MoFe proteins electron transfer

The electrons are subsequently transferred to the MoFe or VFe protein one at a time. The rate of binding of the Fe protein to the MoFe protein has been estimated to occur with a rate constant, k > 5 X 107 dm3 mol-1 sec-1, which is close to the diffusion-controlled limit (72). The Fe protein-MoFe protein electron transfer is followed, when S2042- is the reductant, by the rate-determining dissociation of the two proteins. 

Scheme 1. The catalytic cycle for the reduction of N2 by the Mo nitrogenase. Eq represents the resting state of the MoFe protein of K. pneumoniae and species E -E represent intermediate forms of this protein following sequential one-electron reduction steps. The arrows between each state represent complex formation between the Fe protein and MoFe protein, electron transfer, and protonation, followed by protein dissociation. N2 binds to species 3, accounting for the stoichiometry of Eq. (1) the displacement of N2 from this species accounts for the competitive inhibition of N2 reduction by H2 (see Ref. 50 for a detailed presentation of this scheme).

It is becoming clear that the MgATP hydrolysis is not required to induce protein-protein electron transfer, but its role in nitrogenase function is still undefined. The most likely hypothesis at the moment is that its hydrolysis, on the Fe protein, induces important changes in the MoFe protein, presumably by altering the conformation of the enzyme complex. Nevertheless, the nature of the changes in the MoFe protein remain obscure. 

There is little doubt that MgATP both causes a conformational change in the Fe protein to lower its redox potential and is hydrolyzed during inter-protein electron transfer. However, its possible roles in substrate reduction remain obscure. There is also uncertainty about the site of inhibition by MgADP. Some evidence suggests that it may be associated with the MoFe... 

To a first approximation, the interface between the Fe-protein and MoFe-protein are similar in both complexes. The relative positions of the metalloclusters observed in these complex structures indicate that electron transfer from the Fe-protein to the FeMo-cofactor proceeds through the P-clusters (Figure 2) the edge to edge distance of 14A is compatible with inter-protein electron transfer rates much more rapid (-10 sec (27, 22)) than the... 

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. 

An electron is transferred from the Fe protein to the MoFe protein with concomitant hydrolysis of MgATP to MgADP and Pi... 

Fig. 8. The Fe protein cycle of molybdenum nitrogenase. This cycle describes the transfer of one electron from the Fe protein (F) to one afi half of the MoFe protein (M) with the accompEmying hydrolysis of 2MgATP to 2MgADP + 2Pf. The rate-determining step is the dissociation of F (MgADP)2 from M,rf. Subscript red = reduced and ox = oxidized.

However, some data have been more difficult to incorporate into the mechanism shown in Figs. 8 and 9. As reported 21) in Section II,B the Fe protein can be reduced by two electrons to the [Fe4S4]° redox state. In this state the protein is apparently capable of passing two electrons to the MoFe protein during turnover, although it is not clear whether dissociation was required between electron transfers. More critically, it has been shown that the natural reductant flavodoxin hydroquinone 107) and the artificial reductant photoexcited eosin with NADH 108) are both capable of passing electrons to the complex between the oxidized Fe protein and the reduced MoFe protein, that is, with these reductants there appears to be no necessity for the complex to dissociate. Since complex dissociation is the rate-limiting step in the Lowe-Thorneley scheme, these observations could indicate a major flaw in the scheme. 

Transfer of single electrons from Fe-protein to MoFe-protein in an MgATP-... 

Actual electron transfer to the dinitrogen substrate at the MoFe-protein, with electrons first passing through the MoFe-protein s P-cluster. During this process, dinitrogen is most probably bound to the iron-molybdenum cofactor (FeMoco) of the MoFe-protein.6... 

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. 

Intercomponent electron transfer takes place from reduced Fe-protein to MoFe-protein as Fe-protein s [4Fe-4S] cluster moves to the 2+ oxidation state, [Fe4S4]2+. 

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... 

Researchers studying the stepwise kinetics of nitrogenase electron transfer using stopped-flow kinetic techniques have presented other scenarios. One hypothesis presents kinetic evidence that dissociation of Fe-protein from MoFe-protein is not necessary for re-reduction of Fe-protein by flavodoxins.13 These authors state that the possibility of ADP-ATP exchange while Fe-protein and MoFe-protein are complexed with each other cannot be excluded and that dissociation of the complex during catalysis may not be obligatory when flavodoxin is the Fe-protein reductant. This leads to the hypothesis that MgATP binds to the preformed Fe-protein/... 

MoFe-protein complex, causing the essential conformational changes necessary for electron transfer. 

Fe-protein, the unique, highly specific electron donor to MoFe-protein, mediates coupling between ATP hydrolysis and electron transfer to MoFe-protein and also participates in the biosynthesis and insertion of FeMoco into MoFe-protein. Fe-protein contains one ferredoxin-like [Fe4S 4 2 /1+ cluster as its redox center. There is now evidence for an [Fe4S4]° super-reduced state in which four high-spin iron(II) (S= 2) sites are postulated. These were previously discussed in Section 6.3 and illustrated in Table 6.1.16 The [Fe4S4] cluster in this state bridges a dimer of... 

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