Azide inhibition of chloroplast ATPase is prevented by a high protonmotive force

Ji, Wei; Howlett, Bruce; Jagendorf, A. T.

doi:10.1016/0005-2728(88)90121-1

“…Interestingly, ATP synthesis was not inhibited by azide, whereas ATP hydrolysis was strongly inhibited. Different azide sensitivity of ATP synthesis and ATP hydrolysis was also reported for chloroplast thylakoid membranes (18). This indicates that generation of the MgADP inhibited form is avoided in ATP synthesis reaction, and Syroeshkin et al (17) have suggested a possibility that ATP synthesis catalyzed by submitochondrial particles is not a simple reversal of ATP hydrolysis.…”

mentioning

confidence: 92%

ATP Synthesis by F0F1-ATP Synthase Independent of Noncatalytic Nucleotide Binding Sites and Insensitive to Azide Inhibition

Bald¹,

Amano²,

Muneyuki³

et al. 1998

Journal of Biological Chemistry

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ATP hydrolyzing activity of a mutant ␣ 3 ␤ 3 ␥ subcomplex of F 0 F 1 -ATP synthase (⌬NC) from the thermophilic Bacillus PS3, which lacked noncatalytic nucleotide binding sites, was inactivated completely soon after starting the reaction (Matsui, T., Muneyuki, E., Honda, M., Allison, W. S., Dou, C., and Yoshida, M. (1997) J. Biol. Chem. 272, 8215-8221). This inactivation is caused by rapid accumulation of the "MgADP inhibited form" which, in the case of wild-type enzyme, would be relieved by ATP binding to noncatalytic sites. We reconstituted F 0 F 1 -ATP synthase into liposomes together with bacteriorhodopsin and measured illumination-driven ATP synthesis. Remarkably, ⌬NC F 0 F 1 -ATP synthase catalyzed continuous turnover of ATP synthesis while it could not promote ATP-driven proton translocation. ATP synthesis by ⌬NC F 0 F 1 -ATP synthase, as well as wild-type enzyme, proceeded even in the presence of azide, an inhibitor of ATP hydrolysis that stabilizes the MgADP inhibited form. The time course of ATP synthesis by ⌬NC F 0 F 1 -ATP synthase was linear, and gradual acceleration to the maximal rate, which was observed for the wild-type enzyme, was not seen. Thus, ATP synthesis can proceed without nucleotide binding to noncatalytic sites even though the rate is sub-maximal. These results indicate that the MgADP inhibited form is not produced in ATP synthesis reaction, and in this regard, ATP synthesis may not be a simple reversal of ATP hydrolysis.The F 0 F 1 -ATP synthase is a ubiquitous enzyme in plasma membranes of bacteria, inner membranes of mitochondria, and thylakoid membranes of chloroplasts which utilizes a transmembrane electrochemical potential difference of protons (⌬H ϩ ) 1 for ATP synthesis (1, 2). It can be reversibly separated into a hydrophilic, water-soluble F 1 part and a hydrophobic, membrane-embedded F 0 part. F 0 conducts protons across the membrane. F 1 , also called F 1 -ATPase, has a subunit composition of ␣ 3 ␤ 3 ␥␦⑀ and shows strong activity of ATP hydrolysis. According to the crystal structure of the major part of beef heart mitochondrial F 1 -ATPase (3), ␣ and ␤ subunits are alternatively arranged to form a hexagonal cylinder with a central cavity through which coiled-coil ␣ helices of the ␥ subunit penetrate. F 1 -ATPase was recently proven to be a "rotary motor enzyme", the first ever found in the biological world; the ␥ subunit rotates within an ␣ 3 ␤ 3 hexagon during ATP hydrolysis (4 -6).The F 1 -ATPase has six nucleotide binding sites. Three of them are catalytic and located mainly on the ␤ subunits. The other three, called noncatalytic sites, are mainly located on the ␣ subunits. The function of the noncatalytic site had been obscure but recent studies indicated its regulatory role as follows. During multiple turnover of ATP hydrolysis, MgADP is prone to be entrapped in a catalytic site producing the MgADP inhibited form of the enzyme, which is inactive in ATP hydrolysis. ATP binding to the noncatalytic sites causes release of this inhibitory MgADP from the affected catalytic s...

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“…The photophosphorylation activity of chloroplast was measured by luciferin/luciferase luminescence assay according to Wei et al [17] . Chloroplast photophosphorylation was performed in 1 mL reaction solution, containing 50 mmol/L Tris-HCl (pH 8.0), 5 mmol/L NaCl, 5 mmol/L MgCl 2 , 2 mmol/L Na 2 HPO 4 , 1 mmol/L ADP, 1 mmol/L FeCy (Ferricyanide) or 0.05 mmol/L PMS (phenazine methosulfate) and chloroplast suspension containing 20 g chlorophyll.…”

Section: Methodsmentioning

confidence: 99%

Effects of truncated mutants of the e subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability

ZENG¹

2004

Chinese Sci Bull

0

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The subunit of the chloroplast ATP synthase and the truncated mutants which lack some amino acid residues from the N-terminus or C-terminus were overexpressed in E. coli. When the subunit or the truncated proteins was added to the spinach chloroplast suspension, both the intensity of the fast phase of millisecond delayed light emission (ms-DLE) and the cyclic and noncyclic photophosphorylation activity of chloroplast were enhanced. With an increase in the number of residues deleted from the N-terminus, the enhancement effect of the N-terminal truncated proteins decreased gradually. For the C-terminal truncated proteins, the enhancement effect increased gradually with an increase in the number of residues deleted from the C-terminus. Besides, the ATP synthesis activity of -deficient membrane reconstituted with the subunit or the truncated proteins was compared. The ATP synthesis activity of reconstituted membrane with the N-terminal truncated proteins decreased gradually as the number of residues deleted from the N-terminus increased. For the C-terminal truncated proteins, the ATP synthesis activity of reconstituted membrane increased gradually with an increase in the number of residues deleted from the C-terminus, but was still lower than that of the wild type protein. These results suggested that: (a) the N-terminal domain of the subunit of the chloroplast ATP synthase could affect the ATP synthesis activity of ATP synthase by regulating the efficiency of blocking proton leakage of subunit; and (b) the C-terminal domain of the subunit of the chloroplast ATP synthase had a subtle function in modulating the ATP synthesis ability of ATP synthase.The F 1 F o ATP synthase is located in the cytoplasmic membranes of bacteria, the inner membrane of mitochondria, and the thylakoid membranes of chloroplast. It serves as the central catalyst of ATP synthesis in most organisms. It can catalyze the synthesis of ATP using the transmembrane proton motive force generated by oxidative phosphorylation or photophosphorylation. ATP synthase from different organisms has a similar structure, but has its own speciality. In chloroplast, this enzyme consists of two parts: a membrane-embedded CF o and a cytoplasmic CF 1 (homologous to F 0 and F 1 in bacteria and mitochondria). CF o comprises four types of subunits, which were designated as subunit , , and , respectively. CF o conducts protons flux through the thylakoid membrane and provides the affinity site for CF 1 [1] . CF 1 is the hydrophilic part of ATP synthase and consists of five types of subunits with the stoichiometry of 3 3 . There are nucleotide-bind-ing and catalytic sites on each of the and subunits [2] . A feature of the chloroplast ATP synthase is that it exists in two distinct states: in light, the ATP synthase can generate ATP; in the dark, the ATP synthase is inactive, showing almost no ATP hydrolysis [3] .The subunit is a potent inhibitor of ATPase and necessary for blocking proton leakage, and is also necessary for ATP synthesis [4] . The study of truncated mutants s...

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“…The photophosphorylation activity of chloroplast was meas-ured by luciferin/luciferase luminescence assay according to Wei et al [17] . The photophosphorylation activity of chloroplast was meas-ured by luciferin/luciferase luminescence assay according to Wei et al [17] .…”

Section: Methodsmentioning

confidence: 99%

Effects of truncated mutants of the ε subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability

Zeng

¹

,

Shi

²

2004

Chin.Sci.Bull.

1

0

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The subunit of the chloroplast ATP synthase and the truncated mutants which lack some amino acid residues from the N-terminus or C-terminus were overexpressed in E. coli. When the subunit or the truncated proteins was added to the spinach chloroplast suspension, both the intensity of the fast phase of millisecond delayed light emission (ms-DLE) and the cyclic and noncyclic photophosphorylation activity of chloroplast were enhanced. With an increase in the number of residues deleted from the N-terminus, the enhancement effect of the N-terminal truncated proteins decreased gradually. For the C-terminal truncated proteins, the enhancement effect increased gradually with an increase in the number of residues deleted from the C-terminus. Besides, the ATP synthesis activity of -deficient membrane reconstituted with the subunit or the truncated proteins was compared. The ATP synthesis activity of reconstituted membrane with the N-terminal truncated proteins decreased gradually as the number of residues deleted from the N-terminus increased. For the C-terminal truncated proteins, the ATP synthesis activity of reconstituted membrane increased gradually with an increase in the number of residues deleted from the C-terminus, but was still lower than that of the wild type protein. These results suggested that: (a) the N-terminal domain of the subunit of the chloroplast ATP synthase could affect the ATP synthesis activity of ATP synthase by regulating the efficiency of blocking proton leakage of subunit; and (b) the C-terminal domain of the subunit of the chloroplast ATP synthase had a subtle function in modulating the ATP synthesis ability of ATP synthase.

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Azide inhibition of chloroplast ATPase is prevented by a high protonmotive force

Cited by 17 publications

References 24 publications

ATP Synthesis by F0F1-ATP Synthase Independent of Noncatalytic Nucleotide Binding Sites and Insensitive to Azide Inhibition

ATP Synthesis by F0F1-ATP Synthase Independent of Noncatalytic Nucleotide Binding Sites and Insensitive to Azide Inhibition

Effects of truncated mutants of the e subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability

Effects of truncated mutants of the ε subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability

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