The effect of N,N′-dicyclohexylcarbodiimide on enzymes of bioenergetic relevance

Azzi, Angelo; Casey, Robert P.; Nałȩcz, Maciej J.

doi:10.1016/0304-4173(84)90017-x

Cited by 118 publications

(50 citation statements)

References 124 publications

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“…Our results imply that the search for protein structures involved in this activity should focus on COI and COIl, and that the more than ten years of speculation about a role of COUI in proton pumping (Casey et al, 1980;Prochaska et al, 1981;Wikstrom and Casey, 1985;Capitanio et al, 1990;Prochaska and Wilson, 1990; for reviews, see Azzi et al, 1984;Brunori et al, 1987;Prochaska and Fink, 1987;Chan and Li, 1990) should come to an end. COIII has a distinct role in the assembly of the functional oxidase (for other assembly factors needed at earlier stages of oxidase biosynthesis, see Nobrega et al, 1990;Tzagoloff et al, 1990;Steinrucke et al, 1991).…”

mentioning

confidence: 74%

Subunit III of cytochrome c oxidase is not involved in proton translocation: a site-directed mutagenesis study.

1991

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Subunit III (COIII) is one of the three core subunits of the aa3‐type cytochrome c oxidase. COIII does not contain any of the redox centres and can be removed from the purified enzyme but has a function during biosynthesis of the enzyme. Dicyclohexyl carbodiimide (DCCD) modifies a conserved glutamic acid residue in COIII and abolishes the proton translocation activity of the enzyme. In this study, the invariant carboxylic acids E98 (the DCCD‐binding glutamic acid) and D259 of COIII were changed by site‐directed mutagenesis to study their role in proton pumping. Spectroscopy and activity measurements show that a structurally normal enzyme, which is active in electron transfer, is formed in the presence of the mutagenized COIII. Experiments with bacterial spheroplasts indicate that the mutant oxidases are fully competent in proton translocation. In the absence of the COIII gene, only a fraction of the oxidase is assembled into an enzyme with low but significant activity. This residual activity is also coupled to proton translocation. We conclude that, in contrast to numerous earlier suggestions, COIII is not an essential element of the proton pump.

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confidence: 74%

Subunit III of cytochrome c oxidase is not involved in proton translocation: a site-directed mutagenesis study.

1991

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“…DCCD is a protein modifying reagent that covalently binds to carboxylate residues involved in reversible protonation in hydrophobic environments (17). DCCD has previously been shown to act as a specific inhibitor of qE (14 -16), thus leading to the search for target sites using pH-sensing Glutamates in PsbSmutations E122Q and E226Q each reduce the level of DCCD binding to PsbS by ϳ50% and in the same way decrease the amplitude of qE.…”

Section: Discussionmentioning

confidence: 99%

“…There were no significant differences between npq4-1 and the double mutant or between the npq4-E122Q and npq4-E226Q single mutants. DCCD Binding-DCCD binds to carboxylate residues in hydrophobic environments (17,37). It is known for its ability to inhibit qE (14 -16), which suggests that carboxylate residues are involved in the process.…”

Section: Ph-sensing Glutamates In Psbsmentioning

confidence: 99%

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Regulation of Photosynthetic Light Harvesting Involves Intrathylakoid Lumen pH Sensing by the PsbS Protein

