John D. Mills

Thiol modulation of the chloroplast protonmotive ATPase and its effect on photophosphorylation

Thiol modulation of the chloroplast protonmotive ATPase (CF0-CF1) by preillumination of broken chloroplasts in the presence of dithiothreitol (or preillumination of intact chloroplasts in the absence of added thiols) had the following effects on photophosphorylation. (1) When assayed at pH 8 and saturating light, the initial rate of photophosphorylation was increased by 10–40%. There was an accompanying increase in the rate of coupled electron transport with no significant change in the overall P2e ratio. (2) On lowering the pH of the assay medium to pH 7, the stimulatory effect of thiol modulation on photophosphorylation and coupled electron flow was enhanced. At pH 7, there was also a small increase in P2e ratio. (3) Addition of a non-saturating amount of uncoupler to the assay medium enhanced the stimulatory effect of thiol modulation on photophosphorylation. In the presence of 1 mM NH4Cl, there was only a small increase in coupled electron flow and a correspondingly larger increase in P2e ratio. (4) Lowering the light intensity, or inhibiting electron transport, diminished the stimulatory effect of thiol modulation on photophosphorylation, coupled electron transport and P2e ratio. (5) Under all the above conditions, the ΔpH maintained across the thylakoid membrane was lower after thiol modulation, even when photophosphorylation markedly increased in rate. (6) Thiol modulation of CF0-CF1 increased the observed Michaelis constant for ADP (Km(ADP)) and the apparent maximum rate (Vapp of photophosphorylation by the same factor, so that ratio VappKm was not altered. VappKm was also unaffected by changing the medium pH, but was significantly decreased upon addition of uncouplers to the medium. These results indicate that the observed rate of ATP synthesis catalysed by thiol demodulated chloroplasts is limited kinetically by the fraction (α) of enzyme molecules that are active during photophosphorylation. A model based on a dual pH optimum requirement for activation of CF0-CF1 is presented to explain the dependence of α on ΔpH. Thiol modulation of CF0-CF1 is proposed to stimulate photophosphorylation by causing the enzyme to become active over a lower range of ΔpH, thereby reducing the kinetic limitation on ATP synthesis imposed by the activation process.

Modulation of coupling factor ATPase activity in intact chloroplasts. Reversal of thiol modulation in the dark

Abstract Illumination of intact pea chloroplasts results in both the pH activation and the thiol modulation of the reversible protonmotive ATPase, or CF0-CF1 (Mills, J.D., Mitchell, P. and Schurmann, P. (1980) FEBS Lett. 112, 173–177). We have studied the reversal of the activation and thiol-modulation processes that occurs when illuminated chloroplasts are darkened. (1) Methyl viologen is shown to prevent light-dependent thiol modulation of CF0-CF1 in intact chloroplasts. Studies using methyl viologen indicate that the decline in ATPase activity of intact chloroplasts in the dark is due both to pH deactivation and thiol demodulation of CF0-CF1. Reillumination in the presence of methyl viologen reactivates CF0-CF1 complexes that have undergone pH deactivation, but does not allow thiol modulation to occur. (2) At 20°C, both pH deactivation and thiol demodulation of CF0-CF1 are complete within 10 min darkness. At 3.5°C, both processes are retarded, and require more than 30 min for completion. (3) Lysis of intact chloroplasts greatly retards the thiol demodulation of CF0-CF1. In the dark, addition of oxidants to lysed chloroplasts promotes thiol demodulation. Under these conditions, thioredoxin behaves as a weak oxidant, but glutathione has very little effect. Illumination of intact chloroplasts followed by rapid lysis and storage on ice stabilises the pH-activated, thiol-modulated state of CF0-CF1 for extended periods in the dark and increases the time required for thiol demodulation to well over 2 h. This method may prove to be useful for preparing thiol-modulated chloroplasts without the need to add or remove exogenous thiols. The results suggest that an oxidative system exists in the stroma which actively reverses thiol modulation of CF0-CF1 in the dark. Such a system would be expected if thiol modulation of CF0-CF1 were an important physiological process regulating reversible ATPase activity in vivo.

