Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase

Cordewener, J.; Asbroek, A. ten; Wassink, Hans; Eady, Robert R.; Haaker, H.; Veeger, C.

doi:10.1111/j.1432-1033.1987.tb10594.x

Cited by 23 publications

(14 citation statements)

References 23 publications

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“…The amounts of Av2, Avl and ADP in the nitrogenase.ADP-A1F~ complex were determined: 2.7 + 0.2 Av2/Avl and 2.0 + 0.2 ADP/Avl were present. Cordewener et al [19] observed that two molecules of MgADP bind to Av2, but that only one MgADP molecule binds very tightly to Av2. The less tightly bound MgADP apparently is not present in the nitrogenase.…”

Section: Characterization Of the Nitrogenase Adp Alf 4 Complexmentioning

confidence: 99%

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Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

1996

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Section: Characterization Of the Nitrogenase Adp Alf 4 Complexmentioning

confidence: 99%

“…Cordewener et al [19] observed that two molecules of MgADP bind to Av2, but that only one MgADP molecule binds very tightly to Av2. The less tightly bound MgADP apparently is not present in the nitrogenase.…”

Section: Characterization Of the Nitrogenase Adp Alf 4 Complexmentioning

confidence: 99%

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

1996

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“…This distance is too large to permit direct chemical coupling of electron transfer and ATP hydrolysis. A close structural similarity has been found between the nucleotide-binding regions of the nitrogenase Fe protein and the H-Ras p21 protein 19,201, which has GTPase activity and of which the MgGTPbinding and MgGDP-binding properties have been well characterized. The nucleotide-binding regions of the Fe protein and the H-Ras p21 protein are different with respect to the orientation of the nucleotide.…”

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confidence: 94%

“…Cordewener et al [19] have shown that Av2 tightly binds one molecule of MgATP or MgADP. Later it was found that a single ADP molecule co-crystallized with Av2, bound in the interface region between the two subunits, and was separated by approximately 2 nm from the [4Fe-4S] cluster [9].…”

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Pre‐Steady‐State MgATP‐Dependent Proton Production and Electron Transfer by Nitrogenase from Azotobacter vinelandii

Duyvis

Wassink

Haaker

1994

European Journal of Biochemistry

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MgATP-dependent pre-steady-state proton production by nitrogenase from Azotobacter vinelandii was studied by monitoring the absorbance changes at 572 nm of the pH indicator o-cresolsulphonphtalein in a weakly buffered solution. The absorbance changes are characterized by a constant phase, a single exponential decrease and a linear decrease. The observed rate constant for the single exponential MgATP-dependent proton production by reduced nitrogenase proteins at 20.0 "C is 14 2 4 s-'. No proton production with a rate constant comparable to the observed rate constant of electron transfer (/coba = 100 s-l) was detected. The extent of the observed MgATP-dependent proton production is determined by the redox state of the nitrogenase proteins before mixing with MgATP; less protons are produced when more electrons are transferred from the Fe protein to the MoFe protein. Values of 2.7 +-0.3 mol H&duced/mOl MoFe protein with oxidized Fe protein, and 1.1 2 0.1 mol H&duced/mol MoFe protein with reduced Fe protein, were found. The data are interpreted to mean that protons are taken up after electron transfer from the Fe protein to the MoFe protein; the ratio electronsuansre,,ed~/H,+,,,, was calculated to be 1.2 2 0.2.After mixing the nitrogenase proteins with MgADP, proton production takes place as well. The proton-production curve did not have a constant phase and the observed rate constant of the single exponential reaction is higher, compared to MgATP-dependent proton production (koba = 35 sP).The amount of protons produced depends also on the redox state of the Fe protein; no proton production was observed with the oxidized Fe protein ; with dithionite-reduced Fe protein a value of 3.1 2 0.4 mol H&,duCed/mol MoFe protein was found (or 0.5 2 0.1 mol H+/mol Fe protein). Similar results were obtained when only the Fe protein was mixed with MgADP, but the observed absorbance changes were smaller; mixing of dithionite-reduced Fe protein with MgADP resulted in the production of 0.17 2 0.05 mol H+/mol Fe protein.All reported absorbance changes were absent when the experiments were performed in a buffered solution.The series of events that occur after mixing of the nitrogenase proteins with MgATP will be presented and discussed. In the case of the reduced Fe protein, electron transfer takes place at a rate of 100 s-', which is followed by H' production (kobs = 14 ss'). When there is no electron transfer (oxidized Fe protein) the rate constant of the MgATP-induced proton production decreases. When electrons are transferred, stoichiometrically less protons are produced.Nitrogenase is the enzyme system which catalyses the reaction of nitrogen fixation, in which dinitrogen is reduced to ammonia. Nitrogenase consists of two distinct oxygensensitive metalloproteins, which are both necessary for catalytic activity [l -31. The molybdenum-containing nitrogenase was studied in the present investigation. The largest of the nitrogenase proteins is the MoFe protein (Avl); it contains two types of metal-sulphur clusters, an Fe-, Mo-and Scon...

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“…Sequence analysis [10] and the three-dimensional X-ray crystal structure of the Fe protein [11] has revealed a potential nucleotide-binding site on each subunit. However, the hydrolysis of MgATP by nitrogenase requires both the MoFe protein and the Fe protein, although protein-protein electron transfer is not necessarily required, since hydrolysis is catalysed by dye-oxidized proteins [12]. Thus the MgATP-hydrolysing site is generated when the MoFe protein and the Fe proteins form a complex, which has been physically demonstrated by ultracentrifugation studies [13].…”

Section: Introductionmentioning

confidence: 98%

Energy transduction by nitrogenase: binding of MgADP to the MoFe protein is dependent on the oxidation state of the iron-sulphur ‘P’ clusters

Miller¹,

Smith

Eady

1993

Biochemical Journal

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Hydrolysis of MgATP to MgADP is essential for nitrogenase action. There is good evidence for binding of both nucleotides to the Fe protein of nitrogenase, but data indicating their binding to the MoFe protein have been controversial [see Miller and Eady (1989) Biochem. J. 263, 725-729]. The binding of MgADP to the MoFe protein of nitrogenase of Klebsiella pneumoniae was investigated by non-equilibrium gel-filtration column methods. No binding of MgADP to the dithionite-reduced protein could be detected. Treatment of the MoFe protein with phenosafranine [midpoint potential (Em) -270 mV] did not affect the activity, and oxidized the 'P' clusters but not the iron-molybdenum cofactor (FeMoco) centres. This oxidized species bound 3.9 mol of MgADP with a binding pattern characteristic of low rates of ligand dissociation. These observations suggest that the variability in published data on nucleotide binding to the MoFe protein is related to poor control of the protein oxidation level. Our data, coupled with the observation that 'P' clusters become oxidized during reduction of N2 [Lowe, Fisher and Thorneley (1993) Biochem. J., in the press], led us to propose that the ADP binding sites are transiently filled during enzyme turnover by hydrolysis of ATP originally bound to the Fe protein, and that hydrolysis occurs on a bridging site on the MoFe-Fe-protein complex.

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Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase

Cited by 23 publications

References 23 publications

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

Pre‐Steady‐State MgATP‐Dependent Proton Production and Electron Transfer by Nitrogenase from Azotobacter vinelandii

Energy transduction by nitrogenase: binding of MgADP to the MoFe protein is dependent on the oxidation state of the iron-sulphur ‘P’ clusters

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