Progress 04/01/00 to 03/31/05
Outputs
The biological catalyst, nitrogenase, fixes N2 to ammonia by an unknown mechanism. The Azotobacter vinelandii enzyme consists of the MoFe protein and Fe protein, with substrate reduction occurring on the former. Our specific target is one of the two prosthetic groups within the MoFe protein, the FeMo-cofactor. Curiously, it has homocitrate (function unknown) bound to Mo. We have previously shown that the FeMo-cofactor contains the substrate-reducing site(s). Using site-directed mutagenesis to place amino-acid substitutions in its polypeptide environment, we have isolated altered nitrogenases and examined their changed catalytic properties for insight into how the enzyme works. These studies employed an alternative substrate, acetylene (C2H2), as well as N2. We found that acetylene has two binding sites; one of high affinity and shared with N2, and the other of low affinity. The low-affinity site, when occupied, inhibits overall substrate-reduction activity. Added
carbon monoxide, a nitrogenase inhibitor, did not relieve this inhibition, indicating that carbon monoxide binds to a separate (third) site. Much of these data and interpretations were obtained using labeled acetylene (C2D2) and whether cis or trans proton addition occurred during reduction to C2D2H2. These same studies also implicated a reversible protonation step in the mechanism. A related effort probed how the required protons are transferred to substrate. Using the dependence of activity on pH (activity-pH profiles), all MoFe proteins tested gave distorted bell-shaped profiles for H2 evolution underargon, indicating at least two acid-base groups were required for catalytic activity. The pKa value (6.3 for wild type) of acid-base group(s) that must be deprotonated for H2-evolution activity was affected by neither amino-acid substitution nor different substrates. However, some substitutions did affect the pKa (8.5 for wild type) of the group that must be protonated for activity. Of
these, one altered MoFe protein had citrate replacing homocitrate at Mo and the other had glutamine, which normally is hydrogen-bonded to the homocitrate, replaced by lysine. Both had similar profiles (pKa of 8), but clearly shifted from that of wild type. Thus, replacing either partner in this hydrogen-bonding system on Mo removes a contribution to this pKa and implicates this glutamine-homocitrate system as a component of the proton-delivery system for H2 evolution. Furthermore, both profiles closely resembled that of wild type with CO present. This similarity suggests that disturbing the glutamine-homocitrate system is equivalent to adding CO, indicating that the CO-binding site is spatially closeby and near Mo. When CO is added to the MoFe protein with the Lys-for-Gln substitution, the pKa shifts to ca. 7.0 with a symmetrical profile. This result suggests either that the contributions of two of the total of three acid-base groups have been lost (more likely) or that different
acid-base groups become involved depending on the conditions. The identity of the two latter groups remains unknown. A third project showed that the FeMo-cofactor can assume a variety of conformations and protonation states.
Impacts
Biological nitrogen fixation is Nature's major way of recycling the unusable nitrogen (N2) gas in the atmosphere into usable (fixed) forms of nitrogen as are found in commercial fertilizer. Fixed nitrogen is relatively scarce and it represents the limiting nutrient and most energy-intensive component of US agriculture. The focus of this project has been on the workings of the natural catalyst, an enzyme called nitrogenase. Our probes of its N2-fixation mechanism, using N2 as a direct probe, plus a variety of alternative substrates (like acetylene, azide, and cyanide) and inhibitors (like H2 and carbon monoxide), have indicated that at least two substrate-binding sites exist on a single prosthetic group within the enzyme. Although the detailed mechanism of N2 reduction reamins unsolved, these findings have generated targets for continued investigation and have indicated how this enzyme might be manipulated for potential agricultural benefit in the future.
Publications
- Maskos, Z., K. Fisher,M. Sorlie, W.E. Newton,and B.J. Hales. 2005. Variant MoFe Proteins of Azotobacter vinelandii: Effects of Carbon Monoxide on Electron Paramagnetic Resonance Spectra Generated During Enzyme Turnover, J. Biol. Inorg. Chem., 10: 394-406.
- Fisher, K., and W.E. Newton. 2005. Nitrogenase Proteins from Gluconacetobacter diazotrophicus, a sugarcane-colonizing bacterium, Biochim. Biophys. Acta, 1750: 154-165.
- Newton, W.E. 2005. Recent Progress in the Biochemistry of Molybdenum-containing Nitrogenases, in Biological Nitrogen Fixation for Sustainable Agriculture and the Environment, (Y.-P. Wang, L. Min, Z.-X. Tian, C. Elmerich, and W.E. Newton, Eds.), Springer, Dordrecht, The Netherlands, pp. 25-28.
