Progress 03/01/04 to 02/28/09
Outputs
We are studying the microbial metabolism of one-carbon compounds (CO, CO2, methane) and xenobiotics (e.g., PCBs); oxygen sensing in the human carotid body; and the roles of metal ions in biology, including the mechanisms of nickel, B12, heme, and iron-sulfur enzymes. We use transient and steady-state kinetics, spectroscopy, and molecular biology to uncover mechanistic information. The research is funded by NIH and DOE. Certain anaerobic microbes use chlorinated aromatics like PCBs as electron acceptors, coupling dehalogenation to energy conservation. We are characterizing the mechanisms of the Vitamin B12 containing enzyme (CprA) that catalyzes dehalogenation, and the transcriptional activator (CprK) that regulates expression of proteins involved in metabolizing these xenobiotics through effector binding and a thiol/disulfide redox switch.
Impacts
We use a variety of biological, spectroscopic, structural, and kinetic tools to elucidate the mechanisms of redox enzymes. We are studying methyl coenzyme M reductase, which contains a nickel tetrapyrrole and is responsible for all biologically generated methane. We recently trapped an alkyl-Ni intermediate and are elucidating its role in catalysis. We are characterizing a nickel-iron-sulfur enzyme called CO dehydrogenase (CODH) that enables microbial growth on CO2 and the toxic gas CO. CODH complexes another nickel iron-sulfur enzyme, acetyl-CoA synthase (ACS), forming a macromolecular machine that catalyzes an intriguing condensation reaction between in situ generated CO, Coenzyme A, and a methyl group to generate acetyl-CoA. This is a novel way to generate this important metabolic intermediate and high-energy compound. During catalysis, CO migrates through a 70 angstom channel. We also are studying methyl and proton transfer in a methyltransferase and a vitamin
B12/iron-sulfur protein and characterizing how enzymes like pyruvate ferredoxin oxidoreductase catalyze reactions involving radical intermediates. We are studying the human heme oxygenase-2 (HO-2), which plays an important role in heme homeostasis and in generating CO, a signal molecule that regulates many physiological processes. We recently uncovered a thiol/disulfide redox switch that regulates heme binding and are evaluating the physiological role of this switch. We also are determining how HO-2 regulates a potassium channel, enabling HO-2 control of oxygen levels in the blood stream.
Publications
- Dey, M., Kunz, R. C., Lyons, D. M., and *Ragsdale, S.W. (2007) Characterization of Alkyl-Nickel Adducts Generated by Reaction of Methyl-Coenzyme M Reductase with Brominated Acids Biochemistry, in press.
- Dey M, Telser, J, Kunz R.C., Lees, N.S., Ragsdale, S.W., *Hoffman, B. (2007) Biochemical and spectroscopic studies of the electronic structure and reactivity of a methyl-Ni species formed on Methyl-Coenzyme M Reductase, Journal of the American Chemical Society, in press. Faculty of 1000 Pick.
- Parkin, A., Seravalli, J., Vincent, K. A., Ragsdale, S. W., and *Armstrong, F. A. (2007) Rapid electrocatalytic CO2/CO interconversions by Carboxydothermus hydrogenoformans CO dehydrogenase I on an electrode. Journal of the American Chemical Society 129: 10328-10329.
- Yi, Li and *Ragsdale, S.W. (2007) Evidence that the heme regulatory motifs in heme oxygenase-2 serve as a thiol/disulfide redox switch regulating heme binding. Journal of Biological Chemistry, 282: 21056-21067.
- Doukov, T.I., Hemmi, H., Drennan, C.L., and *Ragsdale, S.W. (2007) Structural And Kinetic Evidence For An Extended Hydrogen Bonding Network In Catalysis Of Methyl Group Transfer: Role Of An Active Site Asparagine Residue In Activation Of Methyl Transfer By Methyltransferases, Journal of Biological Chemistry 282: 6609-6618.
