Properties of the MgATP and MgADP binding sites on the Fe protein of nitrogenase from Azotobacter vinelandii

Cordewener, J.; Haaker, H.; Ewijk, H. van; Veeger, C.

doi:10.1111/j.1432-1033.1985.tb08867.x

Cited by 22 publications

(19 citation statements)

References 28 publications

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“…From a biochemical standpoint, the low cytosolic [ADP] may favor some aspects of cell metabolism, because it promotes the function of MgATP-using enzymes that are competitively inhibited by MgADP, such as nitrogenase (36) and various kinases (37,38).…”

Section: Discussionmentioning

confidence: 99%

Interplay of Mg ²⁺ , ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg ²⁺ in cell respiration

Gout

Rébeillé

Douce

et al. 2014

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

181

156

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In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14) ] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg 2+ concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis. MgATP is the substrate of numerous phosphorylating enzymes and the principal energy source of the cell. Indeed, any increase in metabolic activity increases the rate of MgATP use and, consequently, the rate of ADP and magnesium release, and vice versa. In normoxia, the MgATP concentration should be essentially balanced by the ADP phosphorylation catalyzed by mitochondrial ATP synthase, thereby adjusting oxidative phosphorylation to cell ATP needs. The ADP/ATP carrier (AAC) of the inner mitochondrial membrane, which exchanges free nucleotides, and adenylate kinase (EC 2.7.4.3), which interconverts MgADP and free ADP with MgATP and free AMP in the presence of Mg 2+ (7), participate in this regulation (reviewed in ref. 8). Clearly, to better understand the interplay of free and Mg-complexed ADP and ATP in the regulation of cell respiration it is necessary to know their concentrations, as well as the concentration of Mg 2+ in the cytosol and mitochondrial matrix.Nucleotides can be measured using ] (13). We adapted this noninvasive technique to the simultaneous in vivo measurement of cytosolic and mitochondrial Mg 2+ and free/Mgcomplexed nucleotides concentrations in culture cells.We used homogenous cells cultivated on liquid nutrient media (NM) so as to narrow resonance peaks on in vivo NMR spectra, thus improving the signal-to-noise ratios and the accuracy of chemical shift measurements and limiting peak overlaps. In addition, the heterotrophic sycamore (Acer pseudoplatanus L.) cells of cambial origin used in this study contain no large chloroplasts, but only small plastids (14, 15) with low amounts of nucleotides (16), thus permitting more precise measurement of the cytosolic and mitochondrial nucleotide pools. SignificanceThe respiration of heterotrophic cells, where most of the ATP demand is met by mitochondrial oxidative phosphorylation, is generally thought to be regulated either by the ATP/ADP ratio and/or energy charge or by nucleotide concentration. The way in which ADP and ATP may directly mediate respirat...

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Section: Discussionmentioning

confidence: 99%

Interplay of Mg ²⁺ , ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg ²⁺ in cell respiration

Gout

Rébeillé

Douce

et al. 2014

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

181

156

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“…rnol Av;' at 22°C (J. Cordewener, unpublished results [13]. Under all conditions gel centrifugation experiments showed that approximately 1 mol MgADP or MgATP was bound/mol Av, (see Table 2).…”

Section: Muter Ialsmentioning

confidence: 99%

“…The higher values were observed with Fe protein with higher activity. Since Fe protein has two MgATP biMing sites [13], it means that when each binding site is converted into a catalytic site in the complex, each site has two turnovers in the burst reaction. Since one of the two adenine-nucleotide-binding sites has a low rate of dissociation of MgADP, it might be possible that this site will be constantly occupied by MgADP after the first molecule of MgATP is hydrolyzed in the pre-steady-state burst reaction.…”

Section: Muter Ialsmentioning

confidence: 99%

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Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase

