Nitrogenase EXAFS studies

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. 

Molybdoenzymes other than the nitrogenases are usually termed oxomolybdoenzymes. This prefix relates to the nature of the catalysis effected, i.e. the net effect of the conversion (xanthine to uric acid, sulfite to sulfate, nitrate to nitrite, or aldehyde to carboxylate) corresponds to the transfer of one oxygen atom to or from the substrate. Furthermore, molybdenum X-edge EXAFS studies have established that this metal is coordinated to one or more terminal oxo groups in each enzyme studied by this technique.204... 

EXAFS studies indicate very little change in the coordination of the Mo atom in Mo-nitrogenase during enzyme turnover an indication that N2 probably does not bind to molybdenum but instead to iron. However, this issue has not been completely resolved. 

The smaller protein has a molecular mass of 60000-70000 daltons. Extended X-ray absorption fine structure (EXAFS) studies show that it includes a single Fe4S4 cubane cluster (P-cluster) similar to that in ferredoxins, which are important for various electron transport chains in living organisms. The smaller protein in the nitrogenase system is a strongly reductive reductase that is obviously responsible for the transfer of electrons to the larger protein. 

Molybdenum extended X-ray absorption fine structure (EXAFS) studies yielded the first structural insight into a metallocenter of nitrogenase. The results of these initial analyses delivered compositional and metrical data on the molybdenum environment and led to the identification of the FeMo-cofactor as a Mo-Fe-S cluster. One proposal for an EXAFS-consis-tent local molybdenum environment was a MoFe3S4 eubane model derived from the established Fe4S4 structure. Independent synthetic efforts from Holm and Garner quickly yielded species possessing this motif in the form... 

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

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). 

One of the early triumphs of biological x-ray absorption spectroscopy was the deduction that the nitrogenase M center is an Mo-Fe-S cluster. (It is also worth noting that nitrogenase was the first enzyme to be studied by x-ray absorption spectroscopy.) Early work on lyophilized protein samples indicated the presence of two major contributions to the Mo K-edge EXAFS, which were attributed to Mo-S ligands, plus a more distant Mo-Fe contribution. Subsequently, these conclusions have been confirmed and extended, using samples in solution and with much more sensitive detection systems. 

Several spectroscopic techniques have been used to study nitrogenase proteins both in the isolated forms and during enzymatic activity. These include electron paramagnetic resonance (epr) spectroscopy, Mossbauer spectroscopy, uv-visible spectrophotometry, circular dichroism. X-ray absorption edge and fine structure spectroscopy (EXAFS), and linear electric field effects (LEFE). 

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