FeMo protein

The Fe-protein, whose molecular structure is shown in Figure 31,40,41 acts as a one-electron donor to the FeMo-protein. This electron-donating ability arises from the propensity of the Fe4S4 cluster to undergo a one-electron oxidation. 

As illustrated in Figure 32, the FeMo-protein (of Azotobacter vinelandii) incorporates the P-cluster, which for sake of simplicity can be thought of as composed of two cuboidal Fe4S4 centres each linked by a common sulfur atom, and the FeMoco (sometimes defined as the M-centre) of composition MoFe7S9. A Fe4S3 subunit and a MoFe3S3 subunit form this latter centre.42... 

Figure 33 shows in more detail the two clusters of the FeMo-protein and the sulfur bridges which connect the two cluster subunits. 

Figure 32 X-Ray structure of the active site of FeMo-protein in the nitrogenase of Azotobacter vinelandii...

Even if the extraction of FeMoco in nmf slightly modifies the outer coordination of the MoFe7S9 cluster, in agreement with the experimental observation that the FeMo-protein can exist in the oxidized, semi-reduced and reduced forms, it exhibits two successive one-electron reductions (E st = -0.32 V and is d = -1.00 V,vj. NHE). In the absence of thio-phenol, the first reduction appears to be complicated by self-oxidation processes, whereas the presence of thiophenol apparently stabilizes the different oxidation states. 

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. 

The FeMo-co or M center of the FeMo protein has been identified spectroscoplcally(, 13,30) within the protein and has been extracted from the protein into N-methyl formamlde(31) and other organic solvents(32.33). Its biochemical authenticity can be assayed by its ability to activate FeMo protein from a mutant organism that produces protein that lacks the M center(31). The extracted cofactor resembles the M-center unit spectroscopically and structurally as shown in Table I. It seems reasonable to presume that the differences are due to variation in the ligation of the center between the protein and the organic solvent(34). 

Additionally, Wang and Watt have shown that the FeMo protein alone can act as an uptake hvdrogenase(63). Specifically, H2 in the presence of [FeMo] causes the reduction of oxidizing dyes such as methylene blue or dichlorophenolindophenol in the absence of Fe protein. The hydrogen evolution and uptake behavior of nitrogenase proteins forces us to consider the ways in which hydrogen can interact with transition metal sulfur centers. This we discuss in the following section. 

Interestingly, the splitting parameters for the nitrogen are quite similar to those involving imidazole nitrogen directly bound to low-spin heme(79). We conclude that a protein N binds directly to FeMo-co in the FeMo protein. This conclusion is reinforced by the determination of a non-zero isotropic hyperfine term for the splitting, which unequivocally shows direct (Fermi... 

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... 

Figure 3.4. View of the protein environment between the FeMo-cofactor and P-cluster of the FeMo-protein (Rees and Howard, 2000). Reproduced with permission...

The basic mechanism of nitrogenase with the use of dithionate as an electron donor for the iron protein involves the following steps (Thomeley and Lowe, 1985 Likhtenshtein, 1988a Burgess and Lowe, 1996 Smith, 1999 Seefeldt and Dean, 1997 Rees and Howard, 2000 Syrtsova and Timofeeva, 2001) 1) reduction of Fe-protein with flavodoxin or dithionate and attachment of two ATP molecules to the protein, 2) formation of a complex between the reduced FeP with two bound ATP molecules and FeMo-protein, 3) electron transfer between the reduced [Fe4S4] cluster of FeP to the P-cluster ofFeMoP coupled to the ATP hydrolysis, 4) electron transfer from P-cluster to... 

Figure 3.7. The energy profile of a nitrogenase reaction. Eo is the standard redox potential of the reactants, intermediates and products of the reaction Fd = ferredoxin FeP = Fe protein FeMo = FeMo protein. The arrow indicates the increase of the reduction potential upon ATP hydrolysis (Likhtenshtein 1988a). Reproduced with permission.

Kenneth O. Hodgson, Richard H. Hohn, et al. EXAFS (extended X-ray absorption fine structure) study of structure of FeMo protein component of nitrogenase... 

Figure 11 Cartoon showing CO binding to the FeMo-cofactor of nitrogenase FeMo protein. The S atoms are shown in yellow, the Fe atoms in red, and the Mo atom in blue-green. At lower CO partial pressure, the lo-CO form exists with a single, bound, bridging CO. At higher CO partial pressure, the hi-CO form exists, with two CO molecules bound to the cluster...

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). 

Figure 23,13 Metal centres in the FeMo protein of nitrogenase. (a) P-cluster pair. Each of the four outer Fe atoms is further coordinated to the S of a cysteine group, (b) FeMo cofactor. (Y is probably S, 0 or N.) Fe-Fe bridge distances are in the range 240-260pm, suggesting weak Fe-Fe interactions. The Mo achieves 6-coordination by further bonds to N (of histidine) and two O atoms (of a chelating homocitrate), while the Fe at the opposite end of the cofactor is letrahedrally coordinated by attachment of a cysteine, (c) Possible intermediate in the interaction of Nj with FeMo cofactor.

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