Inhbition of photosystem II-dependent phosphorylation in chloroplasts by mercurials.

Photophosphorylation supported by the coupling site associated with Phostosystem II electron transport (coupling site II) is 50 to 60 times less sensitive to the energy transfer inhibitor HgCl₂ than phosphorylation supported by the coupling site associated with Photosystem I electron transport (coupling site I). Coupling site II phosphorylation is only about 2 times less sensitive to the lipophilic mercurial p-hydroxymercuribenzoate (PHMB), however. Both coupling sites are equally sensitive to CF₁ antiserum. These results suggest that a portion of the energy conserving apparatus associated with coupling site II is in a more hydrophobic environment than the corresponding apparatus associated with coupling site I.     

The phosphorylation site associated with the oxidation of exogenous donors of electrons to Photosystem I

1. The Photosystem I-mediated transfer of electrons from diaminodurene, diaminotoluene and reduced 2,6-dichlorophenolindophenol to methylviologen is optimal at pH 8–8.5, where phosphorylation is also maximal. In the presence of superoxide dismutase, the efficiency of phosphorylation rises from ? 0.1 at pH 6.5 to 0.6–0.7 at pH 8–8.5, regardless of the exogenous electron donor used.2. The apparent K_m (at pH 8.1) for diaminodurene is 6·10^?4 M and for diaminotoluene is 1.2·10^?3 M. The concentrations of diaminodurene and diaminotoluene required to saturate the electron transport processes are > 2 mM and > 5 mM, respectively. At these higher electron donor concentrations the rates of electron transport are markedly increased by phosphorylation (1.5-fold) or by uncoupling conditions (2-fold).3. Kinetic analysis of the transfer of electrons from reduced 2,6-dichlorophenolindophenol (DCIPH₂) to methylviologen indicates that two reactions with very different apparent K_m values for DCIPH₂ are involved. The rates of electron flux through both pathways are increased by phosphorylation or uncoupling conditions although only one of the pathways is coupled to ATP formation. No similar complications are observed when diaminodurene or diaminotoluene serves as the electron donor.4. In the diaminodurene → methylviologen reaction, ATP formation and that part of the electron transport dependent upon ATP formation are partially inhibited by the energy transfer inhibitor HgCl₂. This partial inhibition of ATP formation rises to about 50% at less than 1 atom of mercury per 20 molecules of chlorophyll, then does not further increase until very much higher levels of mercury are added.5. It is suggested that exogenous electron donors such as diaminodurene, diaminotoluene and DCIPH₂ can substitute for an endogenous electron carrier in donating electrons to cytochrome f via the mercury-sensitive coupling site (Site I) located on the main electron-transporting chain. If this is so, there would seem to be no reason for postulating yet another coupling site on a side branch of the electron transport chain in order to account for cyclic photophosphorylation.     

Site-specific inhibition of photophosphorylation in isolated spinach chloroplasts by HgCl2. II. Evidence for three sites of energy conservation associated with non-cyclic electron transport

Energy transfer inhibition by HgCl₂ has been demonstrated to be selective for certain System I partial reactions. On the basis of different HgCl₂ effects on the System I reactions, reduced 2,6-dichlorophenolindophenol → methylviologen, diaminodurene → methylviologen and N-phenazine methosulfate cyclic, two sites of energy conservation associated with System I are proposed. Furthermore, these sites are in parallel with each other, in series with the site closely associated with Photosystem II and are shared between non-cyclic and cyclic electron transport.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Inhibition of energy-transducing functions of chloroplast membranes by lipophilic iron chelators

