New structural insights into the iron-molybdenum cofactor from Azotobacter vinelandii nitrogenase through sulfur K and molybdenum L x-ray absorption edge studies

Hedman, Britt; Frank, Patrick; Gheller, Stephen F.; Roe, A. L.; Newton, William E.; Hodgson, K. O.

doi:10.1021/ja00220a013

Cited by 139 publications

(159 citation statements)

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“…Details of the experimental configuration for low energy studies have been described previously. 23 The energy calibration, data reduction and error analysis follow the methods described in Ref. 24 .…”

Section: Data Collectionmentioning

confidence: 99%

Sulfur K-Edge XAS and DFT Calculations on P450 Model Complexes: Effects of Hydrogen Bonding on Electronic Structure and Redox Potentials

et al. 2005

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Hydrogen bonding is generally thought to play an important role in tuning the electronic structure and reactivity of metal-sulfur sites in proteins. To develop a quantitative understanding of this effect, S K-edge X-ray absorption spectroscopy (XAS) has been employed to directly probe ligand-metal bond covalency, where it has been found that protein active sites are significantly less covalent than their related model complexes. Sulfur K-edge XAS data are reported here on a series of P450 model complexes with increasing H-bonding to the ligated thiolate from its substituent. The XAS spectroscopic results show a dramatic decrease in pre-edge intensity. DFT calculations reproduce these effects and show that the observed changes are in fact solely due to H-bonding and not from the inductive effect of the substituent on the thiolate. These calculations also indicate that the Hbonding interaction in these systems is mainly dipolar in nature. The −2.5 kcal/mole energy of the H-bonding interaction was small relative to the large change in ligand-metal bond covalency (30%) observed in the data. A bond decomposition analysis of the total energy is developed to correlate the pre-edge intensity change to the change in Fe-S bonding interaction on H-bonding. This effect is greater for the reduced than the oxidized state, leading to a 330 mV increase in the redox potential. A simple model shows that E° should vary approximately linearly with the covalency of the Fe-S bond in the oxidized state, which can be determined directly from S K-edge XAS.

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Section: Data Collectionmentioning

confidence: 99%

Sulfur K-Edge XAS and DFT Calculations on P450 Model Complexes: Effects of Hydrogen Bonding on Electronic Structure and Redox Potentials

et al. 2005

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“…However this component, which probably derived from S20: -, was lost after oxidation/reduction of the FeMoco and therefore should not be regarded as an essential component [35].…”

Section: The Mofe Proteins Vfe Proteins and The Third Nitrogenasementioning

confidence: 99%

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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“…[27][28][29][30][31][32] In those studies, where the focus was on the metal-sulfur bonding and its perturbation by the protein environment, it was found that the S K-edge is very sensitive to the Z eff of a ligand, the chemical environment of the ligand, and the Z eff of the metal to which it is bound. 33 In this study we apply XAS and density functional theory (DFT) to a series of crystallographically characterized model complexes to identify signature spectroscopic features associated with thiolate-based ligands having different oxidation and protonation states. We then use these features to identify the ligands present in the active site of the protein NHase in its NO-bound inactive and photolyzed active forms.…”

Section: Introductionmentioning

confidence: 99%

Sulfur K-Edge XAS and DFT Calculations on Nitrile Hydratase: Geometric and Electronic Structure of the Non-heme Iron Active Site

et al. 2005

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The geometric and electronic structure of the active site of the non-heme iron enzyme nitrile hydratase (NHase) is studied using sulfur K-edge XAS and DFT calculations. Using thiolate (RS − )-, sulfenate (RSO − )-, and sulfinate (RSO 2 − )-ligated model complexes to provide benchmark spectral parameters, the results show that the S K-edge XAS is sensitive to the oxidation state of S-containing ligands and that the spectrum of the RSO − species changes upon protonation as the S-O bond is elongated (by ~0.1 Å). These signature features are used to identify the three cysteine residues coordinated to the low-spin Fe III in the active site of NHase as CysS − , CysSOH, and CysSO 2 − both in the NO-bound inactive form and in the photolyzed active form. These results are correlated to geometry-optimized DFT calculations. The pre-edge region of the X-ray absorption spectrum is sensitive to the Z eff of the Fe and reveals that the Fe in [FeNO] 6 NHase species has a Z eff very similar to that of its photolyzed Fe III counterpart. DFT calculations reveal that this results from the strong π back-bonding into the π* antibonding orbital of NO, which shifts significant charge from the formally t 2 6 low-spin metal to the coordinated NO.

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New structural insights into the iron-molybdenum cofactor from Azotobacter vinelandii nitrogenase through sulfur K and molybdenum L x-ray absorption edge studies

Cited by 139 publications

References 1 publication

Sulfur K-Edge XAS and DFT Calculations on P450 Model Complexes: Effects of Hydrogen Bonding on Electronic Structure and Redox Potentials

Sulfur K-Edge XAS and DFT Calculations on P450 Model Complexes: Effects of Hydrogen Bonding on Electronic Structure and Redox Potentials

Metalloclusters of the nitrogenases

Sulfur K-Edge XAS and DFT Calculations on Nitrile Hydratase: Geometric and Electronic Structure of the Non-heme Iron Active Site

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