FeMoco EXAFS

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. 

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... 

Extended X-ray absorption fine structure (EXAFS) studies on the Fe/Mo/S aggregate in nitrogenase have made available structural data that are essential in the design of synthetic analog clusters. Analyses of the Mo K-edge EXAFS of both the Fe-Mo protein and the FeMoco (9) have shown as major features 3-4 sulfur atoms in the first coordination sphere at 2.35 A and 2-3 iron atoms further out from the Mo atom at 2.7 A. The Fe EXAFS of the FeMoco (10,11) shows the average iron environment to consist of 3.4 1.6 S(C1) atoms at 2.25(2) A, 2.3 +0.9 Fe atoms at 2.66(3) A, 0.4 0.1 Mo atoms at 2.76(3) A and 1.2 1.0 0(N) atoms at 1.81(7) A. In the most recent Fe EXAFS study of the FeMoco (11) a second shell of Fe atoms was observed at a distance of 3.75 A. 

The VFe proteins have been shown to contain both the spectroscopic equivalent of the P clusters and an analog of FeMoco called the iron vanadium cofactor (FeVaco). EXAFS spectroscopy has indicated that FeVaco is structurally very similar to FeMoco, with the only real difference being that Mo is replaced by V [1],... 

A cofactor can be extracted from the iron-molybdenum protein, using Af-methylformamide. This cofactor (called FeMoCo) has many spectroscopic properties in common with the native protein, especially the EXAFS spectrum, and activates the inactive large protein derived from Azobacter vinelandii UW45 mutant which cannot incorporate molybdenum. The cofactor contains no protein or peptide, but does contain molybdenum, iron, and sulfur in atomic ratios of 1 6-8 4-9. It is believed to contain the dinitrogen-binding site (presumably molybdenum) but there is no definitive proof of this. 

The structure of FeMoCo (and the dinitrogen-binding protein) is unknown. The environment of the metals is constituted primarily of sulfur atoms, but the interpretation of the X-ray absorption fine structure (EXAFS) data on both the cofactor and protein (117-119) are contentious. It is not known whether the system in any specific part is aqueous or anhydrous, but oxygen destroys the activity. The electron-transfer pathway in the functioning nitrogenase is believed to be as shown in Scheme 22. 

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. 

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). 

One of the earliest successful applications of EXAFS to probe a me-talloenzyme was the study of the molybdenum site of nitrogenase. Studies were made on both the C. pasteurianum and A. vinelandii MoFe-proteins and on isolated FeMoco (116). These studies showed definitively that molybdenum is present as part of a polynuclear cluster containing sulfur and iron, with Mo—S and Mo—Fe distances of —2.36 and —2.72 A, respectively. This work inspired the successful development of many chemical systems containing Mo—Fe—S clusters, and XAS studies of these systems strengthened the basis for the interpretation of corresponding data for the natural system. The most accurate picture of the molybdenum site of FeMoco currently available involves a coordination of about three oxygen (or nitrogen), sulfur, and iron atoms at —2.10, —2.37, and —2.70 A, respectively (117). 

Most EXAFS studies to date have been on the molybdenum K-edge of the protein or of FeMoco, and indicate a very similar Mo environment in both (Table 7.7, Figure 7.30). A consensus of the best available analyses indicates that Mo is coordinated by three or four sulfur atoms at 2.4 A, one to three oxygens or nitrogens at 2.2 A, with approximately three nearby iron atoms at... 

Fe if-edge EXAFS of FeMoco-deficient MoFe protein gives a picture of the P and P° states that is generally consistent with crystallography, although the presence of a large number of iron... 

In the above compounds, the structural motif is the M4S4 cubane, but these closed structures will not incorporate three //2-sulfide bridges easily. A different self-assembly route leads to MoFe clusters that have a different composition but do contain a sulfur-voided cubane held by three //2-sulfides. The clusters MFe4S6Ls are shown as (15). Their Fe4(//3-S)3(//2-S)3 portions structurally resemble the part of the FeMoco farthest from molybdenum. These pentametallic clusters have been used to calibrate Fe EXAFS of the FeMoco. Another important aspect of these clusters is that three iron atoms have trigonal pyramidal geometry like the belt iron atoms of the FeMoco, with three sulfides in the basal positions and a phosphine in the axial position. A series of basket ... 

Progress in the biochemistry and spectroscopy of iron-only nitrogenase has occurred, with the most active, pure preparations reported in 1997. Mossbauer and EXAFS results support electronic and structural analogy between the eight-iron cluster in this enzyme (the FeFeco ) and the FeMoco. One notable difference is that the FeFeco is diamagnetic in its dithionite-reduced form, and thus corresponds to M° in the MoFe protein. Therefore, the oxidation states are thought to be Fe(II)4Fe(III)4 or Fe(II)6Fe(III)2, in analogy to the two most likely oxidation... 

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