Lipophilic metal chelators inhibit various energy-transducing functions of chloroplasts. The following observations were made.1. Photophosphorylation coupled to any known mode of electron transfer, i.e. whole-chain noncyclic, the partial noncyclic Photosystem I or Photosystem II reactions, or cyclic, is inhibited by several lipophilic chelators, but not by hydrophilic chelators.2. The light- and dithioerythritol-dependent Mg²⁺-ATPase was also inhibited by the lipophilic chelators.3. Electron transport through either partial reaction, Photosystem I or Photosystem II was not inhibited by lipophilic chelators. Whole-chain coupled electron transport was inhibited by bathophenanthroline, and the inhibition was not reversed by uncouplers. The diketone chelators diphenyl propanedione and nonanedione inhibited the coupled, whole-chain electron transport and the inhibition was reversed by uncouplers, a pattern typical of energy transfer inhibitors.The electron transport inhibition site is localized in the region of plastoquinone → cytochrome f. This inhibition site is consistent with other recent work (Prince et al. (1975) FEBS Lett. 51, 108 and Malkin and Aparicio (1975) Biochem. Biophys. Res. Commun. 63, 1157) showing that a non-heme iron protein is present in chloroplasts having a redox potential near +290 mV. A likely position for such a component to function in electron transport would be between plastoquinone and cytochrome f, just where our data suggests there to be a functional metalloprotein.4. Some of the lipophilic chelators induce H⁺ leakiness in the chloroplast membrane, making interpretation of their phosphorylation inhibition difficult. However, 1–3 mM nonanedione does not induce significant H⁺ leakiness, while inhibiting ATP formation and the Mg²⁺-ATPase. Nonanedione, at those concentrations, causes a two- to four-fold increase in the extent of H⁺ uptake.5. These results are consistent with, but do not prove, the involvement of a non-heme iron or a metalloprotein in chloroplast energy transduction.     

Effect of light quality on the composition and function of thylakoid membranes in atriplex triangularis

Light quality was shown to exert well-coordinated regulatory effects on the composition and function of the thylakoid membranes as well as on the photosynthetic rates of intact leaves from Atriplex triangularis grown in continuous blue, white and red lights (50 μE · m^?2 · s^?1). The higher photosynthetic rates in plants grown in blue light, as compared to those in white and red lights, resulted from marked changes in both light-harvesting complexes and electron carriers. The concentrations of electron carriers such as atrazine binding sites, plastoquinone, cytochromes b and f and P-700 on a chlorophyll basis were markedly increased in Atriplex grown in blue light; and the apparent light-harvesting antenna unit sizes of Photosystems I and II were greatly reduced. Consequently, the electron transport capacities of Photosystems I and II were also increased as was the coupling factor CF₁ activity. Atriplex grown in red light had lower photosynthetic rates than those grown in blue or white light by incorporating changes in the composition and function of the thylakoids in a direction opposite to those caused by growth in blue light. When these regulatory effects of light quality were compared with those of light quantity [6,7], it is clear that $\text{Chl}\text{a}\text{Chl}\text{b}$ ratios, electron transport capacities of Photosystems I and II, concentrations of plastoquinone, atrazine binding sites, coupling factor CF₁ activity and the apparent antenna unit size of Photosystem II are more affected by light quantity, whereas light quality has a greater influence on the concentration of P-700, the apparent antenna unit size of Photosystem I and the overall photosynthetic rates of intact leaves.     

Electron transport and photophosphorylation in chloroplasts as a function of the electron acceptor. III. A dibromothymoquinone-insensitive phosphorylation reaction associated with Photosystem II

Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) is reputed to be a plastoquinone antagonist which prevents the photoreduction of hydrophilic oxidants such as ferredoxin-NADP⁺. However, we have found that dibromothymoquinone inhibits only a small part of the photoreduction of lipophilic oxidants such as oxidized p-phenylenediamine. Dibromothymoquinone-resistant photoreduction reactions are coupled to phosphorylation, about 0.4 molecules of ATP consistently being formed for every pair of electrons transported. Dibromothymoquinone itself is a lipophilic oxidant which can be photoreduced by chloroplasts, then reoxidized by ferricyanide or oxygen. The electron transport thus catalysed also supports phosphorylation and the $\text{P}\text{e}{}_2$ ratio is again 0.4. It is concluded that there is a site of phosphorylation before the dibromothymoquinone block and another site of phosphorylation after the block. The former site must be associated with electron transfer reactions near Photosystem II, while the latter site is presumably associated with the transfer of electrons from plastoquinone to cytochrome f.     

