MoFe proteins FeMoco centers

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... 

Figure 6.8 The M center (FeMoco) of MoFe- protein as reprinted from Figure 2A of reference 29b. "Y" atom assigned as sulfur in reference 24. (Reprinted with permission from Kim, J. Rees, D. C. Science 1992, 257, 1677-1682. Copyright 1992, American Association for the Advancement of Science.)...

The MoFe proteins exhibit complex redox properties. Each tetra-meric a2/32 molecule of MoFe protein contains two P clusters and two FeMoco centers and, as normally isolated in the presence of sodium dithionite, the FeMoco centers are EPR-active, exhibiting an S = spin state with g values near 4.3 and 3.7 and 2.01 (Fig. 6). The P clusters are EPR silent and there is a wealth of evidence (39) using a variety of techniques that indicates that the iron atoms in these clusters are all reduced to the Fe state. 

Removing two electrons from each P cluster renders each MoFe protein molecule oxidized by four electrons. Further oxidation leads to removal of electrons from the FeMoco centers. The potential of this oxidation is both species and pH dependent. At pH 7.9 the for Kpl is 180 mV, whereas for Cpl it is 0 mV and for Avl -95 mV (46). 

Yet further oxidation removes at least one more electron from each P cluster with an +90 mV to yield a protein oxidized by a total of at least eight electrons and with EPR signals from mixed spin states of S = I and S = I (42, 47). The combined integrations of these signals demonstrated that their intensity was equivalent to that of the FeMoco EPR signals in the same preparations. This provided the first evidence (47) that MoFe proteins contained equivalent numbers of FeMoco centers and P clusters and that P clusters contained 8 Fe atoms. Previously it had been considered that the P clusters were fully reduced Fe4S4 clusters and thus that there were two P clusters for every FeMoco center per molecule. 

In general there are few reproducible data on binding of reducible substrates to the isolated MoFe proteins. However, the S = EPR signal from the FeMoco centers of Kpl is pH dependent, the g values changing with a pKa of 8.7 (50). Of course, the proton is a substrate of nitrogenase however, there is no direct evidence for the proton associated with the pKa being bound directly to FeMoco. Nevertheless, this pKa can be perturbed by addition of the analog substrate acety-... 

From the crystal structure of the complex (Fig. 10) it appears that only minimal conformational changes occur within the MoFe protein on complexation, although it is hard to be dogmatic about these when at 3 A resolution. Nevertheless, ENDOR 116) studies on the FeMoco center demonstrated that at least one class of protons in the vicinity of the FeMoco center is altered in the complex relative to the free protein. 

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. 

Concurrently with the X-ray crystallographic studies, extended X-ray absorption fine structure (EXAFS) studies confirmed many of the bond distances proposed for nitrogenase s FeMoco cluster. The EXAFS data of reference 25 indicate short Fe-Fe distances of 2.61, 2.58, and 2.54 A for M+, M (resting state), and M forms, respectively. The authors believe that the short M center bond lengths indicate Fe-Fe bonds in this cluster. In another study using dithionite-reduced MoFe-protein Fe-S, Fe-Fe, Fe-Mo distances of 2.32, 2.64, and 2.73 A, respectively, were found in the 1 to 3 A region and Fe-Fe, Fe-S and Fe-Fe distances of 3.8, 4.3, and 4.7 A, respectively, were found in the 3 to 5 A region.30... 

X-ray crystallography has revealed that each a2p2 tetrameric MoFe protein contains two each of two types of cluster [13-16], These are the iron molybdenum cofactor (FeMoco) centers and the Fe8S7 P clusters. The two FeMoco centers are bound within the a subunits about 1 nm below the surface of the protein and are separated by about 7 nm. The P clusters are situated at the interface of the a and P subunits, and each is approximately 1.9 nm from one of the FeMoco centers. 

As discussed below, the consensus view of the redox centers present in MoFe proteins is that there are two types (see Ref. 28 for discussion). There are two FeMoco centers (which have the approximate composition one Mo, six to eight Fe, four to nine S, and one homocitrate this is unique to nitrogenase and different from the Mo cofactors of other Mo-containing enzymes), and four [4Fe-4S] centers (the P clusters). If these assignments are correct, then because only 16 Cys residues are invariant among MoFe proteins, conventional Cys ligation to the P ... 

In this model, the FeMoco centers of the MoFe protein are reduced independently by a series of eight one-electron transfers from the Fe protein with concommitant hydrolysis of MgATP. Following each electron transfer, dissociation of the protein-protein complex occurs in a reaction that is rate limiting when dithionite is used as reductant. Following this dissociation, the oxidized Fe protein, with MgADP... 

The P cluster is an FegS cluster that lies at the interface of the a- and /3-subunits of the MoFe protein. It is situated about 15 A from the site at which the Fe protein binds and about the same distance from the FeMoco, suggesting a role in electron transfer. This idea was first supported by a tandem EPR/kinetics study, in which the (small) characteristic spectroscopic change observed corresponds to the rate of FeMoco reduction.More direct evidence comes from the observation in a mutant of a characteristic EPR signal that disappears during turnover and then returns.Therefore, the P cluster is generally viewed as a gateway for electron transfer into the catalytic FeMoco center. The P cluster is observed in several redox states the most common are called P (or P° ), P " ", and P (fully reduced). 

The Mo-Fe component has M, in the range 200,000-270,000, and is tetrameric (02 2)- Each Mo atom forms part of a polynuclear cluster containing Fe, and homocitrate (/f-2-hydroxy-l,2,4-butanetri-carboxylic acid) the cluster takes the form of a distorted octahedron in which the Mo is coordinated by three S atoms, three Fe atoms and three O, N or C atoms all the available evidence indicates that these Fe-Mo coordination centers are the sites of N2 binding and reduction. The FeMo coordination center or cluster can be removed from the denatured protein without causing essential changes in its structure it can then be used to restore activity to inactive MoFe protein from mutants unable to synthesize the FeMo cluster. It is therefore also referred to as the FeMo cofactor or FeMoco. 

The electrode reactions in Equation (1) correspond to those observed previously for undiluted FeMoco at a glassy carbon electrode and to redox state changes at the Mo-Fe-S centers of intact MoFe protein in nitrogenase . 

The iron K-edge EXAFS measurements on AVI " 182) and the extracted FeVaco from AcF 183) show Fe-S and Fe-Fe interactions at 2.32 and 2.64 A, with a longer Fe-Fe distance of 3.7 A very similar again to the EXAFS data on FeMoco. These data emphasize the structural similarities between the cofactor centers of the MoFe and VFe proteins. 

EXAFS studies have provided important clues on the coordination of the metal atoms. At the V K-edge the features of the absorption edge are consistent with V11 or Viv in a distorted octahedral environment with Fe, S, and O or N atoms as the nearest neighbors to the V atom. The Fe K-edge is dominated by Fe—S and Fe—Fe interactions consistent with the structure proposed for FeMoco. These results emphasize the structural similarity between the cofactor centers of MoFe and VFe proteins. 

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