- Newton, W.E. 2005. Ammonia Synthesis by Biological Catalysts, in Encyclopedia of Chemical Technology, John Wiley & Sons, NY, in press.
- Smith, B.E., R.L. Richards, and W.E. Newton. 2004. Catalysts for Nitrogen Fixation. Kluwer Academic Publishers, Dordrecht, The Netherlands, 340p.
- Klipp, W., B. Masepohl, J.R. Gallon, and W.E. Newton. 2004. Genetics and Regulation of Nitrogen Fixation in Free-Living Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands, 300p.
- Palacios, R., and W.E. Newton. 2005. Genomes and Genomics of Nitrogen-fixing Organisms. Springer, Dordrecht, The Netherlands, 246p.
- Werner, D., and W.E. Newton. 2005. Nitrogen Fixation in Agriculture, Forestry, Ecology and the Environment. Springer, Dordrecht, The Netherlands, 347p.
- Wang, Y-P., L. Min, Z-X. Tian, C. Elmerich, and W.E. Newton. 2005. Biological Nitrogen Fixation for Sustainable Agriculture and the Environment. Springer, Dordrecht, The Netherlands, 442p.
- Newton, W.E. 2005. Biological Nitrogen Fixation and Nitrification, in Biological Inorganic Chemistry: Structure and Reactivity (I. Bertini, H.B. Gray, J. Valentine, and E.I. Stiefel, Eds.), University Science Books, Sausalito, CA, 2005, in press.
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Progress 10/01/03 to 09/30/04
Outputs
Mo-nitrogenase consists of two component proteins, the Fe protein and the MoFe protein. The latter is a hetero-tetramer with the site(s) of substrate reduction provided by the FeMo-cofactor, which is located in its alpha-subunit. In this study, the interactions of acetylene (C2H2) with Azotobacter vinelandii Mo-nitrogenase were probed using isotopically labeled C2D2. C2D2 was used because, after reduction and protonation to ethylene (C2D2H2), both stereoisomers are produced; cis-1,2-d2-ethylene accounts for 95 percent and trans-1,2-d2-ethylene for 5 percent of the total product. The major goals were: to determine if C2H2 and N2 share common binding sites on the FeMo cofactor; to gain insight into the phenomenon that causes high concentrations of C2H2 to inhibit its own reduction; and to determine if any of the protonation steps in the C2H2-reduction pathway are reversible. Specifically, the effect on the formation of both cis- and trans-1,2-d2-ethylene from C2D2 were
measured as a function of: (i) increasing either the C2D2 concentration or the flux of electrons through the enzyme; (ii) adding individually either N2 or carbon monoxide (CO); or (iii) changing the pH of the reaction medium. The simplest interpretation of the results from the substrate-concentration-dependent and electron-flux-dependent experiments requires the presence of two C2H2-reduction sites; a higher-affinity site that produces only cis-1,2-d2-ethylene from C2D2 and a lower-affinity site that produces both cis- and trans-1,2-d2-ethylene. Added N2 specifically inhibited the production of cis-1,2-d2-ethylene from C2D2, which indicates that N2 binds to (and is reduced at) the higher-affinity acetylene-binding site only. Added CO (an inhibitor of substrate reduction, but not itself a substrate) did not affect the relative distribution of cis- and trans-isomers, indicating that CO binds to a separate site. The results of the pH-dependence experiments showed that inhibition of its
own reduction at high C2D2 concentrations is enhanced under acidic conditions but is absent under basic conditions. So, a low flux of protons has a similar impact to that of a low flux of electrons; furthermore, both low-flux conditions inhibit cis-1,2-d2-ethylene formation selectively. The pH-dependence experiments also showed production of d1-ethylene (C2DH3) from C2D2, but only at pH values greater than 7, where the supply of protons to accompany the electrons used to reduce C2D2 could become limited. This C2DH3 formation strongly implicates a reversible protonation step as an integral part of the mechanism of acetylene reduction and supports our previous suggestion of a key sigma-alkenyl intermediate, which is responsible for the mix of stereoisomers produced.
Impacts
Biological nitrogen fixation is Nature's major way of recycling the unusable nitrogen (N2) gas in the atmosphere into usable (fixed) forms of nitrogen as are found in commercial fertilizer. Fixed nitrogen is relatively scarce and it represents the limiting nutrient and most energy-intensive component of US agriculture. We are elucidating how the natural catalyst, an enzyme called nitrogenase, works in order to generate targets and methodologies for future agricultural benefit.
