Effects of far-red light on fluorescence induction in infiltrated pea leaves under diminished ΔpH and Δφ components of the proton motive force

Chlorophyll fluorescence induction curves induced by an actinic pulse of red light follow different kinetics in dark-adapted plant leaves and leaves preilluminated with far-red light. This influence of far-red light was abolished in leaves infiltrated with valinomycin known to eliminate the electrical (Δφ) component of the proton-motive force and was strongly enhanced in leaves infiltrated with nigericin that abolishes the ΔpH component. The supposed influence of ionophores on different components of the proton motive force was supported by differential effects of these ionophores on the induction curves of the millisecond component of chlorophyll delayed fluorescence. Comparison of fluorescence induction curves with the kinetics of P700 oxidation in the absence and presence of ionophores suggests that valinomycin facilitates a build-up of a rate-limiting step for electron transport at the site of plastoquinone oxidation, whereas nigericin effectively removes limitations at this site. Far-red light was found to be a particularly effective modulator of electron flows in chloroplasts in the absence of ΔpH backpressure on operation of the electron-transport chain.     

Rethinking the existence of a steady-state Δψ component of the proton motive force across plant thylakoid membranes

Light-driven photosynthetic electron transport is coupled to the movement of protons from the chloroplast stroma to the thylakoid lumen. The resulting proton motive force that is generated is used to drive the conformational rotation of the transmembrane thylakoid ATPase enzyme which converts ADP (adenosine diphosphate) and Pi (inorganic phosphate) into ATP (adenosine triphosphate), the energy currency of the plant cell required for carbon fixation and other metabolic processes. According to Mitchell’s chemiosmotic hypothesis, the proton motive force can be parsed into the transmembrane proton gradient (ΔpH) and the electric field gradient (Δψ), which are thermodynamically equivalent. In chloroplasts, the proton motive force has been suggested to be split almost equally between Δψ and ΔpH (Kramer et al., Photosynth Res 60:151–163, 1999). One of the central pieces of evidence for this theory is the existence of a steady-state electrochromic shift (ECS) absorption signal detected ~515 nm in plant leaves during illumination. The interpretation of this signal is complicated, however, by a heavily overlapping absorption change ~535 nm associated with the formation of photoprotective energy dissipation (qE) during illumination. In this study, we present new evidence that dissects the overlapping contributions of the ECS and qE-related absorption changes in wild-type Arabidopsis leaves using specific inhibitors of the ΔpH (nigericin) and Δψ (valinomycin) and separately using leaves of the Arabidopsis lut2npq1 mutant that lacks qE. In both cases, our data show that no steady-state ECS signal persists in the light longer than ~60 s. The consequences of our observations for the suggesting parsing of steady-state thylakoid proton motive force between (ΔpH) and the electric field gradient (Δψ) are discussed.     

Mechanisms of generation of local ΔpH in mitochondria and bacteria

The concepts of global and local coupling between proton generators, the enzymes of the respiratory chain, and the consumer, the ATP synthase, coexist in the theory of oxidative phosphorylation. Global coupling is trivial proton transport via the aqueous medium, whereas local coupling implies that the protons pumped are consumed before they escape to the bulk phase. In this work, the conditions for the occurrence of local coupling are explored. It is supposed that the membrane retains protons near its surface and that the proton current generated by the proton pumps rapidly decreases with increasing proton motive force (pmf). It is shown that the competition between the processes of proton translocation across the membrane and their dissipation from the surface to the bulk can result in transient generation of a local ΔpH in reply to a sharp change in pmf; the appearance of local ΔpH, in turn, leads to rapid recovery of the pmf, and hence, it provides for stabilization of the potential at the membrane. Two mechanisms of such kind are discussed: 1) pH changes in the surface area due to proton pumping develop faster than those due to proton escape to the bulk; 2) the former does not take place, but the protons leaving the surface do not equilibrate with the bulk immediately; rather, they give rise to a non-equilibrium concentration near the surface and, as a result, to a back proton flow to the surface. The first mechanism is more efficient, but it does not occur in mitochondria and neutrophilic bacteria, whereas the second can produce ΔpH on the order of unity. In the absence of proton retardation at the surface, local ΔpH does not arise, whereas the formation of global ΔpH is possible only at buffer concentration of less than 10 mM. The role of the mechanisms proposed in transitions between States 3 and 4 of the respiratory chain is discussed. The main conclusion is that surface protons, under conditions where they play a role, support stabilization of the membrane pmf and rapid communication between proton generators and consumers, while their contribution to the energetics is not significant.     