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Progress 10/01/05 to 09/30/06
Outputs
In studies on acetate synthesis, we characterized pyruvate ferredoxin oxidoreductase (PFOR), which is central to the anaerobic metabolism of many bacteria and amitochondriate eukaryotes. PFOR contains thiamine pyrophosphate (TPP) and three [4Fe-4S] clusters, which link pyruvate oxidation to reduction of ferredoxin. Our results showed that rapid electron transfer occurs through the electron-transfer chain, which leaves an oxidized proximal cluster poised to accept an electron from the HE-TPP radical in the subsequent reaction step. We also characterized the radical intermediate of PFOR by electron paramagnetic resonance (EPR) spectroscopy at X-band and D-band microwave frequencies. We determined that this radical is a planar, hydroxyethylidene-thiamine pyrophosphate (HE-TPP) pi-radical, in which spin is delocalized onto the thiazolium sulfur and nitrogen atoms. Earlier it had been proposed based on X-ray crystallographic studies that this radical is a nonplanar,
sigma/n-type cation radical. We also characterized the protein that is a methyl carrier in the acetyl-CoA pathway using a variety of spectroscopic methods and identified the ligands to Cobalt in its various oxidation states. In studies on methanogenesis, we studied methyl-coenzyme M reductase (MCR), which catalyzes the final step of methanogenesis in which coenzyme B (HSCoB) and methyl-coenzyme M (CH(3)-SCoM) are converted to methane and the heterodisulfide, CoM-SS-CoB. At the active site of MCR is coenzyme F(430), a nickel tetrapyrrole. There are two mechanisms that are hotly debated: one involves an alklnickel intermediate, the other a radical methyl group. We published results that provide evidence for the intermediacy of an alkylnickel species in the final step in anaerobic methane oxidation and in the initial step of methanogenesis. We also studied the factors that predispose the F430 cofactor to undergo one- versus two-electron reduction. In studies of dehalorespiration, we
determined the crystal structure of the transcriptional activator protein. We also identified four cysteine residues that control dimerization and DNA binding and constitue a redox swich that ensures that dehalorespiration will only occur when the appropriate redox conditions are present in the cell. I also wrote a review articles on the general topic of how enzymes catalyze difficult reactions.
Impacts
Our work over the past year has provided important information about how enzymes use radicals to catalyze difficult reactions. We also have advanced studies of methanogenesis in providing important information supporting one of the two mechanisms proposed for how methane is generated. Evidence for an alkyl-nickel intermediate makes methanogenesis another of the few examples of organometallic chemistry in biology. This provides a better fundamental understanding of how microbes make methane and how metalloenzymes undergo activation. This could lead to better systems for local generation of methane and also to a product that can be used to decrease atmospheric levels of methane and lower the cost of raising livestock. Our dehalorespiration work provides fundamental information about how microbes respond to and metabolize toxic chlorinated compounds that are listed as major pollutants by the EPA and the United Nations. This work also represents the first example of one
of the major classes of transcriptional activators being trapped and visualized at atomic level in both the substrate-bound and -free states. This work also uncovers a previously unrecognized layer of control of dehalorespiration by redox conditions.
Publications
- Kunz, R., Horng, Y.C., and *Ragsdale, S.W. (2006) Interaction and reaction of methyl-coenzyme M reductase with its potent inhibitor, 3-bromopropane sulfonate. Journal of Biological Chemistry, in press.
- Dey, M., Kunz, R., Heuvelen, K. M. V., Craft, J. L., Horng, Y.-C., Tang, Q., Bocian, D. F., George, S. J., Brunold, T. C., *Ragsdale, S. W. (2006) Cofactor- versus Metal-Centered Reduction of the Coenzyme F430. Biochemistry 45:11915-11933.
- Astashkin, A.V., Seravalli, J., Mansoorabadi, S.O., Reed, G.H., and *Ragsdale S.W. (2006) Pulsed Electron Paramagnetic Resonance Experiments Identify the Radical Intermediates in the Pyruvate Ferredoxin Oxidoreductase Catalytic Cycle. Journal of the American Chemical Society 128: 3888-9.
- Stich, T.A., Seravalli, J., Venkateshrao, S., Spiro, T.G., *Ragsdale, S.W. and *Brunold, T.C. (2006) Spectroscopic Studies of the Corrinoid/Iron-Sulfur Protein from Moorella thermoacetica, Journal of the American Chemical Society 128: 5010-5020.
- Mansoorabadi, SO., Seravalli, J., Furdui, C., Krymov, V., Gerfen, GJ., Begley, TP., Melnick, J., Ragsdale, SW., and *Reed, GH. (2006) EPR Spectroscopic and Computational Characterization of the Hydroxyethylidine-Thiamin Pyrophosphate Radical Intermediate of Pyruvate:Ferredoxin Oxidoreductase. Biochemistry 45: 7122-7131.