Publications
- Han, J. and Newton, W.E. 2004. Differentiation of Acetylene-Reduction Sites by Stereoselective Proton Addition During Azotobacter vinelandii Nitrogenase-Catalyzed C2D2 Reduction. Biochemistry 43: 2947-2956.
- Fisher, K. and Newton, W.E. 2004. Nitrogen Fixation, A Historical Perspective. Nitrogen Fixation: Origin, Applications, and Research Progress (B.E. Smith, R.L. Richards, and W.E. Newton, Eds.), Kluwer Academic Press, Dordrecht, The Netherlands, Vol. 1, pp. 1-31.
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Progress 10/01/02 to 09/30/03
Outputs
Nitrogenase fixes N2 to produce ammonia by an unknown reaction mechanism. It consists of two components, the MoFe protein and Fe protein, with the substrate-reduction reaction occurring on the MoFe protein. Many other substrates, in addition to N2, are catalytically reduced by nitrogenase; all interact with the FeMo-cofactor, which is one of the MoFe protein's two metal-cluster centers. There is no clear consensus as to whether different substrates are reduced at one or several sites on the FeMo-cofactor. The catalyzed reduction of acetylene has been an especially useful tool for both measuring activity and characterizing aspects of the reaction mechanism. Current understanding of the nitrogenase-acetylene interaction is: a) acetylene is reduced exclusively to ethylene; b) acetylene, at high concentrations, inhibits its own reduction by slowing the flow of electrons through the enzyme; c) at least two acetylene-reduction sites exist; and d) one acetylene-reduction
site is involved with N2 reduction. The interactions of acetylene with the FeMo cofactor of the MoFe protein of Azotobacter vinelandii nitrogenase were probed using C2D2. Specifically, the effects of changing C2D2 concentration, electron flow, the pH, or the individual presence of N2, ethylene, or CO on the formation rates of the two major products of C2D2 reduction, namely cis-and trans-1,2-d2-ethylene, were measured. An hypothesis, involving two acetylene-reduction sites, was developed to explain the changes observed in their individual rate of formation during substrate-concentration-dependent and electron-flow-dependent experiments. One of these sites is a higher-affinity acetylene-binding site that produces only the cis-isomer from C2D2. The other is a lower-affinity acetylene-binding site, which produces both the cis- and the trans-isomer. Added N2 specifically inhibits the production of the cis-isomer, which indicates that N2 binds to (and is reduced at) the higher-affinity
acetylene-binding site. Added ethylene behaved similarly but also inhibited the electron flow through the enzyme. Added CO, at very low concentrations, did not affect the relative distribution of cis- and trans-isomers, indicating a separate CO-binding site. The results of pH-dependence experiments showed that substrate inhibition at high C2D2 concentrations is enhanced under acidic conditions but is absent under basic conditions, suggesting that low proton availability (at high pH) has a similar impact to low electron availability in inhibiting cis-isomer formation selectively. Apparently, the factors affecting stereoselective protonation during C2D2 reduction could be the same as those that perturb protonation of the FeMo cofactor when acetylene is reduced. An additional product, d1-ethylene, was produced from C2D2, and its production was enhanced with increasing pH. This result implicates a reversible protonation step in the mechanistic pathway.
Impacts
Biological nitrogen fixation is Nature's major way of recycling the unusable nitrogen (N2) gas in the atmosphere into usable 'fixed' forms of nitrogen that can be used directly by plants. Because fixed nitrogen is almost always the limiting nutrient and most energy-intensive component of US agriculture, our experiments are designed to determine how the natural catalyst, an enzyme called nitrogenase, works in order to generate targets and methodologies for future agricultural benefit.
Publications
- K. Vichitphan and W. E. Newton, Acetylene Reduction with Azotobacter vinelandii Mo-Nitrogenase: Role of alpha-Glutamine-191 of the MoFe Protein; in Nitrogen Fixation: Global Perspectives. T. Finan, M.R. OBrian, D.B. Layzell, J.K. Vessey and W.E. Newton, Eds., CABI Publishing, NY, 2002, p. 365.