The rate of ATP synthesis as a function of ΔpH in normal and dithiothreitol-modified chloroplasts

The rate of ATP synthesis catalyzed by normal and by dithiothreitol-modified ATPases is investigated as a function of ΔpH in spinach chloroplasts at constant pH_out. The transmembrane ΔpH was generated by an acid-base transition and the reaction time was limited to 150 ms by using a rapidly mixing quenched-flow apparatus. The result was that the functional dependence of the rate on ΔpH is shifted to lower ΔpH values and that the shape of this curve is altered after dithiothreitol modification. The maximal rate (400 ATP / CF₁ per s) is the same under both conditions.     

Δψ and ΔpH are equivalent driving forces for proton transport through isolated F0 complexes of ATP synthases

The membrane-embedded F₀ part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F₀ complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F₀ part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either synthesis or hydrolysis direction with Δψ or ΔpH as driving forces. The system displayed a high signal-to-noise ratio and can be accurately quantified. In contrast to ATP synthesis in the Escherichia coli F₁F₀ holoenzyme, no significant difference was observed in the efficiency of ΔpH or Δψ as driving forces for H⁺-transport through F₀. Transport rates showed linear dependency on the driving force. Proton transport in hydrolysis direction was about 2400 H⁺/(s × F₀) at Δψ of 120 mV, which is approximately twice as fast as in synthesis direction. The chloroplast enzyme was faster and catalyzed H⁺-transport at initial rates of 6300 H⁺/(s × F₀) under similar conditions. The new method is an ideal tool for detailed kinetic investigations of the ion transport mechanism of ATP synthases from various organisms.     

ATP synthesis from 2,3-diphosphoglycerate by cell-free extract of Methanobacterium thermoautotrophicum (strain ΔH)

Cell-free extracts of Methanobacterium thermoautotrophicum were found to catalyze ATP synthesis from an endogeneous substrate. Synthesis was stimulated under hydrogen atmosphere and inhibited by KCL (K _i =150 mM). Comparison of the properties of a number of cell constituents showed the endogeneous substrate to be 2,3-diphosphoglycerate. The compound is converted into 3-phosphoglycerate, and via 2-phosphoglycerate and phosphoenolpyruvate into pyruvate, at which the latter reaction is linked with ATP synthesis.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme m, 2-(methylthio)ethanesulfonate - HS-HTP 7-mercaptoheptanoyl-l-threonine phosphate - CoM-SS-HTP the heterodisulfide of HS-CoM and HS-HTP - BCFE bolled cell-free extract - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - PEP phosphoenolpyruvate - 2,3-DPG 2,3-diphosphoglycerate - cDPG cyclic 2,3-diphosphoglycerate - 3-PG 3-phosphoglycerate - 2-PG 2-phosphoglycerate     

Do modulators of the mitochondrial K(ATP) channel change the function of mitochondria in situ?

Pharmacological opening of mitochondrial cardiac ATP-sensitive potassium (K(ATP)) channels has the chance to be a promising but still controversial cardioprotective mechanism. Physiological roles of mitochondrial K(ATP) channels in the myocardium remain unclear. We studied the effects of diazoxide, a specific opener of these channels, on the function of rat mitochondria in situ in saponin-permeabilized fibers using an ionic medium that mimics the cytosol. In the presence of NADH-producing substrates (malate + glutamate), neither 100 microm diazoxide nor 100 microm glibenclamide (a K(ATP) channel blocker) changed the mitochondrial respiration in the absence or presence of ADP. Because the K(ATP) channel function could be modified by changes in adenine nucleotide concentrations near the mitochondria, we studied the effects of diazoxide and glibenclamide on the functional activity of mitochondrial kinases. Both diazoxide and glibenclamide did not change the in situ ADP sensitivity in the presence or absence of creatine (apparent K(m) values for ADP were, respectively, 59 +/- 9 and 379 +/- 45 microm). Similarly, stimulation of the mitochondrial respiration with AMP in the presence of ATP due to adenylate kinase activity was not affected by the modulators of K(ATP) channels. However, when succinate was used as substrate, diazoxide significantly inhibited basal respiration by 22% and maximal respiration by 24%. Thus, at a cardioprotective dose, the main functional effect of diazoxide depends on respiratory substrates and seems not to be related to K(ATP) channel activity.     

