Iron <i>K</i>-edge X-ray absorption spectroscopy of the iron-molybdenum cofactor of nitrogenase from <i>Klebsiella pneumoniae</i>

Arber, Judith M.; Flood, Annette C.; Garner, C. David; Gormal, Carol A.; Hasnain, S. Samar; Smith, Barry E.

doi:10.1042/bj2520421

Cited by 56 publications

(21 citation statements)

References 18 publications

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“…3A). The EXAFSderived distances for the average iron environment in MoFe protein-bound FeMoco obtained by this method (see Table 1) are consistent with those derived from both crystallography of wild-type MoFe protein (Fe-S at 2.24 Ϯ 0.03 Å, Fe-Fe at 2.63 Ϯ 0.03 Å, Fe-Mo at 2.70 Ϯ 0.03 Å, and Fe-Fe at 3.70 Ϯ 0.03 Å) (6) and iron K-edge EXAFS of isolated FeMoco (24). The correspondence between the results reported here for MoFe protein-bound FeMoco with those reported previously for FeMoco by other methods indicates the suitability of the subtraction method to the analysis of the NifEN-bound FeMoco precursor cluster.…”

Section: Resultssupporting

confidence: 72%

“…4). Since its initial observation in the EXAFS Fourier transform of isolated FeMoco, this peak has been regarded as a signature feature representative of the unique long-range order in the FeMoco structure (24,(26)(27)(28). The occurrence of this signature peak in the data for the NifEN-bound precursor together with the EXAFS-fit results imply that the precursor has the same long-range order found in FeMoco.…”

Section: Resultsmentioning

confidence: 78%

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Structural insights into a protein-bound iron-molybdenum cofactor precursor

Corbett

Fay

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

103

107

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The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein is a highly complex metallocluster that provides the catalytically essential site for biological nitrogen fixation. FeMoco is assembled outside the MoFe protein in a stepwise process requiring several components, including NifB-co, an iron-and sulfurcontaining FeMoco precursor, and NifEN, an intermediary assembly protein on which NifB-co is presumably converted to FeMoco. Through the comparison of Azotobacter vinelandii strains expressing the NifEN protein in the presence or absence of the nifB gene, the structure of a NifEN-bound FeMoco precursor has been analyzed by x-ray absorption spectroscopy. The results provide physical evidence to support a mechanism for FeMoco biosynthesis. The NifEN-bound precursor is found to be a molybdenum-free analog of FeMoco and not one of the more commonly suggested cluster types based on a standard [4Fe-4S] architecture. A facile scheme by which FeMoco and alternative, non-molybdenum-containing nitrogenase cofactors are constructed from this common precursor is presented that has important implications for the biosynthesis and biomimetic chemical synthesis of FeMoco.biosynthesis ͉ extended x-ray absorption fine structure ͉ nitrogenase ͉ x-ray absorption spectroscopy

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Section: Resultssupporting

confidence: 72%

Section: Resultsmentioning

confidence: 78%

Structural insights into a protein-bound iron-molybdenum cofactor precursor

Corbett

Fay

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

103

107

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“…Iron [37]. Contrary to earlier findings [38], it was not necessary to include any low atomic number (C, N or 0) atoms in fitting the data, implying that it is unlikely that such atoms coordinate to all of the Fe atoms, if any.…”

Section: 24-butanetricarboxylic Acid) Is An Organic Component Ofmentioning

confidence: 63%

“…Contrary to earlier findings [38], it was not necessary to include any low atomic number (C, N or 0) atoms in fitting the data, implying that it is unlikely that such atoms coordinate to all of the Fe atoms, if any. Fe K-edge EXAFS data have also shown an interaction between each Fe atom and, on average, one other at 0.366 nm, thus providing the first evidence of long-range order in FeMoco structure [37]. This type of Fe -Fe distance is present in the Fe6S6 prismane models (Fig.…”

