nitrogenase and hydrazine

Nitrogenase is the metalloenzyme that performs biological nitrogen fixation by catalyzing the reduction of N2 to ammonia. Understanding how the nitrogenase active site metal cofactor (FeMo-cofactor) catalyzes the cleavage of the N2 triple bond has been the focus of intense study for more than 50 years. Goals have included the determination of where and how substrates interact with the FeMo-cofactor, and the nature of reaction intermediates along the reduction pathway. Progress has included the trapping of intermediates formed during turnover of non-physiological substrates (e.g., alkynes, CS2) providing insights into how these molecules interact with the nitrogenase FeMo-cofactor active site. More recently, substrate-derived species have been trapped at high concentrations during the reduction of N2, a diazene, and hydrazine, providing the first insights into binding modes and possible mechanisms for N2 reduction. A comparison of the current state of knowledge of the trapped species arising from non-physiological substrates and nitrogenous substrates is beginning to reveal some of the intricacies of how nitrogenase breaks the N2 triple bond.

Topics: Electron Spin Resonance Spectroscopy; Hydrazines; Imides; Models, Chemical; Models, Molecular; Molybdoferredoxin; Nitrogen; Nitrogenase; Oxidation-Reduction; Protein Conformation

Topics: Aldehydes; Biomimetic Materials; Coordination Complexes; Crystallography, X-Ray; Hydrazines; Models, Molecular; Nitrogenase; Oxidation-Reduction; Phenylhydrazines; Vanadium

Vanadium(III) thiolate complexes, [V(PS3'')(Cl)]- [1a; PS3'' = P(C6H3-3-Me3Si-2-S)3(3-)] and [V(PS3')(Cl)]- [1b; PS3' = P(C6H3-5-Me-2-S)3(3-)], were synthesized and characterized. Complex 1a serves as a precursor for the catalytic reduction of hydrazine to ammonia. The spectroscopic and electrochemical studies indicate that hydrazine is bound and activated in a V(II) state.

Topics: Ammonia; Catalysis; Electrochemistry; Hydrazines; Models, Chemical; Nitrogen Fixation; Nitrogenase; Organometallic Compounds; Oxidation-Reduction; Spectrum Analysis; Vanadium

A major challenge in understanding the mechanism of nitrogenase, the enzyme responsible for the biological fixation of N(2) to two ammonias, is to trap a nitrogenous substrate at the enzyme active site in a state that is amenable to further characterization. In the present work, a strategy is described that results in the trapping of the substrate hydrazine (H(2)N-NH(2)) as an adduct bound to the active site metal cluster of nitrogenase, and this bound adduct is characterized by EPR and ENDOR spectroscopies. Earlier work has been interpreted to indicate that nitrogenous (e.g., N(2) and hydrazine) as well as alkyne (e.g., acetylene) substrates can bind at a common FeS face of the FeMo-cofactor composed of Fe atoms 2, 3, 6, and 7. Substitution of alpha-70(Val) that resides over this FeS face by the smaller amino acid alanine was also previously shown to improve the affinity and reduction rate for hydrazine. We now show that when alpha-195(His), a putative proton donor near the active site, is substituted by glutamine in combination with substitution of alpha-70(Val) by alanine, and the resulting doubly substituted MoFe protein (alpha-70(Ala)/alpha-195(Gln)) is turned over with hydrazine as substrate, the FeMo-cofactor can be freeze-trapped in a S = (1)/(2) state in high yield ( approximately 70%). The presumed hydrazine-FeMo-cofactor adduct displays a rhombic EPR signal with g = [2.09, 2.01, 1.93]. The optimal pH for the population of this state was found to be 7.4. The EPR signal showed a Curie law temperature dependence similar to the resting state EPR signal. Mims pulsed ENDOR spectroscopy at 35 GHz using (15)N-labeled hydrazine reveals that the trapped intermediate incorporates a hydrazine-derived species bound to the FeMo-cofactor; in spectra taken at g(1) this species gives a single observed (15)N signal, A(g(1)) = 1.5 MHz.

Topics: Azotobacter vinelandii; Binding Sites; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Hydrazines; Molybdoferredoxin; Nitrogen; Nitrogenase; Oxidation-Reduction; Protons; Substrate Specificity

Nitrogenase catalyzes biological dinitrogen fixation, the reduction of N(2) to 2NH(3). Recently, the binding site for a non-physiological alkyne substrate (propargyl alcohol, HC triple bond C-CH(2)OH) was localized to a specific Fe-S face of the FeMo-cofactor approached by the MoFe protein amino acid alpha-70(Val). Here we provide evidence to indicate that the smaller alkyne substrate acetylene (HC triple bond CH), the physiological substrate dinitrogen, and its semi-reduced form hydrazine (H(2)N-NH(2)) interact with the same Fe-S face of the FeMo-cofactor. Hydrazine is a relatively poor substrate for the wild-type (alpha-70(Val)) MoFe protein. Substitution of the alpha-70(Val) residue by an amino acid having a smaller side chain (alanine) dramatically enhanced hydrazine reduction activity. Conversely, substitution of alpha-70(Val) by an amino acid having a larger side chain (isoleucine) significantly lowered the capacity of the MoFe protein to reduce dinitrogen, hydrazine, or acetylene.