High concentrations of PEG as a possible uncoupler of the proton motive force: α-Amylase production with Bacillus amyloliquefaciens in aqueous two-phase systems and PEG solutions

Summary -Amylase production with Bacillus amyloliquefaciens was investigated in two different aqueous two-phase systems and in polyethylene glycol (PEG) 600 solutions of different concentrations. The cells did not partition totally to the bottom phases of the aqueous two-phase systems, and the enzyme production was repressed in both systems as well as in PEG 600 solutions. Concomitantly, the cultivation time was prolonged, indicating an increased maintenance metabolism. The surface properties of cells grown in 200 g/kg PEG 600 were investigated by phase partitioning and compared to the surface properties of Bacillus subtilis, which under these conditions showed increased -amylase production. The cells of B. amyloliquefaciens partitioned to the top phase in a PEG-dextran system, whereas the cells of B. subtilis partitioned to the bottom phase. The results are discussed in relation to water activity, oxygen transfer rate and PEG-induced changes of the surface properties of the cells. The possible role of PEG as an uncoupler of the proton motive force at high concentrations is also discussed.     

Storage of light-driven transthylakoid proton motive force as an electric field (Δψ) under steady-state conditions in intact cells of <Emphasis Type="Italic">Chlamydomonasreinhardtii</Emphasis>

Proton motive force (pmf) is physiologically stored as either a ΔpH or a membrane potential (Δψ) across bacterial and mitochondrial energetic membranes. In the case of chloroplasts, previous work (Cruz et al. 2001, Biochemistry 40: 1226–1237) indicates that Δψ is a significant fraction of pmf, in vivo, and in vitro as long as the activities of counterions are relatively low. Kinetic analysis of light-induced changes in the electrochromic shift (ECS) in intact leaves was consistent with these observations. In this work, we took advantage of the spectroscopic properties of the green alga, Chlamydomonas reinhardtii, to demonstrate that light-driven Δψ was stored in vivo over the hours time scale. Analysis of the light-induced ECS kinetics suggested that the steady-state Δψ in 400 μmol photons m⁻² s⁻¹ red light was between 20 and 90 mV and that this represented about 60% of the light-induced increase in pmf. By extrapolation, it was surmised that about half of total (basal and light-induced) pmf is held as Δψ. It is hypothesized that Δψ is stabilized either by maintaining low chloroplast ionic strength or by active membrane ion transporters. In addition to the strong implications for regulation of photosynthesis by the xanthophyll cycle, these results imply that pmf partitioning is important across a wide range of species.     

A conformational change in the isolated NADP(H)-binding component (dIII) of transhydrogenase induced by low pH: a reflection of events during proton translocation by the complete enzyme?

Transhydrogenase couples the reduction of NADP(+) by NADH to inward proton translocation across the bacterial (or mitochondrial) membrane. Conformational changes in the NADP(H)-binding component of the enzyme (dIII) are central to the coupling mechanism. In the "open" state, NADP(H) bound to dIII can readily exchange with nucleotides in the solvent but hydride transfer [to/from NAD(H) bound to dI] is prevented. In the "occluded" state, bound NADP(H) cannot exchange with solvent nucleotides but the hydride transfer reaction is permitted. It was previously found that the conformational state of isolated, recombinant dIII is pH dependent. At neutral pH, the protein adopts a conformation resembling the occluded state, and at low pH, it adopts a conformation resembling the open state. The crystal structure of dIII indicates that the loop E "lid" might be largely responsible for the very high affinity of the protein for NADP(H). In this paper we show, using fluorescence resonance energy transfer, that the distance between the apex of loop E of isolated dIII, and the core of the protein, increases when the solution pH is lowered. This is consistent with the view that the lid is retracted to permit NADPH release during turnover of the complete enzyme.     

Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae

The filamentous fungus Aspergillus oryzae is used as one of the most favored hosts for heterologous protein production due to its ability to secrete large amounts of proteins into the culture medium. We previously generated a hyper-producing mutant strain of A. oryzae, AUT1, which produced 3.2- and 2.6-fold higher levels of bovine chymosin (CHY) and human lysozyme (HLY), respectively, compared with the wild-type strain. However, further enhancement of heterologous protein production by multiple gene disruption is difficult because of the low gene-targeting efficiency in strain AUT1. Here, we disrupted the ligD gene, which is involved in nonhomologous recombination, and the pyrG gene to create uridine/uracil auxotrophy in strain AUT1, to generate a hyper-producing mutant applicable to pyrG marker recycling with highly efficient gene targeting. We generated single and double disruptants of the tripeptidyl peptidase gene AosedD and vacuolar sorting receptor gene Aovps10 in the hyper-producing mutant background, and found that all disruptants showed significant increases in heterologous protein production. Particularly, double disruption of the Aovps10 and AosedD genes increased the production levels of CHY and HLY by 1.6- and 2.1-fold, respectively, compared with the parental strain. Thus, we successfully generated a fungal host for further enhancing the heterologous protein production ability by combining mutational and molecular breeding techniques.     

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)

PutA (proline utilization A) is a large bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains that catalyze the oxidation of l-proline to l-glutamate in two successive reactions. In the PRODH active site, proline undergoes a two-electron oxidation to Δ¹-pyrroline-5-carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, l-glutamate-γ-semialdehyde (the hydrolyzed form of Δ¹-pyrroline-5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred to NAD⁺-producing NADH and glutamate. Here we report the first kinetic model for the overall PRODH-P5CDH reaction of a PutA enzyme. Global analysis of steady-state and transient kinetic data for the PRODH, P5CDH, and coupled PRODH-P5CDH reactions was used to test various models describing the conversion of proline to glutamate by Escherichia coli PutA. The coupled PRODH-P5CDH activity of PutA is best described by a mechanism in which the intermediate is not released into the bulk medium, i.e., substrate channeling. Unexpectedly, single-turnover kinetic experiments of the coupled PRODH-P5CDH reaction revealed that the rate of NADH formation is 20-fold slower than the steady-state turnover number for the overall reaction, implying that catalytic cycling speeds up throughput. We show that the limiting rate constant observed for NADH formation in the first turnover increases by almost 40-fold after multiple turnovers, achieving half of the steady-state value after 15 turnovers. These results suggest that EcPutA achieves an activated channeling state during the approach to steady state and is thus a new example of a hysteretic enzyme. Potential underlying causes of activation of channeling are discussed.     

Analysis of an electrostatic mechanism for ΔpH dependent gating of the voltage-gated proton channel,HV1, supports a contribution of protons to gating charge

Voltage-gated proton channels (H_V1) resemble the voltage-sensing domain of other voltage-gated ion channels, but differ in containing the conduction pathway. Essential to the functions of H_V1 channels in many cells and species is a unique feature called ΔpH dependent gating. The pH on both sides of the membrane strictly regulates the voltage range of channel opening, generally resulting in exclusively outward proton current. Two types of mechanisms could produce ΔpH dependent gating. The “countercharge” mechanism proposes that protons destabilize salt bridges between amino acids in the protein that stabilize specific gating configurations (closed or open). An “electrostatic” mechanism proposes that protons bound to the channel alter the electrical field sensed by the protein. Obligatory proton binding within the membrane electrical field would contribute to measured gating charge. Estimations on the basis of the electrostatic model explain ΔpH dependent gating, but quantitative modeling requires calculations of the electric field inside the protein which, in turn, requires knowledge of its structure. We conclude that both mechanisms operate and contribute to ΔpH dependent gating of H_V1.     

Evidence for two compartments of exchangeable calcium in isolated rat liver mitochondria obtained using a45Ca exchange technique in the presence of magnesium,phosphate, and ATP at 37°C

Summary The distribution of calcium between isolated rat liver mitochondria and the extramitochondrial medium at 37°C and in the presence of 2mm inorganic phosphate, 3mm ATP, 0.05 or 1.1mm free magnesium and a calcium buffer, nitrilotriacetic acid, was investigated using a⁴⁵Ca exchange technique. The amounts of⁴⁰Ca in the mitochondria and medium were allowed to reach equilibrium before initiation of the measurement of⁴⁵Ca exchange. At 0.05mm free magnesium and initial extramitochondrial free calcium concentrations of between 0.15 and 0.5 m, the mitochondria accumulated calcium until the extramitochondrial free calcium concentration was reduced to 0.15 m. Control experiments showed that the mitochondria were stable under the incubation conditions employed. The⁴⁵Ca exchange data were found to be consistent with a system in which two compartments of exchangeable calcium are associated with the mitochondria. Changes in the concentration of inorganic phosphate did not significantly affect the⁴⁵Ca exchange curves, whereas an increase in the concentration of free magnesium inhibited exchange. The maximum rate of calcium outflow from the mitochondria was estimated to be 1.7 nmol/min per mg of protein, and the value ofK _0.5 for intramitochondrial exchangeable calcium to be about 1.6 nmol per mg of protein. Ruthenium Red decreased the fractional transfer rate for calcium inflow to the mitochondria while nupercaine affected principally the fractional transfer rates for the transfer of calcium between the two mitochondrial compartments. The use of the incubation conditions and⁴⁵Ca exchange technique described in this report for studies of the effects of agents which may alter mitochondrial calcium uptake or release (e.g., the pre-treatment of cells with hormones) is briefly discussed.     