Thiol modulation of CF0CF1 stimulates acid/base-dependent phosphorylation of ADP by broken pea chloroplasts

There is now considerable evidence that the reversible protonmotive ATPase of chloroplasts (CFo-CFl) is a highly regulated enzyme both in vivo [1,2] and in vitro [3-l 11. Studies with broken chloroplasts (that lack an outer envelope and stroma) have shown that CFu-CFt in dark-adapted chloroplasts is catalytically inactive, but undergoes activation when a difference in the electrochemical potential of protons (AjIH+) is generated across the thylaloid [3-lo]. This activation is accompanied by the release of 1 mol bound ADP/mol CFl, by which means the activation process has been extensively studied [5-IO]. Deactivation of CFu-CFl occurs upon the collapse of A-H+ and is associated with the rebinding of ADP [8-lo]. Activation of CFc-CFl may simply reflect a dual pH optimum requirement of CFl, whereby the stromal, or N pole, of CFl has an optimum around pH 8 but the intrathylakoid, or P pole, has an optimum around pH 5 [ 121. These differential pH conditions would normally only exist when AT;H+ was present across the thylakoid. Deactivation would occur when the appropriate pH conditions ceased to exist at either the P or N poles of CFt, and its rate would be influenced by the presence of bound nucleotides. We call this type of regulation of CFo-CFl ‘pH (de)activation’. In addition to pH activation, CFo-CFl activity is also modulated by reduced dithiols [3, 13-171. In the absence of reduced dithiols, CFu-CFl is in a demodulated state and is observed to catalyse net synthesis of ATP under the appropriate conditions, but little ATPase or ATP=Pi exchange activity is observed. In contrast, the presence of reduced dithiols modulates the pH-activated CFu-CFt [3] and ATP hydrolysis [3,13] and PiGATP exchange [ 141 are readily observed.

Changes in the apparent Michaelis constant for ADP during photophosphorylation are consistent with delocalised chemiosmotic energy coupling

Abstract The apparent Michaelis constant ( K m ) for ADP has been measured under various conditions of steady-state photophosphorylation in isolated thylakoid membranes. In addition, the steady-state ΔpH has been simultaneously estimated from the fluorescence quenching of 9-aminoacridine. The following results were obtained. (1) The standard procedure for estimating K m , by increasing the concentration of ADP, progressively lowered the steady-state ΔpH, thereby introducing an uncontrolled system variable into the K m analysis. This has the effect of lowering the apparent K m measured. (2) Lowering the light intensity lowered the observed K m , and addition of uncouplers increased the observed K m . The ability of uncouplers to increase K m was enhanced at lowered light intensities. In contrast, the effect of lowered light intensity on the observed K m was diminished and then reversed under progressively more uncoupled conditions. (3) The addition of energy-transfer inhibitors caused an increase in the observed K m for ADP. (4) All of the observations are qualitatively predicted by a mathematical model based on simple delocalised chemiosmotic energy coupling and Michaelis-Menten kinetics for the chloroplast ATPase with respect to ADP. It is concluded that the complex behaviour of the apparent K m for ADP under different conditions arises because ΔpH is an uncontrolled variable during the K m analysis and that the results are entirely consistent with a model of delocalised chemiosmotic energy coupling.

Thiol modulation of the thylakoid ATPase. Lack of oxidation of the enzyme in the presence of ΔμH+ in vivo and a possible explanation of the physiological requirement for thiol regulation of the enzyme