- H. Li, K. Kloos and W. E. Newton, Azotobacter vinelandii Nitrogenase Containing Altered MoFe Proteins with Substitutions at alpha-278-Ser: Interactions among Substrates and Inhibitors; in Nitrogen Fixation: Global Perspectives. T. Finan, M.R. OBrian, D.B. Layzell, J.K. Vessey and W.E. Newton, Eds., CABI Publishing, NY, 2002, p. 366.
- M.J. Dilworth, K. Fisher and W. E. Newton, Mechanistic Insights into the Functioning of Wild-type and Altered Azotobacter vinelandii Nitrogenase MoFe Proteins through their Interactions with HCN and CN-; in Nitrogen Fixation: Global Perspectives. T. Finan, M.R. OBrian, D.B. Layzell, J.K. Vessey and W.E. Newton, Eds., CABI Publishing, NY, 2002, p. 368.
- K. Fisher, D.J. Lowe and W. E. Newton, Pre-Steady-State Analysis of Reduced MoFe Proein Intermediates Generated during Enzyme Turnover from Wild-type and Altered Azotobacter vinelandii Nitrogenase MoFe Proteins; in Nitrogen Fixation: Global Perspectives. T. Finan, M.R. OBrian, D.B. Layzell, J.K. Vessey and W.E. Newton, Eds., CABI Publishing, NY, 2002, p. 369.
- W. E. Newton, K. Vichitphan and K. Fisher, Substrate Reduction and CO Susceptibility of the delta-nifV and alpha-Q191K MoFe Proteins of Azotobacter vinelandii Nitrogenase; in Nitrogen Fixation: Global Perspectives. T. Finan, M.R. OBrian, D.B. Layzell, J.K. Vessey and W.E. Newton, Eds., CABI Publishing, NY, 2002, p. 370.
- K. Fisher and W. E. Newton, Nitrogen Fixation - A General Overview; in Nitrogen Fixation at the Millennium. G.J. Leigh, Ed., Elsevier Science, Amsterdam, The Netherlands, 2002, pp. 1-34.
- W. E. Newton, Nitrogen Fixation and the Enzyme Nitrogenase; in Handbook of Food Enzymology. J.R. Whitaker, A.G.J. Voragen, G. Beldman, and D. Wong, Eds., Marcel Dekker, NY, 2003, pp. 101-116.
- K. Fisher and W. E. Newton, Plant Nutrition - Nitrogen Fixation; in Encyclopedia of Applied Plant Sciences. B.G. Murray, Ed., Elsevier Science, Oxford, UK, 2003, pp. 634-642.
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Progress 10/01/01 to 09/30/02
Outputs
Mo-nitrogenase consists of two component proteins, the Fe protein and the MoFe protein. The site of substrate reduction is provided by the FeMo-cofactor, located in the alpha-subunit of the MoFe protein. The FeMo cofactor's polypeptide environment is intimately involved in control of the enzyme's interactions with its substrates and inhibitors. In this work, the alpha-subunit 278-serine residue was targeted because (i) it is conserved in all nitrogenases; (ii) it is hydrogen-bonded to the 275-cysteine residue that directly ligates the FeMo-cofactor; and (iii) of its proximity to the 277-arginine residue, which may provide the entry/exit route for substrates and products. Altered MoFe proteins of Azotobacter vinelandii nitrogenase, with the 278-Thr, 278-Cys, 278-Ala and 278-Leu substitutions, were studied for their interactions with H+, C2H2, N2 and CO. All but the 278-Leu MoFe protein reduced N2. From measurements of C2H4 formation from C2H2, the 278-Ala and 278-Cys
MoFe proteins apparently bind C2H2 similarly to the wild type, whereas those with 278-Thr or 278-Leu have ten-times less affinity. Unlike wild type, these last two proteins produce some C2H6 directly from C2H2. When reducing C2H2, the 278-Ala and 278-Cys MoFe proteins respond to the inhibitor CO similarly to wild type, whereas 278-Thr is much more sensitive to CO. Under non-saturating CO concentrations, the 278-Leu MoFe protein catalyzes C2H2 reduction with sigmoidal kinetics, consistent with inhibitor-induced cooperativity between at least two C2H4-evolving sites. Together, these data suggest that the MoFe protein has at least two C2H2-binding sites, one of which is located near the 278 residue. N2 binding is similar to that of wild type, suggesting that the 278-serine residue is not directly involved. A second project attempted to find out how the protons necessary to complete all substrate-to-product transformations are transferred to substrate. The dependence of activity on pH
(activity-pH profiles) gave distorted bell-shaped activity-pH profiles for all MoFe proteins tested under argon alone (only H+ as substrate), with C2H2 as substrate, or with CO as inhibitor. At least two acid-base groups are required for catalytic activity. The pKa value (about 6.3 for wild type) of the deprotonated group was not affected either by substitution or by substrate. Most 278 substitutions (Cys, Ala and Leu) also did not affect the pKa (about 8.3 for wild type) of the protonated group, however, the Thr substitution shifted the pKa to about 8, which was the same shift as found for wild type with added CO. However, the pKa values of the altered MoFe proteins were not changed with added CO. These results suggest that the 278-serine residue is not one of the acid-base groups required for activity. Computer modeling suggests that this residue may 'fine-tune' the pKa of the responsible acid-base group(s). The activity-pH profiles under different atmospheres also support the idea
that more than one proton pathway appears to be involved in catalysis, and specific pathway(s) may be used by individual substrates.
