [NiFe]-hydrogenases are found to occur in many bacteria, Cyanobacteria and Archeae, but not amongst any Eucaryotes.

We are using a combination of specific gene amplification probes in combination with DNA microarray and metagenomic analysis to analyze the ecology of hydrogen metabolism and nitrogen fixation from the perspective of the enzymes that catalyze these reactions. These studies involve approximately fifteen sites within Yellowstone National Park for which the geochemical characterization is being conducted in parallel. These environments are diverse in their physiochemical properties, ranging from low pH (~2) to high pH (~10) over a large range of temperatures and mineral contents. It is a goal of the project to examine the ecology of nitrogen fixation and hydrogen metabolism from the perspective of hydrogenase and nitrogenase enzymes, and since these environments contain numerous deeply rooted organisms, the aforementioned questions of origin with respect to these processes will be answered.

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Biomimetic Systems for Light Driven Hydrogen Production

In work supported by the Department of Energy, we are engaged in the application of enzymes and enzyme mimics (with Trevor Douglas) to the production of hydrogen-producing materials for alternative energy solutions. The project involves novel patented solar to hydrogen materials strategies that can be potentially applied in a number of different ways. Since durability is one of the key aspects of enzymes that limit their effective use in many industrial processes, we are investigating enzyme stability, thermal adaptation, and immobilization as mechanisms to promote the use of enzymes as materials. Hydrogen oxidation and hydrogen production are being investigated in solution, on electroactive surfaces, and in matrices. With the Douglas group we are investigating the coupling of different hydrogen producing catalysts to various photcatalysts to harvest light energy for hydrogen production from simple organics. Different aspects of this overall project are support by the Office of Energy Efficiency and Renewable Energy and the Office of Basic Energy Sciences.

In other work supported by the Office of Naval Research as part of the Center for BioInspired Nanomaterials (CBIN), we are investigating the feasibility of using immobilized hydrogen-producing phototrophic bacteria in bioreactors of various configurations for large scale hydrogen production.

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Novel Enzymatic Carboxylation Reactions

The Department of Energy Office of Basic Energy Sciences supports a project to examine mechanisms of novel carboxylase enzymes involved in the microbial metabolism of alkenes and epoxides. Several microorganisms have been shown to utilize short chain alcohols, alkenes, and ketones as sole sources of carbon. The metabolism of these carbon sources involves novel reactions and cofactors. With Scott Ensign's group at Utah State University, we study 2-ketopropyl coenzyme M oxidoreductase / carboxylase (2-KPCC), the enzyme that catalyzes the final step in the metabolism of propylene or propylene oxide in Xanthobacter autotrophicus strain Py2. The enzyme is a unique member of the disulfide oxidoreductase (DSOR) family of enzymes, includes glutathione reductase and dihydrolioamide dehydrogenase, both of which catalyze reductive cleavage of sulfur-sulfur bonds, and mercuric reductase, which catalyzes the thiol-dependent reduction of mercuric ion. Reductive cleavage occurs by a redox-active dithiol that is reduced via an enzyme bound FAD, the latter of which is reduced by NADPH. 2-KPCC is unique in that it catalyzes carbon-sulfur bond cleavage in concert with carboxylation. We are complementing the mechanistic enzymology work conducted in the Ensign lab with crystal structures of the enzyme in defined states along the catalytic pathway. We have determined ~eight structures of the enzyme, and we are beginning to get a vivid image of the mechanism. The hypotheses concerning the details of the mechanism derived from our structural insights provide a powerful basis for experimental design.

One major current thrust of the project is the de novo structure determination of another novel carboxylase from Xanthobacter Py2, Acetone carboxylase (AC). AC is a key enzyme in the metabolism of isopropanol and acetone, and catalyzes the MgATP-dependent conversion of acetone to acetoacetate. AC exists as a large heterohexamer (~350 Kd) and contains manganese. The limited information available from biochemical work in the Ensign group indicates that AC uses a unique and as of yet uncharacterized mechanism of carboxylation that is coupled to the hydrolysis of MgATP to MgAMP and 2Pi. We have had crystals of the project for some time but the structure determination has been difficult. Recently, we have been able to obtain interpretable electron density maps, and so we anticipate having a structure of this novel, cofactor-free carboxylase in the near future.

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Mercury Tolerance in Thermal Environments

In work supported by the Thermal Biology Institute we are investigating mercury-induced stress and tolerance in the hyperthermophilic Archeon Sulfolobus solfataricus. Mercury tolerance has been examined in a variety of organisms but not to an appreciable extent among the Archeae. We are using a multipronged approach to address 1) the initial protein targets of mercury exposure and inhibition of growth, 2) the accompanied stress response as related to the general stress response in the organism, and 3) onset of the mercury resistance machinery and how the machinery relates to that found in bacteria. We are using proteomic approaches and radionuclide sources of mercury to examine specific protein targets hit during mercury exposure. Both proteomic analysis and DNA microarray technologies will be exploited to determine the stress response and its relationship to general stress mechanisms, as well as the changes in gene expression and protein content related to implementation of the mercury resistance mechanism.

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