Abstract (1) Illumination of Dunaliella induced an increase in the activity of CF1-ATPase, and of fructose-1,6-bisphosphatase (FBPase), that could be assayed under standard conditions in subsequently lysed cells. The light-induced, but not the light-independent, activities could be prevented by inclusion of methyl viologen in the preillumination medium. This effect was concluded to be due to the prevention of reduction of ferredoxin by methyl viologen. (2) Markedly higher concentrations of added methyl viologen were required to prevent induction of ATPase than FBPase. Addition of 1 mM methyl viologen to the preillumination stage prior to illumination totally prevented the appearance of light-induced FBPase, whereas a concentration of 20 mM was required to prevent light-induction of ATPase completely. (3) Although methyl viologen added to intact algae in the light subsequent to achievement of steady-state light activation of ATPase and FBPase led to the inactivation of FBPase, light-induced ATPase was completely uninhibited. This effect indicates that whereas FBPase is subject to reversible modification by thiols in the light, thiol reduction of ATPase in vivo is irreversible in the light, and oxidation only occurs upon dissipation of Δ μ H + produced by darkening. (4) Stabilisation of the thiol-reduced form of CF1 by Δ μ H + (Shahak, Y. (1985) J. Biol. Chem. 260, 1459–1464) accounts for the lower reducing pressure necessary to activate thiol-dependent ATPase, compared to FBPase. Implications for the regulation of CO2 fixation are discussed. (5) Complete activation of CF1-ATPase activity was induced by incubation of intact algae with dithiothreitol in the dark. It is suggested that thiol-regulation of the enzyme is necessary in vivo in order to prevent activation of an ATPase activity which would otherwise inevitably result from autocatalytic generation of Δ μ H + in the dark.

Thiol modulation of chloroplast CF0-CF1 in isolated barley protoplasts and its significance to regulation of carbon dioxide fixation

Abstract (1) The extent of thiol-modulation of chloroplastic CF0-CF1 in isolated barley protoplasts was determined by their capacity to hydrolyse ATP in a subsequently lysed assay system and was measured under conditions that vary the rate of CO2 fixation. CF0-CF1 activity could be separated from other ATPases by using the specific inhibitors triphenyltin and tentoxin. (2) Disruption of dark-adapted protoplasts resulted in little or no ATPase activity associated with CF0-CF1. Preillumination of the protoplasts induced CF0-CF1-dependent ATPase activity which was reversible in the dark. (3) Experiments using methyl viologen established that induction of CF0-CF1-dependent ATPase activity resulted from thiol-modulation of oxidised enzyme complexes by the endogenous thioredoxin system. (4) CF0-CF1 was shown to be rapidly thiol-modulated upon illumination ( t 1 2 ≈ 15 s ) well before steady-state rates of CO2-dependent O2 evolution were achieved. (5) The steady-state extent of light-induced thiol-modulation was shown to be light-saturated at 10 W · m−2, a light intensity far below that required to saturate the rate of CO2-dependent O2 evolution. (6) These results are consistent with the idea that thiol modulation of CF0-CF1 acts primarily as an on-off switch in vivo to prevent ATP hydrolysis in the dark.

Light/dark regulation of photosynthetic enzymes within intact cells of the cyanobacterium Nostoc sp. Mac

Light/dark regulation of several photosynthetic enzymes has been studied under near in vivo conditions using the cyanobacterium Nostoc sp. Mac. Cells were grown photoheterotrophically and rendered osmotically fragile by treatment with lysozyme. Treated cells evolved O2 during photosynthesis at 20–80% of the rate of control cells. Enzyme activities were measured following dilution of preilluminated cells into a hypotonic assay medium. Inhibitor studies using methyl viologen, N,N′-dicyclohexylcarbodiimide and triphenyl tin indicate that fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, ribulose-5-phosphate kinase and NADP-linked glyceraldehyde 3-phosphate dehydrogenase are reversibly light-activated in vivo by the cyanobacterial thioredoxin system. In contrast, the protonmotive ATPase F0-F1-type ATPase appears to be fully active in both light- and dark-adapted cells at physiological temperatures, but is reversibly deactivated by cooling the cells on ice. No activity of NADP-linked malate dehydrogenase or glucose-6-phosphate dehydrogenase could be detected in these cells.

