Electrochemical characterization of the iron-molybdenum cofactor from Azotobacter vinelandii nitrogenase

Schultz, Franklin A.; Gheller, Stephen F.; Burgess, Barbara K.; Lough, Samuel M.; Newton, William E.

doi:10.1021/ja00305a007

Cited by 68 publications

(62 citation statements)

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“…Watt has also identified 9-and 12-electronoxidized species of active MoFe protein but these are not well characterized [22]. The S = 3/2 EPR signal of the FeMoco centers integrates to one electron per molybdenum atom [3, 231 and these centers act as one-electron acceptors when extracted from the protein [24]. Therefore it is almost certain that the FeMoco centers in the MoFe protein act as oneelectron redox centers.…”

Section: Discussionmentioning

confidence: 99%

Quantitative EPR of an S= 7/2 system in thionine‐oxidized MoFe proteins of nitrogenase

Hagen¹,

Wassink²,

Eady

et al. 1987

European Journal of Biochemistry

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Thionine-oxidized nitrogenase MoFe proteins from Azotohucter vinelundii. Azotobacter cliroococcurn and Klehsiella pneurnoniae exhibit excited-state EPR signals with g = 10.4, 5.8 and 5.5 with a maximal amplitude in the temperature range of 20-50 K. The magnitude of these effective g values, combined with the temperature dependence of the peak area at g = 10.4 from 12 K to 86 K, are consistent with an S = 7/2 system with spin01 cm-' and g = 2.00. This interpretation predicts nine additional effective g values some of which have been detected as broad features of low intensity at g z 10, z 2.5 and z 1.8. The S = 7/2 EPR is ascribed to the multi-iron exchange-coupled entities known as the P clusters.Quantification relative to the S = 312 EPR signal from dithionite-reduced MoFe protein indicates a stoichiometry of one P cluster per FeMo cofactor. Two possible interpretations for these observations, together with data from the literature, are proposed. In the first model there are two P clusters per tetrameric MoFe protein. Each P cluster encompasses approximately 8Fe ions and releases a total of three electrons on oxidation with excess thionine. In the second model the conventional view of four P clusters, each containing approximately 4Fe, is retained. This alternative requires that following one-electron oxidation, the P clusters factorize into two populations, P, and Pb, only one of which is further oxidized with thionine resulting in the S = 7 / 2 system. Both models require eight-electron oxidation of tetrameric MoFe protein to reach the S = 7/2 state.

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Section: Discussionmentioning

confidence: 99%

Quantitative EPR of an S= 7/2 system in thionine‐oxidized MoFe proteins of nitrogenase

Hagen¹,

Wassink²,

Eady

et al. 1987

European Journal of Biochemistry

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“…FeMoco was isolated from the MoFe protein either by the original procedure of Shah and Brill [2] or by the modified method of Yang et al [3]. NMF extracts of FeMoco were concentrated by vacuum distillation or column chromatography on DEAE-cellulose as described previously [17]. Final concentrations of Mo were 100 -1000 nmol/ml depending on that required for a particular decomposition technique (see below (X = halide) [23].…”