- Pop, S.M., Gupta, N., Raza, A.S., and *Ragsdale, S.W. (2006) Transcriptional activation of dehalorespiration: identification of redox-active cysteines regulating dimerization and substrate binding. Journal of Biological Chemistry 281: 26382-90.
- Joyce, M. G., Levy, C., Gabor, K., Pop, S. M., Biehl, B. D., Doukov, T. I., Ryter, J. M., Mazon, H., Smidt, H., van den Heuvel, R. H. H., Ragsdale, S. W., van der Oost, J., and *Leys, D. (2006) CprK crystal structures reveal mechanism for transcriptional control of halorespiration. Journal of Biological Chemistry: 281: 28318-25.
- Ragsdale, S. W. (2006) Metals and their scaffolds in catalyzing difficult reactions. Chemical Reviews, 106: 3317-37.
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Progress 10/01/04 to 09/30/05
Outputs
With respect to our goals of understanding how anaerobic microbes utilize CO and CO2 as a source of carbon and energy, there have been several accomplishments. In the area of acetyl-CoA synthesis (a) we have shown that this pathway occurs through a radical intermediate on CO dehydrogenase (CODH). In demonstrating the radical intermediate on CODH, we have settled a long-standing controversy in the field. (b) We also identified the general base catalyst in the CODH mechanism. Identification of the general base catalyst in enzymatic CO oxidation by an undergraduate in my laboratory is a major step in understanding the CODH mechanism and opens up a number of experiments that should lead to trapping of a major intermediate in the catalytic cycle. We will use various spectroscopic methods, including stopped flow infrared and crystallography, to trap the early intermediates in the pathway that do not accumulate in the wild type protein. (c) We described the ligands to the
B12 cofactor in the corrinoid protein. Determining that the corrinoid protein contained a water ligand has been a point of controversy. (d) We defined the structure of the radical intermediate in pyruvate oxidation by pyruvate ferredoxin oxidoreductase (PFOR) and determined the iron sulfur mediators that it is coupled to. The structure of the radical revises the current picture that was published in a recent Science paper. In the area of methanogenesis, (a) we have discovered a new protein that appears to be involved in activation of methyl-CoM reductase, the key enzyme in this pathway. Our work helps clarify how this activation takes place. A number of biochemical studies are opening up based on this discovery. (b) We also have isolated a new protein involved in synthesis of a key methanogenic cofactor. The new cofactor biosynthetic enzyme can be targeted for inhibition of methanogenesis, In the area of dehalorespiration, we have shown that the process is under redox and effector
regulation. The redox regulation is at the level of disulfide bonds which were identified as one intermolecular and one intramolecular. Dehalorespiration offers a way to detoxify chlorinated aromates like PCBs, which are listed as major pollutants by the EPA and the United Nations. With respect to our objective to develop inhibitors of methanogenesis, we cloned and purified a new protein that can serve as a target for inhibition and developed assays for this enzyme. The methanogenesis inhibitors we are developing have the promise of use in animal feed to reduce atmospheric methane concentrations, which could alleviate the greenhouse effect, and to lower the cost of raising livestock.
Impacts
With respect to our goals of understanding how anaerobic microbes utilize CO and CO2 as a source of carbon and energy, our work has several impacts. The work has resolved some points of controversy as outlined in the accomplishments section. CO is becoming recognized as a major intermediate in a variety of microbial systems and our work is providing a better understanding of how soil bacteria transfer CO as well as use it as a metabolic intermediate. We expect to develop a better fundamental understanding of how microbes make methane and how metalloenzymes undergo activation. This could lead to better systems for local generation of methane and also to a product that can be used to decrease atmospheric levels of methane and lower the cost of raising livestock. We expect to provide fundamental information about how microbes respond to and metabolize toxic chlorinated compounds. Many of these compounds are listed as major pollutants by the EPA and the United Nations.
Publications
- George, S. J., Seravalli, J., and Ragsdale. S. W. (2005). EPR and infrared spectroscopic evidence that a kinetically competent paramagnetic intermediate is formed when acetyl-coenzyme A synthase reacts with CO. 127: 13500 -13501.