Changes in the apparent affinity of CF0-CF1 for its substrates during photophosphorylation

The apparent Km value for inorganic phosphate (Pi) of the chloroplastic ATPase has been measured under different conditions of light intensity, uncoupler concentration and energy-transfer inhibitor concentration. Systematic changes in the values were observed which were similar to but not identical with reported changes in the Km for ADP under similar conditions (Quick, W.P. and Mills, J.D. (1987) Biochim. Biophys. Acta 893, 197–207). Both sets of experimental data could be simulated using a mathematical model based on: (a) Michaelis-Menten kinetics of the ATPase with respect to ADP and Pi; and (b) delocalised chemiosmotic energy coupling. We conclude that the changes observed in the Km are a result of systematic changes in the protonmotive force during the Km analysis.

Regulation of pigment content and enzyme activity in the cyanobacterium Nostoc sp. Mac grown in continuous light, a light-dark photoperiod, or darkness

Both short-term and long-term adaptations of cyanobacterial metabolism to light and dark were studied in Nostoc sp. Mac. Long-term adaptations were induced by growing cells in the presence of glucose under (a) 30 μE m-2 s-1 continuous white light, (b) under a 14/10 h light/dark cycle, or (c) complete darkness. Short-term regulation of enzyme activities by light was then studied in cells rendered osmotically fragile with lysozyme. Cells were briefly illuminated then enzyme activities were measured following rapid lysis in a hypotonic assay medium. The following results were obtained. (1) Relative to fresh weight, dark-grown cells contained less chlorophyll, much less phycoerythrin, but similar amounts of phycocyanin compared to cells grown under either light regime. Relative to chlorophyll, the higher phycocyanin and much lower phycoerythrin in the dark-grown vs light-grown cells resembles long term changes in pigment content that occur during complementary chromatic adaptation to red vs orange light. (2) Both dark and light/dark grown cells displayed generally lowered photosynthetic activities compared to light-grown cells. The exception to this was the activity of fructose 1,6-bisphosphatase, which was higher in dark-grown cells. However, the photosynthetic induction period was markedly shorter in the light/dark-grown cells indicating an adaptation to changing illumination during growth. (3) Inhibitor studies using methyl viologen show that the fructose 1,6-bisphosphatase is reversibly light-activated in vivo by the cyanobacterial thioredoxin system under all growth conditions. Glucose-6-phosphate dehydrogenase activity was detected in cells grown in all conditions and this activity was reversibly deactivated by light or by dithiothreitol. In contrast, the protonmotive ATPase F0F1-type ATPase was fully active in both light and dark-adapted cells regardless of the light regime used for growth. (4) It is concluded that the Calvin cycle enzymes, their short-term regulatory system, including thioredoxin, glucose-6-phosphate dehydrogenase and an F0F1 ATPase not under thioredoxin control, are expressed in cells grown in complete darkness. Adaptation to heterotrophic growth in this cyanobacterium does not appear to involve synthesis of different enzyme forms lacking thioredoxin control sequences.

Control of CO2 fixation during the induction period. The role of thiol-mediated enzyme activation in the alga, Dunaliella

Abstract (1) Illumination of the unicellular green alga, Dunaliella, produced a 2–3-fold enhancement of ATPase activity in subsequently lysed algae. Using the inhibitor, tentoxin, it was shown that this light-induced activity, but not the light-independent activity, was attributable to the chloroplast coupling factor, CF1. (1) A 4–5-fold increase in fructose-1,6-bisphosphatase activity was measured in Dunaliella lysed subsequent to illumination. (3) Experiments with methyl viologen demonstrated that both light-induced CF1-ATPase and fructose-1,6-bisphosphatase activities were due to thiol-modulation of the enzymes by the algal thioredoxin system. (4) The light-induced increase in fructose-1,6-bisphosphatase activity could be simulated by incubation of intact algae in the dark with dithiothreitol. This thiol-induced increase in enzyme activity was accompanied by a decrease in the induction period of CO2-dependent O2 evolution upon subsequent measurement. (5) The kinetics of induction of both enzyme activities were very similar to the kinetics of induction of CO2-dependent O2 evolution in Dunaliella. As the light intensity was increased to 180 W · m2 the steady-state enzyme activities increased in parallel with the rate of CO2-dependent O2 evolution. (6) The results are consistent with the imposition of a kinetic restraint on CO2 fixation by the extent of enzyme activation under certain conditions in Dunaliella.