Nitrogenases

Nitrogenase reduces N2 and several nitrogen-containing substrates to ammonia and amines. This assay uses a dabsyl precolumn derivatization method in quantitating the products. 

The dabsyl derivatives of ammonia and methylamine are separated from other reaction components by chromatography an Altex Ultrasphere-ODS Cjs column (4.6 mm X 150 mm, 5 /im). An anion guard column from Bio-Rad (Anion-SA, sulfate form, 4.6 mm X 40 mm) was placed ahead of the main column. For ammonia detection only, an Altex Ultrasphere-ODS column 

The reaction was stopped by addition of 0.1 mL of 1 N HC1 in saturated KI03. Following centrifugation, derivatization was performed on a 0.4 mL aliquot by adding 0.4 mL of 0.164 M borate buffer (pH 10.0) followed by 0.4 mL of 1.67 mM dansyl chloride (dissolved in acetone). After 90 minutes of incubation at room temperature, a 20 /xL sample was injected directly into the HPLC. To minimize background ammonia and methyl amine, it was important to prepare all reagents in HPLC-grade water. 

The source of enzyme was a purified nitrogenase preparation from Azoto-bacter vinelandii. 

Nitrogenase (N2ase), like MMO, is another marvel of Nature. It is capable of converting atmospheric dinitrogen to ammonia under ambient conditions, although the strong N-N bond consumes sixteen equivalents of ATP for every mole of N2. 

This complicated but elegant active site cluster provided many more possibilities for the site of N2 reduction such as the apparently three-coordinate iron centres. Despite being coordinatively saturated in the crystal structure, the Mo centre may still be viable in that the homocitrate may release one site for an incoming N2. DFT calculations indicate that the homocitrate ligand of the cofactor can become monodentate on reduction, allowing N2 to bind at Mo [73]. 

Many computational studies appeared purporting to shed light on the site of N2 coordination and the mechanism of its protonation and reduction to ammonia [74-82]. However, the calculations are extremely difficult since it is hard to know what the ionisation state of the cluster is and what is its overall spin state. Even if you get these details right, there is the extra complication that the enzyme uses two more electrons than the six required for N2 reduction, generating a molecule of H2 for every two molecules of NH3. However, the greater problem is that more recent refinements of the X-ray diffraction data [40] have revealed a hitherto unknown atom at the very heart of the cluster bringing into question all the previous computational work where no such atom was present. Experiment cannot distinguish the identity of this atom but both Hinnemann and Norskov [83] and Lovell et 

Nitrogenase.—A review of the recent chemistry of nitrogen fixation has appeared. The iron protein of nitrogenase from Clostridium pasteurianum acts as a one-electron redox system in the sequence  

Two binding sites for acetylene have been detected by e.p.r. studies on the (Fe-Mo) protein of nitrogenase from Klebsiella pneumoniae. The reduction product, ethylene, has a single site and only the stronger acetylene site with an association constant in excess of 6 x 10 M at pH 7.4 and 10 °C is catalytically active. Binding of acetylene or carbon monoxide at the second site results in a dead-end complex. 

Thiol extrusion of the FeS centres from Azobacter vinelandii and Clostridium pasteurianum with alkyl fluorothiols allows identification of at least two Fe4S4 and one FegSj cluster in the (Fe-Mo) protein. A cluster model in which two Fe4S4 units are bridged through a comer iron on each cube by a Mo S4 unit is proposed for the active site of the (Fe-Mo) cofactor. Molecular nitrogen binds axially to the central molybdenum and is Ji-bonded to the iron in the cubes. This weakens and activates the N=N bond and electrons are injected stepwise via the two iron-sulphur cubes with successive protonation at the terminal N. This latter point is consistent with a report that N—NHg is important in nitrogen fixation. 

The synthesis of Mo-Fe-S clusters as models for the active site of the enzyme has been possible.The clusters consist of two M0FC3S4 cubes bridged through the molybdenum corners by three S atoms but cannot be true analogues because they differ in the Fe S Mo ratio. Mononuclear Mo complexes of cysteinyl peptide ligands have also been reported. 

The active site of nitrogenase may involve a cycle between Mo and Mo during catalysis. Reversible binding of acetylene in the molybdenum(iv) complex [OMo(S2CNEt2)2] is a prerequisite to borohydride reduction to ethylene. It is 

Since the time of Daniel Rutherford, who discovered molecular nitrogen about 200 years ago, this gas has served as an example of a very inert substance. Thus, the mechanism of the relatively fast reduction of N2 in the nitrogenase active site with turnover about 0.2 s 1 appears as a mysterious and challenging problem not only for biochemists but for chemists as well. 

Recent developments in this important field have been reviewed in the last decade (Burgess and Lowe, 1996 Howard and Rees, 1996 Seefeldt and Dean, 1997 Smith, 1994, 1999 Smith et al., 1995 Tikhonovich et al., 1995 Likhtenshtein and Themeley, 1995 Thikhonovich et al., 1995 Shilov, 1997 Themeley and Dean, 2000 Rees and Howard, 2000 Chiu et al., 2001 Elmerich, 2001 Syrtsova and Timofeeva, 2001). 

STRUCTURE AND PHYSICO-CHEMICAL PROPERTIES OF THE NITROGENASE ACTIVE SITES. 

