Probing Catalytic Function through Amino-Acid Substitutions in Azotobacter Vinelandii Molybdenum-Dependent Nitrogenase

Newton, William E.; Fisher, Karl; Kim, C.-H.; Shen, Joan; Cantwell, John S.; Thrasher, Kristin S.; Dean, Dennis R.

doi:10.1007/978-94-011-0379-4_12

Cited by 4 publications

(3 citation statements)

References 25 publications

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“…Interestingly, the Gln-α195 mutant appears to bind N # normally, whereas the other mutants examined appeared to be unable to bind N # [31,32]. It has been pointed out [33] that Gln-α195 would be able to hydrogen-bond to S2B via its amide NH # group, whereas the Asn-α195 NH # group would not reach S2B. These results can be accommodated in the present model as follows : although it can hydrogen-bond to S2B, Gln-α195 would not be able to participate in proton transfer in the same way as His-α195.…”

Section: Comparison With Mutagenesis Resultsmentioning

confidence: 98%

Controlled protonation of iron–molybdenum cofactor by nitrogenase: a structural and theoretical analysis

Durrant

2001

Biochemical Journal

115

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Qualitative molecular modelling has been used to identify possible routes for transfer of protons from the surface of the nitrogenase protein to the iron–molybdenum cofactor (FeMoco) and to substrates during catalysis. Three proton-transfer routes have been identified; a water-filled channel running from the protein exterior to the homocitrate ligand of FeMoco, and two hydrogen-bonded chains to specific FeMoco sulphur atoms. It is suggested that the water channel is used for multiple proton deliveries to the substrate, as well as in diffusion of products and substrates between FeMoco and the bulk solvent, whereas the two hydrogen-bonded chains each allow a single proton to be added to, and subsequently depart from, FeMoco during the catalytic cycle. Possible functional differences in the proton-transfer channels are discussed in terms of assessment of the protein environment and specific hydrogen-bonding effects. The implications of these observations are discussed in terms of the suppression of wasteful production of dihydrogen by nitrogenase and the Lowe–Thorneley scheme for dinitrogen reduction.

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Section: Comparison With Mutagenesis Resultsmentioning

confidence: 98%

Controlled protonation of iron–molybdenum cofactor by nitrogenase: a structural and theoretical analysis

Durrant

2001

Biochemical Journal

115

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“…Reduction of C 2 H 2 in D 2 O by both wild type , and α-Gln 195 nitrogenases produces predominantly cis -CHDCHD . Therefore, the C 2 H x fragment being examined must have x ≥ 2.…”

Section: Discussionmentioning

confidence: 99%

Characterization of an Intermediate in the Reduction of Acetylene by the Nitrogenase α-Gln¹⁹⁵ MoFe Protein by Q-band EPR and ¹³C,¹H ENDOR

et al. 2000

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Recent X-band EPR investigations of an altered nitrogenase MoFe protein for which the R-subunit His 195 residue has been substituted by Gln MoFe protein) revealed that it exhibits three new S ) 1 / 2 EPR signals when incubated under turnover conditions in the presence of acetylene (C 2 H 2 ). These three signals are designated S EPR1 , S EPR2 , and S EPR3 . We now report Q-band EPR and 13 C and 1 H ENDOR of the R-Gln 195 MoFe protein when incubated under turnover conditions in either H 2 O or D 2 O buffers with 12 C 2 H 2 , 13 C 2 H 2 , or C 2 D 2 as the substrate. ENDOR measurements from S EPR1 prepared with 13 C 2 H 2 reveal interactions with three distinct 13 C nuclei, indicating that at least two C 2 H 2 -derived species are bound to the cofactor of the R-Gln 195 MoFe protein under turnover conditions. Although distinct, two of these species have approximately isotropic hyperfine tensors, with hyperfine splittings of A(C1,C2) ∼ 2.4 MHz; the third has a smaller hyperfine splitting, A(C3) e 0.5 MHz at g 1 . 1 H ENDOR measurements further show strongly coupled proton signals (A ∼ 12 MHz) that are associated with bound C 2 H x . The observation of this signal from the C 2 H 2 /D 2 O sample indicates that this proton is not exchangeable with solvent in this cluster-bound state. Conversely, the absence of a signal in the C 2 D 2 /H 2 O sample indicates that there is no strongly coupled proton derived from solvent. We propose that we are monitoring a C 2 H 2 species that is bound to the FeMo-cofactor by bridging two Fe ions of a 4Fe4S "face", thereby stabilizing the S ) 1 / 2 cluster state. Q-band EPR also resolves rhombic features in the spectrum of S EPR2 , giving g ) [2.007, 2.000, 1.992], but ENDOR showed no 13 C signals with enriched substrate, confirming an earlier suggestion that this signal is not derived from C 2 H 2 .Nitrogenase, the catalytic component of biological nitrogen fixation, is comprised of the MoFe protein and the Fe protein.During catalysis the Fe protein serves as a MgATP-dependent reductant of the MoFe protein, which provides the site for substrate binding and reduction. 1 The MoFe protein contains two metal centers of biologically unique structure, the P-cluster (Fe 8 -S 7 ) and the FeMo-cofactor (Fe 7 S 9 Mo:homocitrate). 2 There is compelling biochemical, spectroscopic, and genetic evidence that substrate binding and reduction occurs at the FeMo-cofactor site (for a review, see ref 3). In addition to dinitrogen, nitrogenase also reduces other small molecules with multiple bonds, such as C 2 H 2 , HCN, N 3 -, and CS 2 . 4,5 Although X-ray crystallographic modeling has revealed the organization and the architecture of the FeMo-cofactor, as well as its peptide surroundings, 2 the molecular details of the interaction between substrates and FeMo-cofactor remain uncertain. Recently, the interaction between FeMo-cofactor and CO, a small-molecule inhibitor of all nitrogenase substrate reduction activities except proton reduction, was studied by using electron-nuclear double resonance spectrosc...

