Redox centers, MoFe proteins

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. 

To successfully describe the structure and function of nitrogenase, it is important to understand the behavior of the metal-sulfur clusters that are a vital part of this complex enzyme. Metal-sulfur clusters are many, varied, and usually involved in redox processes carried out by the protein in which they constitute prosthetic centers. They may be characterized by the number of iron ions in the prosthetic center that is, rubredoxin (Rd) contains one Fe ion, ferredoxins (Fd) contain two or four Fe ions, and aconitase contains three Fe ions.7 In reference 18, Lippard and Berg present a more detailed description of iron-sulfur clusters only the [Fe4S4] cluster typical of that found in nitrogenase s Fe-protein is discussed in some detail here. The P-cluster and M center of MoFe-protein, which are more complex metal-sulfur complexes, are discussed in Sections 6.5.2. and 6.5.3. 

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... 

Lowe, D.J., Fisher, K., and Thomeley, R.N.F. (1993) Pre-steady-state absorbance changes show that redox changes occur in the Klebsiella pneumonia MoFe-protein that depend on substrate and components ratio a role for P-centers in reducing dinitrogen, Biochem. J. 292, 93-. 

Recent EPR, Mossbauer, and EXAFS studies of the FeFe protein from R. capsulatus have provided spectroscopic evidence that the metal clusters of the FeFe protein bear a certain degree of structural homology to those present in the MoFe and VFe proteins. Therefore, it is likely that the FeFe protein contains redox centers that are homologous to the P cluster and FeMo or FeVa cofactor, respectively. The FeMo or FeVa cofactor homologue in the FeFe protein is termed FeFe cofactor or FeFeco. 

These clusters are each composed of eight iron atoms and seven sulfide ions. In the reduced form, each cluster takes the form of two 4Fe-3S partial cubes linked by a central sulfide ion. Each cluster is linked to the protein through six cysteinate residues. Electrons flow from the P cluster to the FeMo cofactor, a very unusual redox center. Because molybdenum is present in this cluster, the nitrogenase component is also called the molybdenum-iron protein (MoFe protein). The FeMo cofactor consists of two M-3Fe-3S clusters, in which molybdenum occupies the M site in one cluster and iron occupies it in the other. The two clusters are joined by three sulfide ions. The FeMo cofactor is also coordinated to a homocitrate moiety and to the a subunit through one histidine residue and one cysteinate residue. This cofactor is distinct from the molybdenum-containing cofactor found in sulfite oxidase and apparently all other molybdenum-containing enzymes except nitrogenase. 

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 ... 

A variety of spectroscopic and physical techniques have been used to investigate the nature of these redox centers. EPR, Fe Mossbauer spectroscopy, and Mo and Fe X-ray absorption spectroscopy Mo, Fe, and H electron-nuclear double resonance (ENDOR) linear electric field effect and magnetic circular dichroism (MCD) have provided information about the environment of the Mo and Fe nuclei and their interaction with the unpaired spin of electrons in paramagnetic species of the MoFe proteins. 

The VFe proteins of A. chroococcum and A. vinelandii have been purified to homogeneity ill, 27). Preparations with the highest activity contain 2 V, —20 Fe, and —20 g atoms per hexamer and have specific activities comparable to those of the MoFe protein (see Table II). Since their isolation, attention has naturally focused on the types of redox center they contain, particularly on the involvement of vanadium. Progress in this area has been rapid, and despite the presence of the additional subunit type and the probability that the VFe protein preparations currently available are a mixture of species, the spectroscopic and cofactor extrusion data summarized below are consistent with VFe and MoFe proteins having similar types of redox centers. 

Both VFe proteins show an axial signal with g values near 2.04 and 1.93, assigned to an S = 1/2 spin system associated with an Fe-S center (11, 39). This signal, which is not exhibited by MoFe proteins, integrates to 0.2-0.3 spin mol . The redox and pH dependence of the intensity of this signal indicate that the redox potential of the cluster from which it arises is low. However, recent results have shown that the intensity of this signal does not correlate with the activity of the VFe protein and may arise from a protein lacking a full complement of metals (40). 

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). 

In contrast to the other MoFe proteins, epr-monitored oxidation-reduction experiments with isolated Cvl yielded two 1-electron redox processes with midpoint potentials at -260 and 60 mV. Albrecht and Evans (1973) suggested that both centers were present in each molecule of Cvl however, O Donnell and Smith, to bring this observation in line with evidence from the redox titrations of the other MoFe proteins, argued that the two centers could arise from two different forms of the enzyme. 

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 . 

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