The stoichiometry (ATP/2e− ratio) of non-cyclic photophosphorylation in isolated spinach chloroplasts

1. The stoichiometry of non-cyclic photophosphorylation and electron transport in isolated chloroplasts has been re-investigated. Variations in the isolation and assay techniques were studied in detail in order to obtain optimum conditions necessary for reproducibly higher ADP/O (equivalent to ATP/2e^?) and photosynthetic control ratios.2. Studies which we carried out on the possible contribution of cyclic phosphorylation to non-cyclic phosphorylation suggested that not more than 10% of the total phosphorylation found could be due to cyclic phosphorylation.3. Photosynthetic control, and the uncoupling of electron transport in the presence of NH₄Cl, were demonstrated using oxidised diaminodurene as the electron acceptor. A halving of the ADP/O ratio was found, suggesting that electrons were being accepted between two sites of energy conservation, one of which is associated with Photosystem I and the other associated with Photosystem II.4. ATP was shown to inhibit State 2 and State 3 of electron transport, but not State 4 electron transport or the overall ADP/O ratio, thus confirming its activity as an energy transfer inhibitor. It is suggested that part of the non-phosphorylating electron transport rate (State 2) which is not inhibited by ATP is incapable of being coupled to subsequent phosphorylation triggered by the addition of ADP (State 3). If the ATP-insensitive State 2 electron transport is deducted from the State 3 electron transport when calculating the ADP/O ratio, a value of 2.0 is obtained.5. The experiments reported demonstrate that there are two sites of energy conservation in the non-cyclic electron transfer pathway: one associated with Photosystem II and the other with Photosystem I. Thus, non-cyclic photophosphorylation can probably produce sufficient ATP and NADPH “in vivo” to allow CO₂ fixation to proceed.     

The plastoquinone pool as possible hydrogen pump in photosynthesis

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H⁺ + e^?) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H⁺ uptake and the internal H⁺ release led to the following conclusions:(1) A stoichiometric coupling of 1 : 1 : 1 between the external H⁺ uptake, the electron translocation through the plastoquinone pool and the internal H⁺ release (corrected for H⁺ release due to H₂O oxidation) is valid (pH_out = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation.These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H⁺ + e^?) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.     

Purification, molecular weight, amino acid, and subunit composition of arylsulfatase A from human liver.

In isolated hepatic mitochondria, the oxidation of NAD⁺-dependent substrates was decreased after chronic consumption of ethanol or by the addition of acetaldehyde in vitro. Reversed electron transport from succinate to NAD^?, which requires transfer of electrons through the NADH dehydrogenase complex and energy transduction through coupling site 1, was depressed by ethanol feeding and by acetaldehyde in vitro, whereas NADH formation from glutamate, which is mediated directly by substrate oxidation and is not energy-dependent, was slightly increased. By contrast, reactions involving the terminal portion of the phosphorylation chain, e.g., ATP-³²P exchange or dinitrophenolstimulated ATPase activity, were not affected. Adenine nucleotide translocase activity was not altered by chronic consumption of ethanol or the addition of acetaldehyde in vitro. These data suggest that the NADH-ubiquinone oxidoreductase complex of the respiratory chain, a segment which contains several iron-sulfur centers which participate in electron transport and energy transduction, may be impaired by chronic consumption of ethanol and is especially sensitive to inhibition by acetaldehyde in vitro. Neither energy coupling sites II or III, nor the terminal reactions of oxidative phosphorylation share this sensitivity. CO₂ production from various labeled intermediates of the citric acid cycle was depressed after chronic consumption of ethanol and after the addition of acetaldehyde. Acetate had no effect on these reactions, indicating that the inhibition by acetaldehyde is not mediated via acetate. Impairment of the activities of the respiratory chain and the citric acid cycle, or both, may explain the decreases in oxygen uptake and CO₂ production from citric acid cycle intermediates and fatty acids, as well as the increase in ketone body production, found in mitochondria from ethanolfed rats.     