Impacts
Biological nitrogen fixation is Nature's major way of recycling the unusable nitrogen (N2) gas in the atmosphere into usable 'fixed' forms of nitrogen as are found in commercial fertilizer. Fixed nitrogen is relatively scarce and it represents the limiting nutrient and most energy-intensive component of US agriculture. We are elucidating how the natural catalyst, an enzyme called nitrogenase, works in order to generate targets and methodologies for future agricultural benefit.
Publications
- No publications reported this period
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Progress 10/01/00 to 09/30/01
Outputs
Our specific target is one of the two prosthetic group types, the FeMo-cofactor, which we have previously shown to be the substrate-binding and -reducing site, within the MoFe-protein component of nitrogenase. We use directed mutagenesis to mutate one of the nitrogenase structural genes and so place a targeted amino-acid substitution in the polypeptide around this cluster. Then, after growing the mutant bacterial strains, inducing nitrogenase synthesis, and isolating the resultant altered nitrogenases, we use the observed changes in catalytic properties as an indicator of how the enzyme works. In this period, we have continued our studies on the effects of substitutions at residues, either glutamine-191 or histidine-195, in the alpha-subunit of the MoFe protein with particular emphasis on the interaction with nitrogen gas. In contrast to the wild-type MoFe protein, neither the 195Asn (with asparagine replacing histidine) nor the 191Lys (with lysine replacing
glutamine) MoFe protein catalyzed N2 reduction to ammonia, when complemented with wild-type Fe protein. However, N2 was bound by the 195Asn MoFe protein and inhibited the reduction of both protons and C2H2. The 191Lys MoFe protein did not interact with N2. With the 195Asn MoFe protein, removing the N2 reversed the N2-induced inhibition of substrate reduction. Surprisingly, even though added H2 (as expected) also relieved N2 inhibition of substrate reduction, the 195Asn MoFe protein did not catalyze HD formation under a N2/D2 atmosphere. This observation is the first indication that these two reactions have different chemical origins. The observed reactivity changes are correlated with likely structural changes caused by the amino-acid substitutions and provide important details about the interaction of N2 and H2 with nitrogenase. In a second project, rapid-freezing experiments elicited two transient EPR signals, designated 1b and 1c, from the wild type nitrogenase during turnover. The
first of the signals (1b) to form exhibited g values of 4.21 and 3.76. Its formation was at the expense of the EPR signal (1a, with g = 4.32, 3.66 and 2.01) of the resting state of the enzyme. The second signal (1c, with a g value of 4.69) formed very slowly and was of low intensity. Both signals occurred independently of the substrate being reduced. Increasing the flux of electrons through the MoFe protein caused these signals to form more rapidly. Moreover, after pre-treating a MoFe-protein solution to set up an equimolar mixture of its resting state and one-electron reduced state, these signals appeared even more rapidly when exposed to an excess of Fe protein. We have simulated the kinetics of formation of these EPR signals using a published kinetic model (Lowe, D.J. and Thorneley, R.N.F. 1984. Biochem. J. 224:887-909) and propose that they arise from reduced states of the MoFe protein and reflect different conformations of the FeMo-cofactor with different protonation states.
Impacts
Biological nitrogen fixation is Nature's major way of recycling the unusable nitrogen gas in the atmosphere into usable 'fixed' forms of nitrogen as are found in commercial fertilizer. Fixed nitrogen is relatively scarce and it represents the limiting nutrient and most energy-intensive component of US agriculture. We are elucidating how the natural catalyst, an enzyme called nitrogenase, works in order to generate targets and methodologies for future agricultural benefit.
