L-1-N-methyl-4-mercaptohistidine disulfide, a potential endogenous regulator in the redox control of chloroplast coupling factor 1 in Dunaliella.

A water-soluble, highly polar, heat-stable, small molecule has been isolated from cell-free extracts of the halotolerant green alga Dunaliella salina. This compound, soluble inhibitory factor (SIF), when added to in vivo light-activated, thylakoid membrane-bound preparations of the D. salina coupling factor 1 (CF1), causes a rapid inactivation of the ATPase activity. SIF must be in its oxidized form to inactivate the CF1 ATPase and probably functions by oxidizing the reduced form of the light-activated enzyme. SIF has been purified to homogeneity and characterized by UV-visible and IR absorption spectroscopy, 1H and 13C NMR spectroscopy, and mass spectrometry. SIF has five different kinds of nonexchangeable protons and seven different kinds of carbon atoms. Three of the carbon atoms and one proton are part of a heterocyclic (imidazole) ring. One carbon atom is a carbonyl (carboxylic acid). One carbon atom and three protons form a methyl group attached to the aromatic ring. One carbon atom and two protons are a methylene group, and one carbon atom (an alpha-amino carbon) is attached to a single proton. In addition, in its reduced form, SIF contains a thiol group attached to the heterocyclic ring. From high resolution mass spectrometry, the molecular weight of SIF was determined to be 401 (M + H+) and is consistent with the composition being C14H21N6O4S2. The UV absorption of SIF shows a large increase at 240 nm upon reduction. An effective difference extinction coefficient for this absorbance change has been calculated to be 6.84 meq/cm. A comparison of SIF with the oxidized form of ovothiol A (1-N-methyl-4-mercaptohistidine disulfide) shows the two compounds to be identical in all respects. In addition, ovothiol A disulfide is as effective as SIF in inhibiting the light-triggered, CF1 ATPase activity. It is concluded, therefore, that SIF and L-1-N-methyl-4-mercaptohistidine disulfide are identical.

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ.

Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3-5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF1). This activity, approximately 150-300 mumol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF1 is due to the reduction of the enzyme in vivo in the light.

The partial purification of a factor from Dunaliella salina that causes the rapid in situ inactivation of light-activated chloroplast coupling factor 1 (CF1).

A factor having the expected properties of the in vivo oxidant responsible for inactivating the in vivo light-activated chloroplast coupling factor 1 (CF1) has been partially purified from cell-free extracts of Dunaliella salina. This factor is highly polar, weakly acidic, and relatively temperature stable. The ability of this factor to inactivate light-activated CF1 is prevented if it is pretreated with reductants such as dithiothreitol. The factor has virtually no effect on the ethanol-induced, Mg2+-dependent ATPase activity of the isolated CF1.

Characterization of nucleotide-binding sites on the chloroplast coupling factor 1 using two photolabile analogs.

Nucleotide-binding sites on the chloroplast coupling factor 1 (CF1) have been probed using two photoreactive ADP analogs: 2-azido-ADP (2-N3-ADP) and 2',3'-O-(4-benzoyl)benzoyl-ADP (Bz-ADP). Photolabeling of the isolated CF1 with 2-N3-ADP results in incorporation of the analog exclusively into the beta-subunit of the enzyme. The location of the nucleotide-binding site(s) within the beta-subunit of the CF1 was investigated using peptide mapping. Within the discrimination limits of this technique, it is concluded that the azido- and benzoyl-modified analogs both bind to the same conformation of the nucleotide-binding site(s) of soluble CF1. Bz-ADP, however, labels the binding site(s) on membrane-bound CF1 in a slightly different manner.

N-Ethylmaleimide inhibition of the catalytic activities of the Dunaliella salina coupling factor 1 (CF1) and the restoration of the inhibition of the CF1 ATPase activity by N-ethylmaleimide.

The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF1) to chemical modification by N-ethylmaleimide has been investigated. When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF1 ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF1 as shown by the time-course for the inhibition and the maximal extent of inhibition. Treatment of the purified, latent, D. salina CF1 with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I50 approximately 50 microM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF1 is partially reversed by either prolonged incubation at low concentrations of N-ethylmaleimide or short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF1 that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF1 are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.

Evidence for solvent-induced conformational changes of the soluble Dunaliella chloroplast coupling factor 1 (CF1).