Two pathways of electron transfer in quinol-mediated cyclic phosphorylation in spinach chloroplasts

Low potential quinones are mediators of cyclic phosphorylation in washed spinach thylakoid membranes if they are prereduced to provide the proper redox poise. Cyclic phosphorylation catalyzed by different quinols varies in its sensitivity to the electron transfer inhibitor 2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether (DNPINT), which is thought to inhibit electron flux from the bound plastoquinone (B) to the plastoquinone pool (Trebst, A., Wietoska, H., Draber, W. and Knops, H.J. (1978) Z. Naturforsch. 33c, 919–927). Cyclic phosphorylation catalyzed by uncharged quinols is extremely sensitive to DNPINT, whereas cyclic phosphorylation catalyzed by negatively charged quinols is approximately two orders of magnitude less sensitive. Many quinols have pK₁ values in the physiological range (pH 7–9). Increasing the concentration of the deprotonated quinol either by raising the assay pH, increasing the mediator concentration, or increasing the fractional reduction of the quinone results in a decrease in the sensitivity of cyclic phosphorylation to DNPINT. At very high DNPINT concentrations, cyclic phosphorylation catalyzed by all quinols (and ferredoxin) is inhibited, but not phenazine methosulfate catalyzed cyclic phosphorylation.These data suggest that the deprotonated form of the quinol can donate electrons directly to the plastoquinone pool, whereas the uncharged quinol most obligately transfer electrons through the bound plastoquinone ‘B’. A second site of DNPINT action after the plastoquinone pool is also observed, which requires much higher DNPINT concentrations for inhibition of phosphorylation.     

EGTA, a calcium chelator, inhibits electron transport in photosystem II of spinach chloroplasts at two different sites

A fifteen minute incubation of spinach chloroplasts with the divalent Ca²⁺ chelator, EGTA, in concentrations 50–250 μM, inhibits electron transport through both photosystems. All photosystem II partial reactions, including indophenol, ferricyanide and the DCMU-insensitive silicomolybdate reduction are inhibited from 70–100%. The photosystem II donor reaction, diphenyl carbazide → indophenol, is also inhibited, indicating that the inhibition site comes after the Mn²⁺ site, and that the first Ca²⁺ effect noted (site II) is not on the water oxidation enzyme, as is commonly assumed, but between the Mn²⁺ site and plastoquinone A pool. The other photosystem II effect of EGTA (Ca²⁺ site I), occurs in the region between plastoquinone A and P700 in the electron transport chain of chloroplasts. About 50% inhibition of the reaction ascorbate + TMPD → methyl viologen is given by incubation with 200 μM EGTA for 15 min. Ca²⁺ site II activity can be restored with 20 mM CaCl₂. Ca²⁺ site I responds to Ca²⁺ and plastocyanin added jointly. More than 90% activity in the ascorbate + TMPD → methylviologen reaction can be restored. Various ways in which Ca²⁺ ions could affect chloroplast structure and function are discussed. Since EGTA is more likely to penetrate chloroplast membranes than EDTA, which is known to remove CF₁, the coupling factor, from chloroplast membranes, and since Mg²⁺ ions are ineffective in restoring activity, it is concluded that Ca²⁺ may function in the electron transport chain of chloroplasts in a hitherto unsuspected manner.     

