Cn acetone thf and even benzene should facilitate

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CN, acetone, THF, and even benzene should facilitate attempts at further characterization and crystallization, 259,259a The biochemical authenticity of FeMoco has been assayed by its ability to activate the FeMo protein from the cofactor-less mutant organism. 258 The stoi- chiometry of the cofactor is MoFe6_8S7_1O, with the variability likely due to sample inhomogeneity. The extracted cofactor resembles the M-center unit spec- troscopically and structurally as shown in Table 7.7, The differences are pre- sumed to result from differences in the peripheral ligands of the metal-sulfide center between the protein and the organic solvent. 260 Strong evidence to support FeMoco as the site of substrate binding and reduction comes from the study of nif V mutants.261-263 (The V designation is somewhat unfortunate, as nifV has nothing to do with vanadium.) The NifV mutants do not fix nitrogen in vivo, and have altered substrate specificity in
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422 7 / FERREDOXINS, HYDROGENASES, AND NITROGENASES: METAL·SULFIDE PROTEINS vitro. Dihydrogen evolution by isolated nif V nitrogenase is inhibited by CO, in contrast to the wild type, where H 2 evolution is insensitive to CO. FeMoco can be extracted from the nif V protein and used to reactivate the FeMoco-deficient mutants, such as nif B or UW-45. Remarkably, the reconstituted FeMo protein has CO-sensitive H 2 evolution, which is characteristic of nif V; i.e., the nif V phenotype is a property of FeMoco and not of the protein. 263 This result clearly implicates the FeMoco site as an important part of the substrate reactions of the nitrogenase enzyme complex. Recently, a heat-stable factor called the V-factor has been discovered that restores the wild-type phenotype when added to nif V mutants during in vitro FeMoco assembly reactions. 264 The V-factor has been shown to be homocitrate (see Scheme 7.15) and 14C labeling strongly suggests that homocitrate (or a part of it) is a component of the cofactor center. Interestingly, the far more meta- bolically common citrate appears to be present in the nif V mutant. 265a Replace- ment of homocitrate by analogues that differ in structure or stereochemistry yields modified FeMoco sites that have altered substrate specificities. 265b Thus, as is true for many cofactors (e.g., heme = porphyrin + iron; B 12 = corrin + cobalt; F430 corphin + nickel; Moco, the molybdenum cofactor = Mo + molybdopterin), both inorganic and organic components are present in FeMoco. Hamacitrate CH 2 -GOOH I Ho-e-eOOH I CH 2 -GOOH Citrate (7.15) The biosynthesis of the cofactor and its insertion into [FeMo] apparently requires the presence of [Fe] and ATP in A. vinelandii. 266,266a Whether this involves redox or conformational change in [FeMo] induced by [Fe] is un- known, but the fact that inactive versions of [Fe] are effective would seem to favor the nonredox mechanism. An attractive idea 266 is that [Fe]' MgATP binds to [FeMo], producing a state that is conformationally accessible for cofactor insertion.
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