MoFeS Clusters Research Articles

Redox behavior of the inactive MoFe cluster from Av nitrogenase

Redox behavior of the inactive MoFe cluster from Av nitrogenase

Large scale isolation and characterization of the molybdenum-iron cluster from nitrogenase.

Here we report the large scale isolation and characterization of a species, designated MoFe cluster, that exhibits an S = 3/2 EPR signal, and the comparison of this entity to isolated FeMo cofactor in N-methylformamide and to the active site of the enzyme nitrogenase. MoFe cluster is isolated from purified nitrogenase by extraction into acidic methyl ethyl ketone and it is stable in that solvent in the absence of thiols. As initially isolated, MoFe cluster solutions exhibit an S = 1/2 EPR signal that arises from an oxidized species that can be reduced by dithionite or thiols to an EPR silent state and then to a state that exhibits an S = 3/2 EPR signal. The S = 3/2 signal is as sharp as the signal exhibited by the protein and much sharper than the signal exhibited by isolated FeMo cofactor. Circular dichroism experiments indicate that unlike the last two species, MoFe cluster does not contain the endogenous ligand R-homocitrate and thus, the sharpness of the S = 3/2 signal is an intrinsic property of the metal center and does not depend upon specific interactions with this organic ligand or with the protein. Metal analyses indicate that the metal core responsible for the S = 3/2 signal contains 6 Fe atoms per molybdenum. X-ray absorption spectroscopy experiments show that although the molybdenum atom in MoFe cluster retains its pseudo-octahedral geometry, its first coordination shell has one less iron atom than that of FeMo cofactor and there has been a significant change in the long range order of the cluster.

Comparison of redox and EPR properties of the molybdenum iron proteins of Clostridium pasteurianum and Azotobacter vinelandii nitrogenases

Both heterologous crosses of the Clostridium pasteurianum and Azotobacter vinelandii nitrogenase components are completely inactive, although the reasons for this incompatibility are not known. We have compared a number of properties of the MoFe proteins from these organisms (Cp 1 and av 1, respectively) in an attempt to find differences that could explain this lack of functional activity Optical and CD spectroscopie titrations are similar for both av 1 and Cp 1, but EPR titrations are significantly different, suggesting different chemical reactivity patterns and/or magnetic interaction behavior Similarly, reduction measurements on the six-electron-oxidized state of Cp 1 and av 1 at controlled potentials indicate a difference in both the relative reduction sequence of the redox centers and the numerical values for their measured midpoint potentials. EPR measurements as a function of temperature also demonstrate that the relaxation behavior of the S = 3/2 MoFe centers associated with the proteins differ markedly. The Cp 1 EPR signal only begins to undergo broadening above 65 K, whereas the Av 1 signal is severely broadened above 25 K. These variations in the EPR properties for the two proteins are not likely to be due to differences in the stoichiometry and/or geometry of the MoFe cluster units themselves since similar EPR studies of the isolated cofactors showed them to be essentially identical. Thus, the different EPR behavior of the two proteins seems to arise either from protein constraints imposed on identical cofactors, and/or from magnetic interactions due to neighboring metal clusters.

MULTIELECTRON REDUCTION OF ALKYLAZIDE BY A (n-Bu4N)3 [Mo2Fe6S8 (SPh)9]-MODIFIED GLASSY CARBON ELECTRODE

Abstract A multielectron reduction of RN3 (R=CH3, HOC2H4) giving NH3, N2H4, and RNH2 has been succeeded for the first time by the electrochemically reduced species of (n-Bu4N)3[Mo2Fe6S8(SPh)9] not only dissolved in MeOH/THF (1:1 v/v) containing CH3N3 but also modified on a glassy carbon electrode in water containing HOC2H4N3. The turnover number for the formation of NH3 in the latter system reached more than 1×104 in 2 h, based on the Mo–Fe cluster.

Novel MoFeS Clusters from [(C5Me5)2Mo2S4] and Fe(CO)5 or Fe2(CO)9

Novel MoFeS Clusters from [(C₅Me₅)₂Mo₂S₄] and Fe(CO)₅ or Fe₂(CO)₉

Structural homologies between the amino acid sequence of Clostridium pasteurianum MoFe protein and the DNA sequences of nifD and K genes of phylogenetically diverse bacteria

The complete amino acid sequence of the larger (α-) subunit and about 70% of the total sequence of the smaller (β-) subunit of the MoFe protein from Clostridium pasteurianum was determined by analyses of peptides derived from BrCN cleavage and by digestions with trypsin, staphylococcal protease and lysylendo-peptidase of the separated subunits. The α-subunit has 529 amino acid residues, giving an M r value of 58 774. This is the first complete sequence for the α-subunit of an isolated MoFe protein. In comparing the sequences of both subunits to those from other sources, 5 out of 9 cysteines in the α-subunit and 3 out of 6 in the β-subunit are invariant, thus suggesting a function as ligands to FeS and MoFeS clusters in the MoFe protein. All of these cysteines are located in the amino terminal halves of both subunits.

Iron-57 ENDOR of the nitrogenase molybdenum-iron protein

In order to obtain a more complete characterization of the metal atoms in the EPR-visible center of MoFe, an /sup 75/F ENDOR study was performed and is examined in this report. Despite the complexity of the MoFe cluster, a spectra was obtained from six individual and distinct iron sites. Hyperfine coupling tensor of each were determined. Results show that the FeMo-co cluster must have an extraordiarily complex structure, for no two iron sites have the same characteristics. Since the site inequivalence must have a structural origin, this result will provide a stringent test of any postulated model for the cluster. ENDOR results also give a starting point for discussions of the cluster charge. They also suggest that no more than 3 equiv can be associated with the formally ferric/ferrous iron couple during reduction (or superoxidation) of the molybdenum cofactor of MoFe.

Isolation of a molybdenum--iron cluster from nitrogenase.

A molybdenum-iron cluster (Mo-Fe cluster) containing 6 Fe atoms per Mo was isolated by methyl ethyl ketone extraction of component I of nitrogenase from Azotobacter vinelandii. The cluster has no EPR signal in the g = 4 region but has an intense signal at g = 2.05 and 2.01. After the cluster was transferred from methyl ethyl ketone to N-methylformamide, the signal in the g = 2 region disappeared and a signal similar to that found with Fe-Mo cofactor appeared. The Mo-Fe cluster is the EPR-active center that undergoes reversible oxidation-reduction during catalytic turnover at the active site of the enzyme. In contrast to the Fe-Mo cofactor, which contains 8 Fe atoms per Mo, the Mo-Fe cluster failed to activate either inactive component I in extracts of A. vinelandii mutant strain UW45 or tungsten-containing component I from wild-type A. vinelandii. On the other hand, the Mo-Fe cluster showed as much acetylene-reduction activity with sodium borohydride as the reductant as did the Fe-Mo cofactor. Like nitrogenase-dependent and Fe-Mo cofactor-dependent acetylene reduction, the Mo-Fe cluster-dependent acetylene reduction is strongly inhibited by carbon monoxide.