Sites of inhibition by disulfiram in thylakoid membranes

Disulfiram (tetraethylthiuram disulfide), a metal chelator, inhibits photosynthetic electron transport in broken chloroplasts. A major site of inhibition is detected on the electron-acceptor side of photosystem II between Q_A, the first plastoquinone electron-acceptor, and the second plastoquinone electron-acceptor, Q_B. This site of inhibition is shown by a severalfold increase in the half-time of Q⁻_A oxidation, as monitored by the decay of the variable chlorophyll a flourescence after an actinic flash. Another site of inhibition is detected in the functioning of the reaction center of photosystem II; disulfiram is observed to quench the room temperature variable chlorophyll a fluorescence, as well as the intensity of the 695 nm peak, relative to the 685 nm peak, in the chlorophyll a fluorescence spectrum at 77 K. Electron transport from H₂O to the photosystem II electron-acceptor silicomolybdate is also inhibited. Disulfiram does not inhibit electron flow before the site(s) of donation by exogenous electron donors to photosystem II, and no inhibition is detected in the partial reactions associated with photosystem I.     

Mercury ions inhibit photosynthetic electron transport at multiple sites in the cyanobacteriumSynechococcus 6301

Inhibition of electron transport activities in the spheroplasts ofSynechococcus 6301 by HgCl² is dependent on the concentration of mercury ions. The inhibition of whole chain electron transport activity occurs at low concentration of Hg²⁺ (6 ΜM@#@). This inhibition occurs mostly due to interaction of Hg²⁺ on plastocyanin. At an elevated concentration (24 ΜM@#@), mercury induces inhibition chiefly in photosystem II catalyzed electron transport. At this concentration it also alters both the absorption and emission characteristics of the phycocyanin. The photosystem I catalyzed electron transport was inhibited by 50% only at high concentrations (36 ΜM@#@) of HgCl₂. However, electron transport catalyzed by photosystems I and II from reduced duroquinone to methylviologen which involves intersystem electron transport is extremely sensitive to mercury (low concentration 6–9 ΜM) like that of whole chain assay indicating that the observed inhibition in whole chain electron transport at low concentrations is mostly contributed by the damage involving other intersystem electron transport carrier(s) like plastocyanin. Thus mercury ions depending on the concentration affects the electron transport at multiple sites in the spheroplasts ofSynechococcus.     

A Calcium-Selective Site in Photosystem II of Spinach Chloroplasts

After acid-treatment of spinach (Spinacia oleracea) chloroplasts, various partial electron transport reactions are inactivated from 25 to 75%. Divalent cations in concentrations from 10 to 50 millimolar can partially restore electron transport rates. Two cation-specific sites have been found in photosystem II: one on the 3-(3,4-dichlorophenyl)-1, 1-dimethylurea-insensitive silicomolybdate pathway, which responds better to restoration by Mg²⁺ than by Ca²⁺ ions, the other on the forward pathway to photosystem I, located on the 2,5-dimethylbenzoquinone pathway. This site is selectively restored by Ca²⁺ ions. When protonated chloroplasts are treated with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aziridine, a carboxyl group modifying reagent, presumed to react with glutamic and aspartic acid residues of proteins, restoration of electron transport at the Ca²⁺-selective site on the 2,5-dimethylbenzoquinone pathway is impaired, while no difference in restoration is seen at the Mg²⁺ site on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive silicomolybdate pathway.

Trypsin treatment of chloroplasts modifies the light-harvesting pigment-protein complex, destroys the dibromothymoquinone-insensitive 2,5-dimethyl-benzoquinone reduction, but does not interfere with the partial restoration of activity of this pathway by Ca²⁺ ions, implying that the selective Ca²⁺ effect on photosystem II (selective Ca²⁺ site) is different from its effects as a divalent cation on the light-harvesting pigment-protein complex involved in the excitation energy distribution between the two photosystems.

     

Valinomycin inhibition of chloroplast electron transport at or near plastoquinone

Valinomycin inhibits the coupled portion of whole-chain noncyclic electron transport and phosphorylation and also inhibits the anaerobic menadione-dependent cyclic phosphorylation. Both of these electron transport systems are thought to involve plastoquinone. Electron transport and phosphorylation in various photosystem I and II partial reactions not involving plastoquinone or phosphorylation using other cyclic cofactors that are not dependent on plastoquinone were relatively insensitive to valinomycin. Single-beam ultraviolet spectrophotometric measurements showed that valinomycin decreased the apparent first-order rate constant for plastohydroquinone oxidation by more than twofold and had no observable effect on the reduction rate constant. The valinomycin inhibitions did not require K⁺ addition to the media. However, it was shown that extensive washing or dialysis did not reduce the chloroplast endogenous K⁺ content below 0.1 μmol K⁺/mg of chloroplasts.     