Section: Muterialsmentioning

confidence: 99%

Elicitation of thiomolybdates from the iron‐molybdenum cofactor of nitrogenase

Newton

Gheller

Hedman

et al. 1986

European Journal of Biochemistry

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Aerial oxidation of the iron-molybdenum cofactor (FeMoco) of Azotobacter vinelandii nitrogenase has been shown to yield either the tetrathiomolybdate ion ([MoS4I2-) or the oxotrithiomolybdate ion ([MoOS3l2-), depending on the reaction conditions. Thus, when N-methylformamide (NMF) solutions of FeMoco either were titrated with measured aliquots of air or were diluted with air-saturated NMF, [MoOS3I2-was found to be the predominant product while dilution of NMF solutions of FeMoco with air-saturated methanol produced [MoS4I2-almost exclusively. Similar aerial oxidation of solutions of chemically synthesized Fe-Mo-S clusters showed that significant information about the molybdenum environment in these species could be deduced from the nature of the elicited thiomolybdates. The differences in decomposition products as a function of solvent are postulated to be due to the loss through precipitation of the reducing agent sodium dithionite on addition of methanol but not NMF. These overall decomposition results are discussed in the context of recent X-ray absorption spectroscopic data which suggest the presence of an 'MoS3' core in FeMoco. A possible mechanism whereby [MoS4I2-might be rapidly formed from this core is presented.The iron-molybdenum cofactor (FeMoco) is the extruded, putative Nz-reducing site of nitrogenase [I], obtained by acid treatment of the molybdenum-iron protein of that enzyme followed by extraction with the organic solvent N-methylformamide (NMF) [2, 31. Nitrogenase contains two such cofactors, each of which accounts for one Mo and 6 -8 atoms of Fe and probably 8 -9 atoms of sulfur arranged in a cluster [2-41 so far unrecognized elsewhere in biology [3-91. Although numerous attempts have been made to simulate the structure and/or chemical properties of FeMoco via synthesis and study of Mo-Fe-S cluster compounds, a close chemical model of the site has remained elusive [lo-131. The most incisive structural information on FeMoco has come from Xray absorption spectroscopy (XAS), both near edge spectra (XANES) and fine structure analysis (EXAFS), with the recent data [14, 151 suggesting a molybdenum environment which is comprised of three 0 (or N) and three S atoms from anionic ligands. However, although EXAFS is very precise in identifying the type of donor atom and its distance from the absorber (Mo), it is very much less precise in defining the number of such atoms. Thus, in order to obtain independent, complementary insight into the nature of the molybdenum environment in FeMoco, especially with respect to the number of S donor atoms, we have carried out oxidative decomposition studies of this entity and herein report our results, which Correspondence to W. E.

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“…The FeMoco ox state was obtained by storing the fresh, concentrated extract under dinitrogen for 24-48 h, as described by Schultz and co-workers. [15] The EPR measurements were performed at 10 K, 100 mW using a Bruker ER 200 D-SRC instrument.…”

Section: Methodsmentioning

confidence: 99%

“…[14] In contrast, under an inert atmosphere, dithionite is slowly consumed and the cofactor is (reversibly) oxidised to the EPRsilent FeMoco ox form, as described by Schultz and co-workers. [15] This oxidised form or its thiophenolate derivative, which has PhS À ligated to the terminal Fe atom, provides the starting point for the studies described herein (Figure 1). Figure 2a shows the change in the cyclic voltammetry of FeMoco(SPh) ox with increasing cyanide concentration.…”

Section: Introductionmentioning

confidence: 99%

Synergic Binding of Carbon Monoxide and Cyanide to the FeMo Cofactor of Nitrogenase: Relic Chemistry of an Ancient Enzyme?

Pickett¹,

Vincent²,

Ibrahim³

et al. 2004

Chemistry A European J

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The first electrochemical and infra-red data on the binding of cyanide to the isolated iron-molybdenum cofactor of nitrogenase, FeMoco, is described. It is shown that cyanide stabilises a hitherto unrecognised, low-spin, EPR-active (S= 1/2), superoxidised form of FeMoco, and we provide the first evidence that carbon monoxide and cyanide bind synergically to the oxidised and semireduced states of the isolated cofactor, states which are unreactive to carbon monoxide alone.

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Electrochemical characterization of the iron-molybdenum cofactor from Azotobacter vinelandii nitrogenase

Cited by 68 publications

References 1 publication

Quantitative EPR of an S= 7/2 system in thionine‐oxidized MoFe proteins of nitrogenase

Quantitative EPR of an S= 7/2 system in thionine‐oxidized MoFe proteins of nitrogenase

Elicitation of thiomolybdates from the iron‐molybdenum cofactor of nitrogenase

Synergic Binding of Carbon Monoxide and Cyanide to the FeMo Cofactor of Nitrogenase: Relic Chemistry of an Ancient Enzyme?

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