Nitrogenase substrate reduction

As indicated in Fig. 1, nitrogenase can reduce substrates other than Na. In the absence of other reducible substrates it will reduce protons to dihydrogen, but it can also reduce a number of other small triple-bonded substrates, as indicated in Section V,E,1. Large substrates are not reduced efficiently, indicating physical limitations on access to the enzyme s active site. CO is a potent inhibitor of all nitrogenase substrate reductions except that of the proton to Ha. In the presence of CO the rate of electron transfer is generally not inhibited, but all electrons go toward the production of Ha. 

Mo-Nitrogenase Substrate Reduction. Adenosine triphosphate (MgATP), a low potential reductant (usually dithionite in vitro and either... 

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... 

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). 

Certainly, all three of the bands observed with SF-FTIR must arise from different species, since they appear and disappear with different time courses. The peak at 1904 cm probably corresponds with that observed by ENDOR under low CO conditions, but the relationship of the other two bands to those observed under high CO is not clear, since the ENDOR technique will only detect CO molecules bound to paramagnetic species, whereas FTIR should detect all species. The SF-FTIR technique has the potential to observe the binding and reduction of a wide range of nitrogenase substrates, provided that the appropriate spectroscopic range can be accessed. This will be technically difficult, but well worth the effort. 

Spectroscopic developments such as stopped-fiow FTIR may allow direct observation of the binding and reduction of substrates during turnover, and this may help to narrow down the possible pathways of substrate reduction. However, the complexity of the interactions of substrates with nitrogenase is such that it would probably be unwise to extrapolate from the behavior of any other substrate to that of N2. Only direct observations of N2 binding and reduction will solve this problem. 

Substrate reduction by vanadium nitrogenase has not been investigated as extensively as has molybdenum nitrogenase, but there are clear differences. Acetylene is a poor substrate and N2 does not compete as effectively with protons for the electrons available during turnover. Therefore, high rates of H2 evolution are observed in the presence of these substrates. Furthermore, acetylene is reduced to both ethylene and a minor product, ethane (172). Equation (2) summarizes the most efficient N2 reduction data yet observed for vanadium nitrogenase. 

Substrate reduction by the iron nitrogenase is very similar to that observed with vanadium nitrogenases. Acetylene is a relatively poor substrate, and N2 reduction is accompanied by considerable H2 evolution. Acetylene reduction leads to the production of some ethane as well as ethylene. Beyond this, little has been investigated. Under optimal conditions for N2 reduction, the ratio of N2 reduced to H2 produced was 1 7.5 compared with 1 1 for molybdenum nitrogenase 192). 

These data confirm the sequence similarity of the three nitrogenases and indicate that cofactor exchange experiments are relatively straightforward. However, the environment of the cofactor clearly affects the substrate reduction activity, as observed with mutations in... 

Fe-protein interacts with MoFe-protein. Correct docking of Fe-protein to MoFe-protein is associated with conformational changes taking place during step 1. Steps 1 and 2 are prerequisites for all following nitrogenase reactions and for substrate reduction. 

The so-called midpoint potential, Em, of protein-bound [Fe-S] clusters controls both the kinetics and thermodynamics of their reactions. Em may depend on the protein chain s polarity in the vicinity of the metal-sulfur cluster and also upon the bulk solvent accessibility at the site. It is known that nucleotide binding to nitrogenase s Fe-protein, for instance, results in a lowering of the redox potential of its [4Fe-4S] cluster by over 100 mV. This is thought to be essential for electron transfer to MoFe-protein for substrate reduction.11 3... 

In addition to the physiological reaction of N2 reduction, nitrogenase catalyzes a wide variety of reactions involving small unsaturated molecules(56). Table III lists key reactants and products for FeMo nltrogenases. All substrate reductions involve minimally the transfer of two electrons. Multielectron substrate reductions may involve the accretion of such two-electron... 

Table 1 Nitrogenase Substrates and Their Reduction Products...

Nitrogenases are very versatile enzymes. They reduce, in addition to N2, a lot of other substrates, for example, protons, acetylene, azide, nitriles, and isonitriles. All of these substrates are reduced by multiples of [2 H+/2 e ] reductions. Both CO and NO inhibit nitrogenase activity. The apparent [2 H+/2 e ] multiplicity of substrate reductions and a couple of other findings strongly suggest diazene and hydrazine to be intermediates of the N2 —> NH3 reduction. 

Combining Eqs. 62a and b demonstrates that the formation of HD from D2 and water protons (which are the hydrogen source for HD) can be mediated by a diazene species. Diazene, on the other hand, is the most plausible intermediate of a [2 H+/2e ] reduction of N2, and it is a reduction intermediate that can form only from N2 and not from any other nitrogenase substrate. 

Helleren, C.A., McMahon, C.N., and Leigh, G.J. (2000) The use of chemical models to probe die mechanisms of substrate reduction reactions of nitrogenases Current Plant Science and Biotechnology in Agriculture 38 (Nitrogen Fixation From Molecules to Crop Productivity), 55-56. 

Vanadium nitrogenase is produced by certain bacteria grown in molybdenum-deficient environments. It is effective in the reduction of N2 and other nitrogenase substrates, although with less activity than the Mo—Nase. The enzyme resembles the Mo analogue (see Sections 17-E-10 and 18-C-13) in the construction and structure of the prosthetic groups, as well as in its functions.101 It consists of a FeV protein, FeVco, and an iron protein (a 4Fe—4S ferredoxin). 

VFe3S4X3] (X = Cl, Br, and I), (Me4N)2[TpVFeS4Cl3], and (Me4N)[(NH3)-(bipy)Fe3S4Cl3]. A study of the catalytic reduction of hydrazine (a nitrogenase substrate) to ammonia in the presence of an external source of electrons and protons shows that the rate of reduction decreases as the number of labile solvent molecules coordinated to the V atom decreases but does not depend on the nature of the atom attached to the Fe atoms. 

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