Cordewener¹,

Asbroek²,

Wassink³

et al. 1987

Eur J Biochem

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The pre-steady-state ATPase activity of nitrogenase from Azotobucter vinelundii was investigated. By using a rapid-quench technique, it has been demonstrated that with the oxidized nitrogenase complex the same burst reaction of MgATP hydrolysis occurs as observed with the reduced complex, namely 6 -8 mol orthophosphate released/mol MoFe protein. It is concluded that the pre-steady-state ATPase activity is independent of electron transfer from Fe protein to MoFe protein. Results obtained from gel centrifugation experiments showed that during the steady state of reductant-independent ATP hydrolysis there is a slow dissociation of one molecule of MgADP from the nitrogenase proteins (kOff < 0.2 s-'); the second MgADP molecule dissociates much faster (kOff >, 0.6 s-I). Under the same conditions orthophosphate was found to be associated with the nitrogenase proteins. The rate of dissociation of orthophosphate from the nitrogenase complex, as estimated from the gel centrifugation experiments, is in the same order of magnitude as the steady-state turnover rate of the reductantindependent ATPase activity (0.6mol Pi formed . s-' . mol Av;' at 22°C). These data are consistent with dissociation of orthophosphate or MgADP being rate-limiting during nitrogenase-catalyzed reductant-independent ATP hydrolysis.

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“…The calculated dissociation constants reported vary considerably. For the oxidized Fe protein from Azotobacter vinelandii, the K m values (ATP) are between 49 and 290 M; the corresponding values determined for the reduced protein vary between 220 and 1,710 M. Saturation of binding sites is achieved between 0.5 and 1 mM (6). Considering the effective nucleotide concentration range shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

Proteolytic degradation of dinitrogenase reductase from Anabaena variabilis (ATCC 29413) as a consequence of ATP depletion and impact of oxygen

et al. 1996

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Both components of nitrogenase, dinitrogenase and dinitrogenase reductase, are rapidly inactivated by oxygen. To investigate the proteolytic degradation of dintrogenase reductase irreversibly destroyed by high oxygen concentrations, we carried out in vitro experiments with heterocyst extracts from Anabaena variabilis ATCC 29413. The results indicate a direct dependence of degradation on the applied oxygen concentration. Although the degrees of degradation were similar for both the modified and unmodified subunits of dinitrogenase reductase, there was a significant difference with respect to the cleavage products observed. The pattern of effective protease inhibitors suggests the involvement of serine proteases with chymotrypsin-and trypsin-like specificity. A protective effect was obtained by saturation of the nucleotide binding sites of dinitrogenase reductase with either ATP or ADP. As shown by gel filtration experiments, the adenylates prevented the nitrogenase subunits from extensive noncovalent aggregation, which is usually considered evidence for a denaturing process. The in vitro degradation of dinitrogenase reductase is discussed in connection with previous reports on degradation of nitrogenase in cyanobacteria under oxygen stress and/or starvation.

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Properties of the MgATP and MgADP binding sites on the Fe protein of nitrogenase from Azotobacter vinelandii

Cited by 22 publications

References 28 publications

Interplay of Mg ²⁺ , ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg ²⁺ in cell respiration

Interplay of Mg ²⁺ , ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg ²⁺ in cell respiration

Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase

Proteolytic degradation of dinitrogenase reductase from Anabaena variabilis (ATCC 29413) as a consequence of ATP depletion and impact of oxygen

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Properties of the MgATP and MgADP binding sites on the Fe protein of nitrogenase from Azotobacter vinelandii

Cited by 22 publications

References 28 publications

Interplay of Mg 2+ , ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg 2+ in cell respiration

Interplay of Mg 2+ , ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg 2+ in cell respiration

Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase

Proteolytic degradation of dinitrogenase reductase from Anabaena variabilis (ATCC 29413) as a consequence of ATP depletion and impact of oxygen

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Product

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Interplay of Mg ²⁺ , ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg ²⁺ in cell respiration

Interplay of Mg ²⁺ , ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg ²⁺ in cell respiration