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“…There is a progressive lowering of specific activity for C 2 H 2 reduction by the MoFe protein in going from the wild type (100% activity) through the α-Gln 195 mutant (68%) to the α-Asn 195 mutant (8%) (). The wild-type α-His 195 residue is hydrogen bonded to S2B of the cofactor and is part of a putative proton relay to that atom (), while the α-Gln 195 mutant retains a hydrogen bond to S2B (), but is unable to relay a proton, and the α-Asn 195 mutant presumably lacks even the ability to hydrogen bond to S2B ( , ). These observations suggest that the rate of C 2 H 2 reduction is correlated to the electronic condition of the cofactor.…”

Section: Discussionmentioning

confidence: 99%

An Atomic Level Model for the Interactions of Molybdenum Nitrogenase with Carbon Monoxide, Acetylene, and Ethylene

Durrant

2004

Biochemistry

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A combination of density functional theory and molecular mechanics calculations has been used to study the possible interactions of CO, C(2)H(2), and C(2)H(4) with the central Fe and terminal Mo sites of the iron-molybdenum cofactor of nitrogenase. The most favorable binding mode for CO on the central section of the FeMoco appears to be end-on to a single Fe and results in a change from high to low spin for the ligating Fe atom. If a coordination site for CO is available on the Mo, this becomes the preferred CO binding site. Calculated nu(CO) infrared frequencies are compared with the experimental values given in the literature. C(2)H(2) binds weakly in a side-on orientation to a single Fe site; addition of a single H(+)/e(-) couple to the substrate results in spontaneous migration of the resulting -CH=CH(2) group from Fe to a central S atom of the cofactor. Further reduction liberates C(2)H(4) or alternatively can give an S=CHCH(3) intermediate, which then goes on to produce C(2)H(6). A model for C(2)H(2) reduction by nitrogenase is proposed, based on the results of the calculations and the extensive literature on this process.

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Probing Catalytic Function through Amino-Acid Substitutions in Azotobacter Vinelandii Molybdenum-Dependent Nitrogenase

Cited by 4 publications

References 25 publications

Controlled protonation of iron–molybdenum cofactor by nitrogenase: a structural and theoretical analysis

Controlled protonation of iron–molybdenum cofactor by nitrogenase: a structural and theoretical analysis

Characterization of an Intermediate in the Reduction of Acetylene by the Nitrogenase α-Gln¹⁹⁵ MoFe Protein by Q-band EPR and ¹³C,¹H ENDOR

An Atomic Level Model for the Interactions of Molybdenum Nitrogenase with Carbon Monoxide, Acetylene, and Ethylene

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Probing Catalytic Function through Amino-Acid Substitutions in Azotobacter Vinelandii Molybdenum-Dependent Nitrogenase

Cited by 4 publications

References 25 publications

Controlled protonation of iron–molybdenum cofactor by nitrogenase: a structural and theoretical analysis

Controlled protonation of iron–molybdenum cofactor by nitrogenase: a structural and theoretical analysis

Characterization of an Intermediate in the Reduction of Acetylene by the Nitrogenase α-Gln195 MoFe Protein by Q-band EPR and 13C,1H ENDOR

An Atomic Level Model for the Interactions of Molybdenum Nitrogenase with Carbon Monoxide, Acetylene, and Ethylene

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Characterization of an Intermediate in the Reduction of Acetylene by the Nitrogenase α-Gln¹⁹⁵ MoFe Protein by Q-band EPR and ¹³C,¹H ENDOR