The ATPase activity of the chloroplast coupling factor 1 (CF1) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca2+-dependent activity. The Ca2+-dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca2+ to Mg2+. Both the Ca2+-dependent and Mg2+-dependent ATPase activities of heat-treated Dunaliella CF1 are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF1. However, when assayed under identical conditions, the Ca2+-dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg2+-dependent activity. These data are interpreted as indicating that soluble Dunaliella CF1 can exist in a variety of conformations, at least one of which catalyzes a Ca2+-dependent ATPase and two or more of which catalyze an Mg2+-dependent ATPase.

Purification and Characterization of a Glycerol-Resistant CF(0)-CF(1) and CF(1)-ATPase from the Halotolerant Alga Dunaliella bardawil.

The isolation of the chloroplast ATP synthase complex (CF(0)-CF(1)) and of CF(1) from Dunaliella bardawil is described. The subunit structure of the D. bardawil ATPase differs from that of the spinach in that the D. bardawil alpha subunit migrates ahead of the beta subunit and epsilon-migrates ahead of subunit II of CF(0) when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF(1) isolated from D. bardawil resembles the CF(1) isolated from Chladmydomonas reinhardi in that a reversible, Mg(2+)-dependent ATPase is induced by selected organic solvents. Glycerol stimulates cyclic photophosphorylation catalyzed by D. bardawil thylakoid membranes but inhibits photophosphorylation catalyzed by spinach thylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-P(i) exchange catalyzed by D. bardawil CF(0)-CF(1) proteoliposomes but inhibits the activity with the spinach enzyme. The ethanol-activated, Mg(2+)-ATPase of the D. bardawil CF(1) is more resistant to glycerol inhibition than the octylglucoside-activated, Mg(2+)-ATPase of spinach CF(1) or the ethanol-activated, Mg(2+)-dependent ATPase of the C. reinhardi CF(1). Both cyclic photophosphorylation and ATP-P(i) exchange catalyzed by D. bardawil CF(0)-CF(1) are more sensitive to high concentrations of NaCl than is the spinach complex.

Isolation, purification, and characterization of coupling factor 1 from Chlamydomonas reinhardi.

Chloroplast thylakoid particles were prepared from wild-type Chlamydomonas reinhardi by gentle sonication. These particles catalyzed phenazine methosulfate dependent photophosphorylation with rates ranging from 300 to 700 mumol of adenosine 5'-triphosphate (ATP) formed (mg of chlorophyll)-1h-1. Photophosphorylation was not sensitive to tentoxin but was sensitive to an anticoupling factor 1 (CF1) antiserum preparation made against spinach CF1. The C. reinhardi chloroplast CF1 was isolated from thylakoid particles by either chloroform or ethylenediaminetraacetic acid extraction. The former enzyme appeared to be missing the gamma subunit and did not reconstitute with partially resolved thylakoid particles. The latter enzyme reconstituted with partially resolved particles and had a specific activity at 37 degrees C of 2-5 umol of ATP hydrolyzed (mg of protein)-1 min-1. The enzyme utilized both MnATP and MgATP. CaATP was a poor substrate, and SrATP was not hydrolyzed. The enzyme was not activated by heat or proteolysis but was stimulated approximately 2-fold by 50 mM dithiothreitol. Alcohols reversibly stimulated the ATPase activity of the enzyme 5-25-fold. Ethanol, 20%, dramatically lowered the temperature optimum from approximately 75 to approximately 45 degrees C and slightly lowered the pH optimum from 8.5 to 8.2. Ethanol had no effect on the activation energy of the ATPase reaction (17 +/- 1.7 kcal/mol). The kinetics of the ATPase reaction catalyzed by the C. reinhardi enzyme are complex. Both free divalent cations and divalent cation ATP inhibited the activity of the enzyme. The apparent Km for MgTAP (55 uM free Mg2+) was approximately 0.2 mM.

The steady state kinetics of photophosphorylation.

The steady state kinetics of phenazine methosulfate-catalyzed, photosystem I-driven photophosphorylation with spinach thylakoid membranes has been measured. The Km for ADP is 85 +/- 20 microM and the Km for phosphate is 0.58 +/- 0.11 mM. Adenosine-5'-O-(thiodiphosphate) (beta-ThioADP) is a competitive inhibitor with respect to ADP at high phosphate concentrations (Kis = 105 +/- 14 microM), but is a noncompetitive inhibitor with respect to phosphate (at all ADP concentrations) and ADP at low phosphate concentrations. Thiophosphate is a competitive inhibitor with respect to phosphate (Kis = 95 +/- 9 microM), but an uncompetitive inhibitor with respect to ADP. ADP also inhibits the rate of phosphorylation at high ADP to phosphate ratios. An ordered Bi Uni mechanism with ADP preceding the binding of phosphate is proposed to explain the kinetic data.