Publications
- Fisher, K., Dilworth, M.J. and Newton, W.E. 2001. Differential effects on N2 binding and reduction, HD formation, and azide reduction with alpha-195His- and alpha-191Gln-substituted MoFe proteins of Azotobacter vinelandii nitrogenase. Biochemistry 39:15570-15577.
- Fisher, K., Newton, W.E. and Lowe, D.J. 2001. Electron paramagnetic resonance analysis of different Azotobacter vinelandii nitrogenase MoFe protein conformations generated during enzyme turnover: Evidence for S=3/2 spin states from reduced MoFe-protein intermediates. Biochemistry 40:3333-3339.
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Progress 10/01/99 to 09/30/00
Outputs
Our specific target is one of the two prosthetic group types, the FeMo-cofactor, which we have previously shown to be the substrate-binding and -reducing site, within the MoFe-protein component of nitrogenase. We use directed mutagenesis to mutate one of the nitrogenase structural genes and so place a targeted amino-acid substitution in the polypeptide around this cluster. Then, after growing the mutant bacterial strains, inducing nitrogenase synthesis, and isolating the resultant altered nitrogenases, we use the observed changes in catalytic properties as an indicator of how the enzyme works. In this period, we have studied the effects of substitutions at residues, glutamine-191 or histidine-195, in the alpha-subunit of the MoFe protein. We found that acetylene has two binding sites; one site exhibits high affinity and is shared with nitrogen, whereas the other is of low affinity. These two sites can also bind ethylene in the absence of acetylene. The reduction of
acetylene and ethylene share a common intermediate. The low-affinity binding site, when occupied by either acetylene or ethylene, inhibits overall substrate-reduction activity and also impacts the nature of the products formed from other nitrogenase substrates, like HCN, when mixtures of substrates are used. Added carbon monoxide, a well-known inhibitor of nitrogenase activity, did not relieve the inhibitory effects of acetylene/ethylene indicating that carbon monoxide manifests its effects through a separate (third) site. Molecular modeling indicates that a hydrogen bond between the histidine-195 residue and a central sulfide of the FeMo-cofactor may be important for substrate reduction. When glutamine replaces this histidine, the hydrogen-bond can still form but the resulting altered MoFe protein supports only extremely low rates of nitrogen reduction. However, it does support high rates of both acetylene and HCN reduction. Substitution with asparagine, where the hydrogen-bond
cannot form and where N2 is not reduced, also supports both acetylene and HCN reduction. Thus, if histidine-195 is involved in (and necessary for) protonation of N2, it is clearly not necessary for proton delivery to HCN, which must utilize a different proton-delivery channel into the FeMo-cofactor. Unlike the MoFe proteins substituted at position-195, the lysine-191 MoFe protein (with glutamine-191 substituted by lysine) was ineffective in both nitrogen and HCN reduction and suggests an alternative proton channel utilizing the 191 residue. We speculate that HCN, C2H2, and CO, unlike N2, either do not bind in the vicinity of the histidine-195 residue or do not depend on this residue for effective binding, protonation and/or reduction. We further suggest that the residue occupying the -191 position will have a critical impact on the binding, protonation and/or reduction of all nitrogenase substrates, whereas the residue at the -195 position will play a less important, but still
mechanistically insightful, role.
Impacts
Biological nitrogen fixation is Nature's major way of recycling the unusable nitrogen gas in the atmosphere into usable 'fixed' forms of nitrogen as are found in commercial fertilizer. Fixed nitrogen is relatively scarce and it represents the limiting nutrient and most energy-intensive component of US agriculture. We are elucidating how the natural catalyst, an enzyme called nitrogenase, works in order to generate targets and methodologies for future agricultural benefit.
Publications
- Newton, W.E. 2000. Nitrogen fixation in perspective. In: Nitrogen Fixation: From Molecules to Crop Productivity. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 3-8.
- Fisher, K., Dilworth, M.J., Kim, C.-H., and Newton, W.E. 2000. Azotobacter vinelandii nitrogenases containing altered MoFe proteins: Effects on catalyzed reduction of acetylene and ethylene. Biochemistry 39:2970-2979.
- Fisher, K., Dilworth, M.J., Kim, C.-H., and Newton, W.E. 2000. Azotobacter vinelandii nitrogenases with substitutions in the FeMo-cofactor environment: Effects on interactions with H, HCN and CN. Biochemistry 39:10855-10865.
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