Mechanism of Molybdenum Nitrogenase.

Biological nitrogen fixation is mediated by the nitrogenase enzyme system that catalyses the ATP dependent reduction of atmospheric dinitrogen to ammonia. Nitrogenase consists of two component metalloproteins, the MoFe-protein with the FeMo-cofactor that provides the active site for substrate reduction, and the Fe-protein that couples ATP hydrolysis to electron transfer. An overview of the nitrogenase system is presented that emphasizes the structural organization of the proteins and associated metalloclusters that have the remarkable ability to catalyse nitrogen fixation under ambient conditions. Although the mechanism of ammonia formation by nitrogenase remains enigmatic, mechanistic inferences motivated by recent developments in the areas of nitrogenase biochemistry, spectroscopy, model chemistry and computational studies are discussed within this structural framework.

Structural Basis of Biological Nitrogen Fixation.

The nitrogenase enzyme system catalyzes the ATP (adenosine triphosphate)-dependent reduction of dinitrogen to ammonia during the process of nitrogen fixation. Nitrogenase consists of two proteins: the iron (Fe)-protein, which couples hydrolysis of ATP to electron transfer, and the molybdenum-iron (MoFe)-protein, which contains the dinitrogen binding site. In order to address the role of ATP in nitrogen fixation, the crystal structure of the nitrogenase Fe-protein from Azotobacter vinelandii has been determined at 2.9 angstrom (A) resolution. Fe-protein is a dimer of two identical subunits that coordinate a single 4Fe:4S cluster. Each subunit folds as a single alpha/beta type domain, which together symmetrically ligate the surface exposed 4Fe:4S cluster through two cysteines from each subunit. A single bound ADP (adenosine diphosphate) molecule is located in the interface region between the two subunits. Because the phosphate groups of this nucleotide are approximately 20 A from the 4Fe:4S cluster, it is unlikely that ATP hydrolysis and electron transfer are directly coupled. Instead, it appears that interactions between the nucleotide and cluster sites must be indirectly coupled by allosteric changes occurring at the subunit interface. The coupling between protein conformation and nucleotide hydrolysis in Fe-protein exhibits general similarities to the H-Ras p21 and recA proteins that have been recently characterized structurally. The Fe-protein structure may be relevant to the functioning of other biochemical energy-transducing systems containing two nucleotide-binding sites, including membrane transport proteins.

https://science.sciencemag.org/content/sci/257/5077/1653.full.pdf

Crystallographic Structure of the Nitrogenase Iron Protein from Azotobacter vinelandii

A method for the isolation of an iron-molybdenum cofactor (FeMoCo) from component I of nitrogenase is described. This method is used to isolate FeMoCo from aerobic, anaerobic, facultative, and photosynthetic nitrogen-fixing organisms. The Fe/Mo ratio in the FeMoCo from Azotobacter vinelandii and Clostridium pasteurianum is 8:1. The FeMoCo contains six atoms of acid-labile sulfide per eight Fe atoms. Crystalline component I from A. vinelandii contains 2 Mo, 33 Fe, and 27 acid-labile sulfide atoms per molecular weight of 250,000. The specific activity of FeMoCo is 425 nmol of C(2)H(4) formed/min per nmol of Mo. There is better than 98% reconstitution between FeMoCo and inactive component I in A. vinelandii mutant strain UW45. The FeMoCo yield from component I is about 90%. FeMoCo from nitrogenase component I of C. pasteurianum, Klebsiella pneumoniae, Bacillus polymyxa, and Rhodospirillum rubrum activates inactive component I in an extract from mutant strain UW45 and follows saturation kinetics. The FeMoCo in various nitrogen-fixing organisms seems to be very similar. Wild-type A. vinelandii derepressed for nitrogenase synthesis in tungsten-containing medium and K. pneumoniae mutant strain UN109 are also activated in vitro by FeMoCo. The FeMoCo is very sensitive to oxygen, but is stable even at room temperature as long as it is kept anaerobic and in N-methylformamide, the solvent used for its isolation. FeMoCo is unstable in an aqueous environment, even though it is kept strictly anaerobic. Knowledge of the structure of this cofactor should be useful for understanding the role of molybdenum at the active site of nitrogenase, role of ligands close to molybdenum in electron and proton transfer, and the catalytic mechanism of nitrogen fixation. The FeMoCo might be used as a model for synthesizing catalysts for chemical nitrogen fixation.

https://www.pnas.org/content/pnas/74/8/3249.full.pdf

Isolation of an iron-molybdenum cofactor from nitrogenase

https://science.sciencemag.org/content/sci/188/4188/633.full.pdf

Nitrogen fixation research: a key to world food?