The subcellular localization of glutamate dehydrogenase (GDH): is GDH a marker for mitochondria in brain?

Glutamate dehydrogenase (GDH, EC 1.4.1.2) has long been used as a marker for mitochondria in brain and other tissues, despite reports indicating that GDH is also present in nuclei of liver and dorsal root ganglia. To examine whether GDH can be used as a marker to differentiate between mitochondria and nuclei in the brain, we have measured GDH by enzymatic activity and on immunoblots in rat brain mitochondria and nuclei which were highly enriched by density-gradient centrifugation methods. The activity of GDH was enriched in the nuclear fraction as well as in the mitochondrial faction, while the activities of other mitochondrial enzymes (fumarase, NAD-isocitrate dehydrogenase and pyruvate dehydrogenase complex) were enriched only in the mitochondrial fraction. Immunoblots using polyclonal antibodies against bovine liver GDH confirmed the presence of GDH in the rat brain nuclear and mitochondrial fractions. The GDH in these two subcellular fractions had a very similar molecular weight of 56,000 daltons. The mitochondrial and nuclear GDH differed, however, in their susceptibility to solubilization by detergents and salts. The mitochondrial GDH could be solubilized by extraction with low concentrations of detergents (0.1% Triton X-100 and 0.1% Lubrol PX), while the nuclear GDH could be solubizeded only by elevated concentrations of detergents (0.3% each) plus KCl (>150mM). Our results indicate that GDH is present in both nuclei and mitochondria in rat brain. The notion that GDH may serve as a marker for mitochondria needs to be re-evaluated.     

Partial characterization of placental 3ß-hydroxysteroid dehydrogenase (EC 1.1.1.145), Δ4–5isomerase (EC 5.3.3.1) in human term placental mitochondria

A partial characterization of human term placental 3ß-HSDH in mitochondria is reported. Apparent K_M of pregnenolone: 70 nM. A dose-dependent stimulation of 3ß-HSDH by NAD⁺ or NADP⁺ was observed in the range from 10⁻⁶ to 10⁻³ M (K_M value of NAD⁺: 20 μM). At equimolar concentrations NAD⁺ is more than 10-fold as effective a cofactor of the 3ß-HSDH than NADP⁺. pH optimum: 9.5 (glycine-NaOH buffer). Temperature optimum 40–45°C. A rapid loss of 3ß-HSDH activity was found after preincubation of the enzyme at 37°C after 30 min: less than 50% of initial enzyme activity is present. No inhibition was obtained by Mg²⁺, Ca²⁺ Sr²⁺ and Ba²⁺ (1–100 mM). A strong inhibition was achieved with 1 mM Zn²⁺, Cd²⁺, Cu²⁺ and 10 mM and 100 mM Fe²⁺, Mn²⁺, Co²⁺ and Ni²⁺.     

Evidence for involvement of protein kinase C in regulation of intracellular pH by Cl−/HCO 3 − antiport

Summary The activity of the main base-extruding mechanism in Vero cells, the Na⁺-independent Cl^–/HCO ₃ ^– antiport, increases 5- to 10-fold when the cytosolic pH (pH _i ) is increased over a narrow range close to neutrality. We have studied the effect on this regulation of stimulation and inhibition of protein kinase C by short-term and long-term treatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). After short-term treatment with TPA to stimulate the kinase, the threshold value for activation of the antiport is shifted to a more acidic pH. After prolonged treatment with TPA to downregulate protein kinase C the sensitivity of the antiport to variation in proton concentration was lowered, possibly by reducing the number of essential protonbinding sites. Concomitantly, the steady state pH _i of the cells was increased. The data indicate that protein kinase C is involved in the regulation of the Na⁺-independent Cl^–/HCO ₃ ^– antiport.