Section: 24-butanetricarboxylic Acid) Is An Organic Component Ofmentioning

confidence: 65%

Metalloclusters of the nitrogenases

Smith

Eady

1992

European Journal of Biochemistry

123

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Biological nitrogen fixation, the reduction of dinitrogen to ammonia, is catalysed by nitrogenases. These enzymes are found in relatively few groups of bacteria and until recently it appeared that N2 fixation occurred by a single route involving a molybdenum-containing enzyme. However, it is now clear that Mo is not an essential metal for N2 fixation. The soil bacterium Azotobacter chroococcum has an Mo-containing nitrogenase with properties very similar to those found in many other diazotrophic organisms, but it also has a vanadium-containing nitrogenase which functions in the presence of V if Mo is not available. The closely related species A . vinelandii has, in addition to Mo and V nitrogenases, a third nitrogenase which functions only under conditions where Mo and V levels are very low [l].The three nitrogenase systems of A. vinelandii are genetically distinct, being encoded by different structural genes. These genes for the three nitrogenase systems have been cloned and sequenced and have considerable similarity. The nitrogenases have been purified and shown to have similar requirements for activity, viz. a low-potential electron donor, MgATP and the absence of oxygen. They can each be separated into two essential component proteins. All three have a distinct Fe-containing protein (component 2) which, together with either an MoFe protein, a VFe protein, or a homologous protein which contains Fe but only low levels of Mo or V (component l), make up the active nitrogenases.Studies on the structure and function of nitrogenase proteins have benefitted significantly from information provided from studies on the biochemical genetics of nitrogen fixation. The genetics of nitrogen fixation is complex. In Klebsiella pneumoniae, which is the most intensively studied diazotroph and for which there is no evidence for the presence of additional Mo-independent nitrogenases, the 20 nif' genes required solely for nitrogen fixation and its regulation are clustered on a 23-kb region of the chromosome. The entire n[f gene cluster has been sequenced [2]. In addition to the structural genes of nitrogenase (nif'HDK), genes involved in the activation of nitrogenase Fe protein ( n i j w , iron-molybdenum cofactor (FeMoco) biosynthesis (nijii V3ENH) (see below), electron donation ( n i j l f l , several genes of unknown function and the regulatory genes nif'AL are located in this cluster (see Fig. 1 and [3, 41).A . vinelandii and A . chroococcum have a comparable gene cluster, except that they contain additional open reading frames (ORFs) of unknown function and nifABQ map outside the major nif'gene region. The Azotobacter nitrogen fixation genes associated with Mo-independent nitrogenases have also been cloned and sequenced. They include the structural genes of the V nitrogenase (vnf: vanadium nitrogen fixation) and, in A . vinelandii only, the structural genes of the third nitrogenase (an8 alternative nitrogen fixation). In addition to these structural genes, specific regulatory nif'A-like genes and a reiteration of nif'EN-like ...

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“…X-ray absorption spectroscopy was also used to probe for chemical reactivity (19) and electronic structural changes at the Mo site (20), but little evidence of any significant effects or direct involvement of Mo was observed in these studies. Fe-K edge EXAFS was used to confirm the results obtained from the Mo-K studies and provided evidence for longer-range interactions in FeMoco (21)(22)(23).…”

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confidence: 93%

Selenol binds to iron in nitrogenase iron-molybdenumcofactor: an extended x-ray absorption fine structure study.

Conradson

Burgess

Newton

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The biological N2-flxation reaction is catalyzed by the enzyme nitrogenase. The metal cluster active site of this enzyme, the iron-molybdenum cofactor (FeMoco), can be studied either while bound within the MoFe protein component of nitrogenase or after it has been extracted into N-methylformamide. The two species are similar but not identical. For example, the addition ofthiophenol or selenophenol to isolated FeMoco causes its rather broad S 3/2 electron paramagetic resonance signal to sharpen and more closely approach the signal exhibited by protein-bound FeMoco. The nature of this thiol/selenol binding site has been investigated by using Se-K edge extended x-ray absorption frne structure (EXAFS) to study selenophenol ligated to FeMoco, and the results are reported here. EXAFS data analysis at the ligand Se-K edge was performed with a set of software, GNXAS, that provides for direct calculation ofthe theoretical EXAFS signals and least-squares fits to the experimental data. Data analysis results show definitively that the selenol (and by inference thiol) binds to Fe at a distance of 2.4 A. In contrast, unacceptable fits are obtained with either Mo or S as the liganded atom (instead of Fe). These results provide quantitative details about an exchangeable thiol/selenol binding site on FeMoco in its isolated, solution state and establish an Fe atom as the site of this reaction. Furthermore, the utility of ligand-based EXAFS as a probe of coordination in polynuclear metal clusters is demonstrated.The biological N2-fixation reaction is catalyzed by the enzyme nitrogenase. The active site of this enzyme is a metal cluster of stoichiometry Mo:Fe7:.

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Iron K-edge X-ray absorption spectroscopy of the iron-molybdenum cofactor of nitrogenase from Klebsiella pneumoniae

Cited by 56 publications

References 18 publications

Structural insights into a protein-bound iron-molybdenum cofactor precursor

Structural insights into a protein-bound iron-molybdenum cofactor precursor

Metalloclusters of the nitrogenases

Selenol binds to iron in nitrogenase iron-molybdenumcofactor: an extended x-ray absorption fine structure study.

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