The oxidation-reduction properties of azoferredoxin, molybdoferredoxin, and the inactive species of molybdoferredoxin, all iron-sulfur proteins purified from Clostridium pasteurianum, were studied by potentiometry combined with electron paramagnetic resonance spectroscopy at low temperature. The technique, evaluated with spinach ferredoxin for which a midpoint potential of -446 mV was determined (pH 7.9, showed that azoferredoxin has a midpoint potential of -294 ± 20 mV (pH 7.5). In the presence of MgATP2−, azoferredoxin undergoes a negative shift of 110 mV in its oxidation-reduction potential. The resulting potential of approximately -400 mV is likely to be effective under physiological conditions. The potential shift is consistent with a proposed conformational change of the protein caused by the binding of two molecules of ATP to the protein (dimer). Molybdoferredoxin and its inactive species, which has an incomplete iron-sulfur and molybdenum centre, have midpoint potentials of approximately −20 mV and −395 mV, respectively, both at pH 7.5. All nitrogenase proteins are characterized by the transfer of one electron per redox step.



Kinetic studies of the reduction of azoferredoxin and molybdoferredoxin by dithionite gave half times of <10 ms and 3–5 min, respectively. Hence, the latter process must be excluded from being part of the physiological reaction. The complex formation of ATP with azoferredoxin is a relatively slow process, which is markedly accelerated by molybdoferredoxin in the reconstituted nitrogenase system. Changes in the electron paramagnetic resonance signals of reconstituted nitrogenase were followed by a rapid-freeze technique as a function of mixing sequence and ratio of the two proteins. The observed signal changes were independent of the mixing sequence. The steady-state level of the molybdoferredoxin signal was also independent of the ratio of the two proteins and was less than 15% of the original resonance. The results indicate that the role of the azoferredoxin · ATP complex may not be confined solely to the function of an electron carrier for the nitrogenase reaction.

Electron-Paramagnetic-Resonance Studies on Nitrogenase

Electron paramagnetic resonance studies on nitrogenase. II. Interaction of adenosine 5'-triphosphate with azoferredoxin

The nitrogen-fixing complex of bacteria.

Electron paramagnetic resonance studies on nitrogenase. III. Function of magnesium adenosine 5′-triphosphate and adenosine 5′-diphosphate in catalysis by nitrogenase

Molybdoferredoxin, the high-molecular-weight component of clostridial nitrogenase has been separated into two components by gradient chromatography on DEAE-cellulose. One component when combined with azoferredoxin, the low-molecular-weight component of nitrogenase, does not reduce acetylene, evolve hydrogen from dithionite or hydrolyse ATP; the other does. Compared with the active component, the inactive species lacks molybdenum and approximately two thirds of the iron. Both species can be identified by their electron paramagnetic resonance spectra. Active molybdoferredoxin has resonances at g= 4.27, 3.78 and 2.01, whereas the inactive species has signals at g= 2.05 and 1.92. Apart from the difference in their catalytic centre, the protein moiety of both species seems to be identical as based on subunit composition, sedimentation and electrophoretic behaviour, amino acid composition and immunological reaction with a molybdoferredoxin antiserum. The method currently employed for purification of nitrogenase proteins and the fractionation of molybdoferredoxin is described. The consequence of this finding influences previously reported physico-chemical data of molybdoferredoxin.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1973.tb02852.x

Walter G. Zumft

Papers

Electron-Paramagnetic-Resonance Studies on Nitrogenase

Electron paramagnetic resonance studies on nitrogenase. II. Interaction of adenosine 5'-triphosphate with azoferredoxin

The nitrogen-fixing complex of bacteria.

Electron paramagnetic resonance studies on nitrogenase. III. Function of magnesium adenosine 5′-triphosphate and adenosine 5′-diphosphate in catalysis by nitrogenase

Evidence for a Catalytic-Centre Heterogeneity of Molybdoferredoxin from Clostridium pasteurianum