Topics: Acetylene; Alanine; Azotobacter vinelandii; Binding Sites; Catalysis; Dose-Response Relationship, Drug; Hydrazines; Hydrogen-Ion Concentration; Iron; Isoleucine; Kinetics; Macromolecular Substances; Models, Chemical; Models, Molecular; Molybdoferredoxin; Nitrogen; Nitrogenase; Oxidation-Reduction; Protein Binding; Substrate Specificity

During the enzymic reduction of N2 to NH3 by Mo-nitrogenase, free hydrazine (N2H4) is not detectable, but an enzyme-bound intermediate can be made to yield N2H4 by quenching the enzyme during turnover [Thorneley, Eady & Lowe (1978) Nature (London) 272, 557-558]. In contrast, we show here that the V-nitrogenase of Azotobacter chroococcum produces a small but significant amount of free N2H4 (up to 0.5% of the electron flux resulting in N2 reduction) as a product of the reduction of N2. The amount of N2H4 formed increased 15-fold on increasing the assay temperature from 20 degrees C to 40 degrees C. Activity cross-reactions between nitrogenase components of Mo- and V-nitrogenases showed that the formation of free N2H4 was associated with the VFe protein. These data provide the first direct evidence for an enzyme intermediate at the four-electron-reduced level during the reduction of N2 by V-nitrogenase.

Topics: Azotobacter; Hydrazines; Kinetics; Models, Biological; Nitrogen; Nitrogenase; Thermodynamics; Vanadium

Micromethods of direct chemical coupling have been developed for several different enzyme reactions, using the principles of flow injection analysis. Samples of 1-25 microliters are injected into a flowing stream of color-forming reagents and the peak of color change is measured after about 1 min. Alternatively, continuous slow infusion of a reacting system (5-100 microliters/min) gives a continuous change of color which can be monitored to derive enzyme reaction rates. These techniques are highly sensitive, requiring a few nanomoles of the substance being detected. Phosphate, ammonia, dithionite, creatine, and hydrazine have been measured. Consumption of reagents is less than 75 ml per hour; typical sample throughout is 30-40 samples per hour by the injection method, and 5 samples per hour by continuous infusion. The procedure has been applied to nitrogenase, continuously monitoring creatine produced from creatine phosphate by creatine kinase which is used to supply a constant level of ATP for nitrogenase. In this way nitrogenase activity can be determined over a wide range of enzyme concentrations. Production of inorganic phosphate directly from ATP, by injection of formaldehyde-quenched samples, was used when coupling to creatine kinase was not possible. Both injection of aliquots and continuous infusion were used for detection of hydrazine during nitrogenase reduction of azide, and the injection method has been used for ammonia assay during dinitrogen reduction. Dithionite oxidation was measured directly from decolorization of iodine, after trapping both dithionite and bisulfite with formaldehyde.

Topics: Ammonia; Chemical Phenomena; Chemistry; Creatine; Dithionite; Hydrazines; Indicators and Reagents; Nitrogenase; Phosphates; Spectrophotometry

Klebsiella pneumoniae nitrogenase reduced azide, at 30 degrees C and pH 6.8-8.2, to yield ammonia (NH3), dinitrogen (N2) and hydrazine (N2H4). Reduction of (15N = 14N = 14N)-followed by mass-spectrometric analysis showed that no new nitrogen-nitrogen bonds were formed. During azide reduction, added 15N2H4 did not contribute 15N to NH3, indicating lack of equilibration between enzyme-bound intermediates giving rise to N2H4 and N2H4 in solution. When azide reduction to N2H4 was partially inhibited by 15N2, label appeared in NH3 but not in N2H4. Product balances combined with the labelling data indicate that azide is reduced according to the following equations: (formula: see text); N2 was a competitive inhibitor and CO a non-competitive inhibitor of azide reduction to N2H4. The percentage of total electron flux used for H2 evolution concomitant with azide reduction fell from 26% at pH 6.8 to 0% at pH 8.2. Pre-steady-state kinetic data suggest that N2H4 is formed by the cleavage of the alpha-beta nitrogen-nitrogen bond to bound azide to leave a nitride (= N) intermediate that subsequently yields NH3.

Topics: Ammonia; Azides; Carbon Monoxide; Hydrazines; Kinetics; Klebsiella pneumoniae; Mass Spectrometry; Nitrogen; Nitrogenase; Oxidation-Reduction