Uncouplers enhance photosynthetic electron transport from water to NADP+ in the presence of plastoquinone inhibitors

Uncouplers have been previously observed to relieve appreciably the inhibition of photosynthetic electron transport from water to NADP⁺ by the plastoquinone analogues, dibromothymoquinone (DBMIB) and dinitrophenyl ether of iodonitrothymol (DNP-INT). These results were now extended by demonstrating that the reversal by uncouplers of DBMIB and DNP-INT inhibition occurred under conditions when the uncouplers did not stimulate or inhibit NADP⁺ reduction in control treatments without the plastoquinone analogues. Since effects of uncouplers on photosynthetic electron transport depend on external pH, we determined for each of the four uncouplers, gramicidin, nigericin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and SF 6847 (a ditertiary phenol derivative) its effect on oxygenic electron transport (H₂O to NADP⁺) over a range of external pH from 6.7 to 8.7. The effect of each uncoupler on counteracting the inhibition of DBMIB and DNP-INT was then measured at its crossover external pH at which the uncoupler had little or no effect on electron transport in the uninhibited controls. Under these controlled conditions, uncouplers increased the rate of plastoquinone-inhibited electron transport, in some cases by almost 300%. To explain these results, a role for plastoquinone in processing protons released by the oxidation of water is postulated.     

Regulation of Human Cytidine Triphosphate Synthetase 2 by Phosphorylation

Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the K_m and IC₅₀ values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. Metabolic labeling of hCTPS2 with [³²P]H₃PO₄ demonstrated that Ser⁵⁶⁸ and Ser⁵⁷¹ were two major phosphorylation sites, and additional studies demonstrated that Ser⁵⁶⁸ was phosphorylated by casein kinase 1 both in vitro and in vivo. Interestingly, mutation of Ser⁵⁶⁸ (S568A) but not Ser⁵⁷¹ significantly increased hCTPS2 activity, demonstrating that Ser⁵⁶⁸ is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the V_max of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser⁵⁶⁸ as a major site of CTPS2 regulation by phosphorylation.     

Interactions of herbicides and azidoquinones at a Photosystem II binding site in the thylakoid membrane

6-Azido-5-decyl-2,3-dimethoxy-p-benzoquinone (6-azido-Q₀C₁₀) was found to replace the native plastoquinone at B (the second stable electron acceptor to Photosystem II (PS II)). The 6-azido-Q₁₀C₁₀ would accept electrons from the primary electron-accepting quinone, Q, thus allowing electron transport through PS II to the plastoquinone pool in thylakoids. The synthetic azidoquinone also competes with the PS II herbicides ioxynil and atrazine for binding. This observation strongly favors the hypothesis that PS II herbicides block electron transport by replacing the native quinone which acts as the second electron carrier on the reducing side of PS II (termed B). Covalent linkage of 6-azido-Q₀C₁₀ to its binding environment by ultraviolet irradiation greatly reduces herbicide-binding affinity but does not lead to a loss in herbicide-binding sites. We take this as evidence that covalent attachment of 6-azido-Q₀C₁₀ allows some freedom of quinone head-group movement such that the herbicides can enter the binding site. This indicates that the protein determinants which regulate quinone and herbicide binding are very closely related, but not identical. A compound somewhat related to 6-azido-Q₀C₁₀ is 2-azido-3-methoxy-5-geranyl-6-methyl-p-benzoquinone (2-azido-Q₂). This compound was found to be an ineffective competitor with respect to herbicide binding. Thus, interactions with protein-binding determinants are highly dependent on the molecular structure of quinones. The 2-azido-Q₂ was an inhibitor of electron flow in the intersystem portion of the chain.