FeMo cofactor, FeMoco

The MoFe proteins are all a2 2 tetramers of 220-240 kDa, the a and (3 subunits being encoded by the nifD and K genes, respectively. The proteins can be described as dimers of a(3 dimers. They contain two unique metallosulfur clusters the MoFeTSg homocitrate, FeMo-cofactors (FeMoco), and the FesSy, P clusters. Neither of these two types of cluster has been observed elsewhere in biology, nor have they been synthesized chemically. Each molecule of fully active MoFe protein contains two of each type of cluster 2-7). 

Nitrogen Fixation in Nature The nitrogenase enzyme is a two-component protein that consists of an electron-transfer Fe protein and a catalytic protein [85]. Three different nitrogenase enzymes are known, which differ primarily in the nature of the putative active site within the catalytic protein. The most common form is the MoFe protein, in which the active site for nitrogen reduction, the so-called FeMo cofactor (FeMoco), is composed of seven irons, one molybdenum, and nine sulfides... 

The iron protein contains a single [Fe4S4] cluster, which mediates electron transfer to the FeMo-containing protein. The FeMo protein contains two 8Fe-7S clusters referred to as P-clusters and two lMo7Fe-9S clusters referred to as FeMo cofactors (FeMocos). 

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. 

Fig. 1. New structure of the FeMo-cofactor found by Rees and collaborators in 2002 (5). (Top structure of the cluster Bottom FeMoco and surrounding ligands and amino acids in the protein pocket.)...

The MoFe protein is an aifli tetramer of Mr 220 kDa, and its a and subunits are encoded by the nifD and nifK gene, respectively (Figure 1(a) and Table 1). It contains, in preparations with the highest activity, 2 molybdenum (Mo), 30 to 34 iron (Fe), and an approximately equivalent number of acid-labile sulfur (S ) atoms (Table 1). This metal content is consistent with the presence of two different types of unique metal clusters in the protein, that is, the [8Fe-7S] cluster (P cluster), which is bridged between each a/3 subunit pair, and the [Mo-7Fe-9S-homocitrate] cluster (FeMo cofactor or FeMoco), which is located within each a subunit (Figure la). ... 

The second example is the measurement of the kinetics of a substrate binding to extracted FeMo-cofactor of nitrogenase by a double-mixing stopped-flow approach. In a typical experiment, solutions of FeMoco-L and CN are rapidly mixed and held together for a known length of time. Subsequently, this solution is mixed with a solution of [NEt4]SPh whereupon the thiolate reacts with the cofactor. 

The first electron acceptor from the external donor is the Fe4S4 cluster situated between two subunits of Fe protein. From Fe4S4 an electron travels first to a P-cluster, then to FeMo cofactor, and finally to an activated substrate molecule. According to the X-ray data the shortest distance from the Fe4S4 cluster of the Fe protein to the P cluster of the MoFe protein is ca 18 A, that from P cluster to FeMo cofactor is ca 14 A, and the distance from Fe4S4 to FeMoco is ca 32 A. The P-cluster lies between Fe4S4 and FeMoco. Thus, electron transfer in nitrogenase can be presented as ... 

As we have already seen, FeMo cofactor of nitrogenase is a polynuclear complex of composition Fc7MoS9 (homocitrate), and all the available evidence implies it is the active site at which dinitrogen and other nitrogenase substrates are activated and reduced. Yet despite its first isolation in 1977 the catalytic activity of FeMoco was detected only in 1997, i.e. 20 years later. 

Fig. 2. The fe -weighted EXAFS data associated with the iron K-edge of the iron-molybdenum cofactor (FeMoco) extracted from the FeMo-protein of the nitrogenase of Klebsiella pneumoniae, and its Fourier transform (19). 

However, this model does not explain why, in the comparable experiment performed under HD, no D2 forms, nor why substrates other than N2 do not promote HD formation. Also, if H2 can interact with the active site, why is a substrate of any kind needed to promote HD formation Displacement of H2 is not a necessity for binding N2, but why does HD form only when N2 is being reduced One simple answer proposed by Helleren et al. is that HD formation and N2 binding occur at different places.54 It is possible that different substrates bind to and are transformed at different parts of the large FeMoco (FeMo cofactor) site of N-ases discussed below. CO inhibits nitrogen fixation in N-ases but not H2 evolution. A single site that binds H2 and N2 equivalently should be poisoned by CO for both H2 and N2 activity, and evidence increasingly points to multisite processes in the FeMoco cluster. However, a possible model (10) for HD formation at the same site as N2 activation is discussed below. 

In a more recent study, DFT and QM/MM calculations were applied by Cao et al to obtain information on the interactions of the FeMoco with CO and N2, and to the problem of whether the interstitial atom X of the FeMoco is N, C or 0. On the basis of frequencies calculated for metal-bound CO, QM/MM-op-timized geometries and calculated redox potentials, the authors arrived at the conclusion that the central atom X=0. Calculations on the interactions of the FeMo cofaetor with CO and N2 revealed a strong dependence of the binding energy on the binding site and the interstitial atom. Moreover, it was found that in general Fe2 has stronger interactions with CO and N2 than to Fe6 cf. Section 6.1) and that both Fe2 and Fe6 in the N-centered and O-centered clusters of the FeMo cofactor effectively bind N2. The coordination of N2 to the Fe6 site of the C-centered active cluster is unfavorable. 

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. 

FeMoco, both as a constituent of the FeMo protein and an isolated entity, has been the subject of detailed spectroscopic examination. 57Fe Mossbauer and EPR studies of the cofactor have been interpreted in terms of an S = centre that contains one molybdenum and ca. six irons in a spin-coupled structure. The EPR signal serves as a valuable fingerprint of FeMoco furthermore, release of FeMoco from the FeMo protein produces an EPR spectrum with broader features, but the same profile, thereby indicating that the core of this cluster is little changed by the extraction procedure. Treatment of FeMoco with ca. one equivalent of... 

The detailed characterization of the FeMoco site has involved parallel studies of the site within the protein and in its extracted form. The authentication of the extracted FeMoco involves the production and use of mutant organisms that make an inactive FeMo protein that contains all subunits and P clusters, but lacks the FeMoco sites.A mutant of Azotobacter vinelandii called UW-45 (UW = C/niversity of Wisconsin) was first used to assay for isolated FeMoco.Since several genes are involved in specifying FeMoco biosynthesis, mutants lacking these genes produce FeMo protein either lacking FeMoco or having a defective version of FeMoco. Mutants such as NifB of Klebsiella pneumoniae lack cofactor, and an inactive apo protein can be isolated from them. 

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