Gilmore

Caffarri

et al. 2004

Journal of Biological Chemistry

504

410

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The biochemical, biophysical, and physiological properties of the PsbS protein were studied in relation to mutations of two symmetry-related, lumen-exposed glutamate residues, Glu-122 and Glu-226. These two glutamates are targets for protonation during lumen acidification in excess light. Mutation of PsbS did not affect xanthophyll cycle pigment conversion or pool size. In conditions of excess light, photosynthetic light harvesting is regulated by a feedback de-excitation mechanism termed energy-dependent quenching (qE), 1 which increases thermal dissipation of excess absorbed light energy in photosystem II (PSII). The qE mechanism is triggered by conditions that limit photosynthetic carbon fixation and result in increased acidification of the chloroplast thylakoid lumen (1-4). The thermal dissipation of excess excitation energy is most commonly measured and referred to as nonphotochemical quenching (NPQ) of PSII chlorophyll (Chl) a fluorescence. Although there are several components of NPQ, in higher plants qE can account for the major part of NPQ and is characterized by its relatively fast induction and relaxation kinetics, on a physiological time scale of seconds to minutes. The decrease in the intensity of Chl fluorescence is the result of the decrease in the electronic excited state lifetime of Chl caused by an increased thermal dissipation rate constant (5). The rapid response of the qE process is chemically associated with changes in the trans-thylakoid membrane pH gradient (⌬pH). The ⌬pH change has at least two functions in qE. First, it activates the violaxanthin de-epoxidase that converts violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) (6). A and/or Z are essential elements of qE (7-9). Second, the lower pH in the lumen results in protonation of PSII proteins, including the 22-kDa PSII subunit, PsbS, which plays a key role in qE (10). When both pH-induced changes occur together it is believed that Chls in PSII can transfer their excess energy to Z, which can return to the ground state via thermal decay (7,11,12). Plants containing PsbS mutations of both glutamatesThe pH-sensing mechanism of the PsbS protein is influenced by two pairs of symmetrically arranged glutamate residues, each located within or close to the two lumen-exposed loops of the protein (13). Dicyclohexylcarbodiimide (DCCD), a well known inhibitor of qE (14 -16) is a carboxylate-modifying agent (17) that binds to PsbS (18). Although it was suggested that the DCCD binding site is in the lumenal loops of PsbS, the exact binding site has not been determined. Importantly, site-directed mutagenesis experiments indicated that two of the PsbS glutamates, Glu-122 and Glu-226, are necessary for the function of PsbS (13).In this article we used single and double mutations of PsbS (E122Q/E226Q) to make a detailed biochemical and biophysical analysis of the role of these two glutamates in pH sensing and DCCD binding. We probed the role of the Glu-122 and Glu-226 residues by monitoring the changes in the PSII Chl a fluores-

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“…In the presence of 1 mM DCCD the V-ATPase did not bind to the column, indicating that the binding site for bafilomycin is near the binding site(s) for DCCD and may thus be located in the proteolipid, subunit c. However, because DCCD, especially at the rather high concentration of 1 mM, may bind to sites different from that in the proteolipid and may bind not only to carboxyl groups but also to sulfhydryl groups or to tyrosine residues (9), and since the 100-kDa subunit a was not clearly resolved in this study, the bafilomycin binding subunit was still not identified. Strong hints in favor of subunit c representing the bafilomycin binding subunit were provided by recent results from analysis of Neurospora crassa strains, indicating that those with point mutations in subunit c show a higher tolerance to bafilomycin (I 50 80 -400 nM) compared with the wild type (I 50 6.3 nM); interestingly, the tolerance to concanamycin was not changed simultaneously (10).…”

mentioning

confidence: 93%

Concanamycin A, the Specific Inhibitor of V-ATPases, Binds to the Vo Subunit c

Huss

Ingenhorst

König

et al. 2002

Journal of Biological Chemistry

254

244

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The effect of N,N′-dicyclohexylcarbodiimide on enzymes of bioenergetic relevance

Cited by 118 publications

References 124 publications

Subunit III of cytochrome c oxidase is not involved in proton translocation: a site-directed mutagenesis study.

Subunit III of cytochrome c oxidase is not involved in proton translocation: a site-directed mutagenesis study.

Regulation of Photosynthetic Light Harvesting Involves Intrathylakoid Lumen pH Sensing by the PsbS Protein

Concanamycin A, the Specific Inhibitor of V-ATPases, Binds to the Vo Subunit c

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