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Progress 10/01/03 to 09/30/04
Outputs
In research on the key enzymes involved in carbon monoxide metabolism, we have examined the active site of the CO dehydrogenase (CODH) from Carboxydothermus hydrogenformans using Ni K-edge X-ray absorption near edge spectroscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy. The enzyme was studied under three conditions: as-isolated and after treatment with CO or Ti(III). We found that the active site (C cluster) undergoes structural rearrangement after CO treatment, and the observed changes help reconcile the two different crystal structures. Wwe studied the bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) that plays a central role in anaerobic autotrophic CO2 fixation. We studied enzyme preparations with a wide range of Ni and Cu contents and determined the effects of metal chelators on ACS activity. Combined with spectroscopic and computational studies, we found that the CuNi enzyme is inactive and that the NiNi enzyme is active, and
that the NiNi enzyme is responsible for the characteristic EPR spectoscopic signal of the enzyme. The vulnerability of the active site to substitution with different metals appears to underlie the heterogeneity observed in samples that has confounded studies of CODH/ACS for many years. In studies of the methanogenic enzyme responsible for the final step of methane reduction, we characterized the redox state of the active site metal cluster as a Ni(I) species and also another state that involves reduction of the tetrapyrrole ring, not the metal. We studied the enzyme involved in the first committed step in biosynthesis of the essential cofactor for methanogens, methanopterin, 4-(beta-D-ribofuranosyl) amino-benzene-5'-phosphate (RFA-P) synthase. We purified this enzyme and determined the kinetic mechanism including the order of binding of substrates and release of products. We found that this enzyme lacks any chromogenic cofactor and ruled out the presence of pyridoxal phosphate and the
mechanistically related pyruvoyl cofactor. In studies of anaerobic dehalogenation of toxic chlorinated aromatics, we cloned, actively overexpressed in Escherichia coli, and purified to homogeneity the transcriptional activator, called CprK, of the cpr gene cluster. We found that CprK binds its effector molecule, 3-chloro-4-hydroxyphenylacetate (CHPA) with high affinity, which promotes its specific interaction with a DNA sequence (TTAAT-N(4)-ACTAA) located upstream of the promoter regions of several cpr genes and activates transcription of these genes. We determined the requirement for binding of different chlorinated compounds. Lacking metals, CprK was found to be oxygen sensitive. Oxidation by diamide, which converts thiols to the disulfide, inactivates CprK and reduction of the oxidized protein by dithiothreitol fully restores DNA binding, indicating that CprK is redox regulated and is active only when reduced. This is the first reported characterization of a transcriptional
regulator of anaerobic dehalorespiration.
Impacts
We expect to provide information about how microbes respond to and metabolize toxic chlorinated compounds. We expect to develop a product that can be used to decrease atmospheric levels of methane and lower the cost of raising livestock. We expect to determine the mode of cofactor reduction by methane producing bacteria. We expect to develop a better understanding of the mechanism by which anaerobic microbes use complex metallocenters to generate and utilize carbon monoxide.
Publications
- Pop, S., Kolarik, R.J., and Ragsdale, S.W. (2004) Regulation of anaerobic dehalorespiration by the transcriptional activator CprK. Journal of Biological Chemistry: 279: 49910-49918.
- Dumitru, R. V., and S. W. Ragsdale (2004) Mechanism of Beta-RFA-P Synthase, a Key Enzyme in the Methanopterin Biosynthetic Pathway. Journal of Biological Chemistry 279: 39389-39395.
- Craft, J. L., Y.-C. Horng, S. W. Ragsdale, and T. C. Brunold (2004) Nickel Oxidation States of F430 Cofactor in Methyl-Coenzyme M Reductase. Journal of the American Chemical Society: 126: 4068 - 4069
- Craft, J.L., Horng, Y.-C., Ragsdale, S.W., and Brunold, T.C. (2004) Spectroscopic and computational studies of the nickel-containing F430 cofactor in methyl-coenzyme M reductase. Journal of Biological Inorganic Chemistry 9: 77-89.
- Gu, W., J. Seravalli, S. W. Ragsdale, and S. P. Cramer (2004) CO-Induced Structural Rearrangement of the C-cluster in Carboxydothermus hydrogenoformans CO Dehydrogenase - Evidence from Ni K-Edge X-ray Absorption Spectroscopy. Biochemistry: 43: 9029-9035.
- Seravalli, J., Xiao, Y., Gu, W., Cramer, S.P., Antholine, W.E., Krymov, V., Gerfen, G.J., and Ragsdale, S. W. (2004) Evidence That Ni-Ni Acetyl-CoA Synthase Is Active And That The Cu-Ni Enzyme Is Not. Biochemistry: 43: 3944 - 3955.
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