Extensive endor investigations 274275275a have been

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Extensive ENDOR investigations 274,275.275a have been reported using protein samples enriched with the stable magnetic isotopes 2H, 33S, 57Fe, 95Mo, and 97Mo. The 57Fe couplings have been investigated in the most detail. Individual hyperfine tensors of five coupled 57Fe nuclei are discernible, and were evaluated by simulation of the polycrystalline ENDOR spectrum. 275 The data from 33S and 95Mo were analyzed in less detail; 33S gave a complex ENDOR spectrum, evi- dently with quite large hyperfine couplings, although no quantification was at- tempted because of the complexity of the spectrum. 274 On the other hand, 95Mo was shown to possess a small hyperfine coupling, indicating that the molybde- * Recent x-ray crystallographic results show that, if Fe4S4 clusters are present, they are very close together in two pairs,379.380 which may account for their unusual properties.
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424 7 I FERREDOXINS, HYDROGENASES, AND NITROGENASES: METAL-SULFIDE PROTEINS Table 7.7 Comparison of the FeMo protein and isolated FeMoco. a EPR g'values EXAFS" Mo-S Mo-Fe Mo-O or N Fe-S Fe-Fe Fe-Mo Fe-O or N XANES M00 3 S 3 fits best f FeMo protein (M center) 4.27 3.79 2.01 2.36 (4)b 2.69 (3)b 2.18(l)b FeMoco (inNMF) 4.8 3.3 2.0 2.37 (3.1)C 2.70 (2.6)C 2.10 (3.I)C 2.25 (3A)d 2.20 (3.0)° 2.66 (2.W 2.64 (2.2)e 3.68 (0.8)" 2.76 (OA)d 2.70 (0.8)C 1.81 (1.2)d a Distance in A with number of atoms in parentheses. b From 287; earlier study reported in 286. C Data from 373; earlier study reported in 372. d Data from 290. e Data from 291. f Data from 288, 289. num possesses very little spin density (although the quantitative aspects of the conclusions of the 95Mo ENDOR study have recently been shown to be in error 276 ). Although no nitrogen splittings were reported in any of the ENDOR studies, evidence for involvement of nitrogen as a cluster component has been forthcom- ing from ESEEM spectroscopy. 277-279 14N modulations are observed in the ESEEM of the M center. The observed 14N is not from the substrate (N 2 ), or from an intermediate or product of nitrogen fixation, because enzyme turnover using 15N as a substrate does not change the ESEEM spectrum. The isolated cofactor (FeMoco) does not show the modulation frequencies observed for the M center in the protein. These experiments suggest that the M-center 14N ESEEM arises from a nitrogen atom that is associated with the M center, and probably from an amino-acid side chain (most likely a histidine) ligated to the cluster. 279 Re- cent evidence from site-directed mutagenesis of the Azotobacter vinelandii protein 280 provides strong support for the presence of histidine ligation, and points specifically to His-195 of the Q' subunit as the N ligand. 8. M6ssbauer studies Extensive Mossbauer investigations of nitrogenase 271,281-283 and FeMoc0 283a have been reported. Unlike EPR and EPR-based spectroscopies, which can be
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II. MULTISITE REDOX ENZYMES 425 used to investigate only the EPR-active S = ~ oxidation state, all three available M-center oxidation states are accessible to Mossbauer spectroscopy. The fully reduced site was found to be diamagnetic with S = 0 (but see Reference 284), whereas the oxidized site was found to have S 2:: I. The zero-field spectrum of reduced C.
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