Reductions substrates

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... 

Substrate reduction is accompHshed by a series of sequential associations and dissociations of the two proteias, and duting each cycle, two molecules of MgATP are hydroly2ed and a single electron is transferred from the Fe proteia to the MoFe proteia (11,133), with the dissociation step being rate-limiting at about 6 (H)- Although the kinetics of aU. the partial reactions have been measured, Httie is known about the physical details of the... 

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). 

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. 

Homocitrate is bound to the molybdenum atom by its 2-carboxy and 2-hydroxy groups and projects down from the molybdenum atom of the cofactor toward the P clusters. This end of FeMoco is surrounded by several water molecules (5, 7), which has led to the suggestion that homocitrate might be involved in proton donation to the active site for substrate reduction. In contrast, the cysteine-ligated end of FeMoco is virtually anhydrous. 

It is clear from these data that homocitrate is intimately involved in the mechanism of substrate reduction and that close homologs such as citrate cannot entirely fill this role. Rationalization of this phenomenon is difficult, but comparisons of the reactivity of extracted FeMoco from the MoFe protein from wild type and NifV strains have led to an intellectually satisfying explanation (see Section V,E,2). 

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... 

Carbon isotope fractionation was examined during the aerobic degradation of TCE by Burkholderia cepacia strain G4 that possesses toluene monooxygenase activity (Barth et al. 2002). There were substantial differences in values of isotope shifts during degradation, from 57 to 17 ppm, and when the data were corrected to correspond to the same amount of substrate reduction the Releigh enrichment factor was 18.2. 

The mechanism and sequence of events that control delivery of protons and electrons to the FeMo cofactor during substrate reduction is not well understood in its particulars.8 It is believed that conformational change in MoFe-protein is necessary for electron transfer from the P-cluster to the M center (FeMoco) and that ATP hydrolysis and P release occurring on the Fe-protein drive the process. Hypothetically, P-clusters provide a reservoir of reducing equivalents that are transferred to substrate bound at FeMoco. Electrons are transferred one at a time from Fe-protein but the P-cluster and M center have electron buffering capacity, allowing successive two-electron transfers to, and protonations of, bound substrates.8 Neither component protein will reduce any substrate in the absence of its catalytic partner. Also, apoprotein (with any or all metal-sulfur clusters removed) will not reduce dinitrogen. 

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... 

The Fe-protein has the protein fold and nucleotide-binding domain of the G-protein family of nucleotide-dependent switch proteins, which are able to change their conformation dependent on whether a nucleoside diphosphate (such as GDP or ADP) is bound instead of the corresponding triphosphate (GTP or ATP). However, nucleotide analogues, which induce the conformational switch of the Fe-protein, do not allow substrate reduction by the MoFe-protein, nor does reduction of the MoFe-protein by other electron-transfer reagents (whether small proteins or redox dyes) drive substrate reduction. Only the Fe-protein can reduce the MoFe-protein to a level that allows it to reduce substrates such as... 

Substrate Reduction conditions % yield (% ratio of product)... 

This caveat notwithstanding, the Dominant Hypothesls( ) designates [FeMo] as the protein responsible for substrate reduction. The FeMo protein contains an 02fi2 subunit structure due to expression of the nifD and nlfK genes(24,25). Its overall M.W. of about 230,000 reflects the 50-60,000 M.W. of each of its four subunits. The nonprotein composition of 30 Fe, 2 Mo, and 30 s2- betokens the presence of transition metal sulfide clusters, which are presumed to be the active centers of the protein. 

Clearly, the spectroscopic properties of the P clusters in the proteins do not reveal their structural nature. However, extrusion of these clusters from the protein leads to the clear identification of 3-4 Fe S clusters(13.291. Despite the uncertainties inherent in the extrusion procedure (due to possible cluster rearrangement) the extrusion result supports the Dominant Hypothesis, which designates the P centers as Fe S units, albeit highly unusual ones. The P clusters are thought to be involved in electron transfer and storage presumably providing a reservoir of low potential electrons to be used by the M center (FeMo-co) in substrate reduction. 

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... 

The mechanism of substrate reduction by sulfite reductase has not been established. The close contact between the 4Fe 4S cluster and the siroheme could provide an efficient pathway for multielectron transfer from the enzyme to the substrate (McRee et al., 1986). Of special significance is the possibility that the cluster—siroheme overlap could stabilize high-oxidation states of the siroheme that might be involved in the catalytic mechanism. With the availability of genetic, biochemical, spectroscopic, and crystallographic approaches, it is anticipated that rapid progress will be made in working out the details of substrate reduction by sulfite reductase. 

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