More detailed information about structure and spectral properties of the nitrogenase Fe-clusters were obtained by a combination of physical methods. The structure suggested at that time and variation of spectra parameters is presented in Fig. 3.1, which was plotted on the basis of the data obtained in the works of Ohrme-Johnson s and Miinck s groups cited above. Subsequent investigations have confirmed the main parameters and added some important details. 

The principle advances in the area has been made using x-ray structural analysis. Crystallographic data have been first produced for the nitrogenase complex of FeP (A2) and FeMoP (Al) from Azotobacter vinelandii (Kim and Rees, 1992) and for the corresponding complex of Cp2 and Cpl from Clostridium pasterianum (Bolen et al., 1993), ). A 1.6 A resolution X-ray crystallographic structure of Klebsiella pneumoniae proteins has been recently reported (Mayer et al., 1999) It was shown that FeMoco sites in Al, Cpl, and Kpl are 70 A apart and FeMoco and P clusters are separated by about 19 A. X-ray structures of the nitrogenase complex and the active site clusters are presented in (Figs. 3.2-3.4). 

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Many key protein ET processes have become accessible to theoretical analysis recently because of high-resolution x-ray stmctural data. These proteins include the bacterial photosynthetic reaction centre [18], nitrogenase (responsible for nitrogen fixation), and cytochrome c oxidase (the tenninal ET protein in mammals) [19, 20]. Although much is understood about ET in these molecular machines, considerable debate persists about details of the molecular transfonnations. 

The nitrogenase system reduces hundreds of millions of kilograms of nitrogen gas to ammonia each year, catalysing tire reaction at ambient temperatures and atmospheric pressure. Nitrogenase consists of two proteins tliat contain... 

Schindeiin N, Kisker C, Sehiessman J L, Howard J B and Rees D C 1997 Structure of ADP center dot AiF(4)(-)-stabiiized nitrogenase compiex and its impiications for signai transduction Nature 387 370-6... 

Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedraHy coordinated by a combination of thiolate and sulfide donors. Of the 10 or more stmcturaHy characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein mbredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane stmctures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane stmcture (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH at a MoFe Sg homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

Molybdenum. Molybdenum is a component of the metaHoen2ymes xanthine oxidase, aldehyde oxidase, and sulfite oxidase in mammals (130). Two other molybdenum metaHoen2ymes present in nitrifying bacteria have been characteri2ed nitrogenase and nitrate reductase (131). The molybdenum in the oxidases, is involved in redox reactions. The heme iron in sulfite oxidase also is involved in electron transfer (132). 

Free-living bacteria are, however, used as the source of the enzyme nitrogenase, responsible for N2 fixation (1,4,26,80), for research purposes because these ate easier to culture. The enzyme is virtually identical to that from the agriculturally important thizobia. These free-living N2-fixets can be simply classified into aerobes, anaerobes, facultative anaerobes, photosynthetic bacteria, and cyanobacteria. 

AH three nitrogenases comprise two separately puriftable component proteins. Each has a specific homodimeric Fe protein. The Fe... 

Fig. 4. Requirements, substrates, and products of Mo-nitrogenase catalysis, where I is the MoFe protein II the Fe protein and Pi is inorganic phosphate. The generating system is composed of creatine phosphate and creatine phosphokinase to recycle the inhibitory MgADP produced during catalysis to...

In contrast to the situation with the alternative nitrogenases, but with the notable exception of the C. pasteurianum proteins, the component proteins from aU. Mo-based nitrogenases interact as heterologous crosses to form catalyticaHy active enzymes (52). Carbon monoxide, CO, is a potent inhibitor of aU. nitrogenase-cataly2ed substrate reductions, with the exception of reduction (126). Molecular hydrogen has a unique involvement with Mo-nitrogenase... 

Fig. 6. View of the nitrogenase MoFe protein P-cluster pair where ( ) represents Fe, (O) S, and (Q) C as modeled (153). The side chain of one of the...

Fig. 7. View of the FeMo-cofactor prosthetic group of the nitrogenase MoFe protein with some of the surrounding amino acid residues where ( ) represents the molybdenum coordinated to a-His-442 and homocitrate (at the top), ( ) represents the iron, interspersed with the sulfur (O) and carbon...

Although FeMo-cofactor is clearly knpHcated in substrate reduction cataly2ed by the Mo-nitrogenase, efforts to reduce substrates using the isolated FeMo-cofactor have been mosdy equivocal. Thus the FeMo-cofactor s polypeptide environment must play a critical role in substrate binding and reduction. Also, the different spectroscopic features of protein-bound vs isolated FeMo-cofactor clearly indicate a role for the polypeptide in electronically fine-tuning the substrate-reduction site. Site-directed amino acid substitution studies have been used to probe the possible effects of FeMo-cofactor s polypeptide environment on substrate reduction (163—169). Catalytic and spectroscopic consequences of such substitutions should provide information concerning the specific functions of individual amino acids located within the FeMo-cofactor environment (95,122,149). 

Acetylene-reduction assay Estimates nitrogenase activity by measuring the rate of acetylene reduced to ethylene. 

A number of nitrogen-fixing bacteria contain vanadium and it has been shown that in one of these, Azotobacter, there are three distinct nitrogenase systems based in turn on Mo, V and Fe, each of which has an underlying functional and structural similarity.This discovery has prompted a search for models and the brown compound [Na(thf)]+[V(N2)2(dppe)2] (dppe = Pli2PCH2CH2PPh2) has recently been prepared by reduction of VCI3 by sodium naphthalenide... 

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