Movement of the Rieske iron-sulfur protein in the p-side bulk aqueous phase: effect of lumenal viscosity on redox reactions of the cytochrome b6f complex

Based on the atomic structures of the mitochondrial cytochrome bc(1) complex, it has been proposed that the soluble domain of the [2Fe-2S] Rieske iron-sulfur protein (ISP) must rotate by ca. 60 degrees and translate through an appreciable distance between two binding sites, proximal to cytochrome c(1) and to the lumen-side quinol binding site. Such motional freedom implies that the electron-transfer rate should be affected by the lumenal viscosity. The flash-induced oxidation of cytochrome f, the chloroplast analogue of cytochrome c(1), was found to be inhibited reversibly by increased lumenal viscosity, as was the subsequent reduction of both cytochrome b(6) and cytochrome f. The rates of these three redox reactions correlated inversely with lumenal viscosity over a viscosity range of 1-10 cP. Reduction of cytochrome b(6) and cytochrome f was not concerted. The rate of cytochrome f reduction was observed to be approximately half that of cytochrome b(6) regardless of the actual viscosity, implying that the path length traversed by the ISP in reduction of cytochrome f is twice that of cytochrome b(6). This suggests that upon initiation of electron transfer by a light flash, cytochrome b(6) reduction requires movement of reduced ISP from an initial position predominantly proximal to cytochrome f, apparently favored by the reduced ISP, to the quinol binding site at which the oxidant-induced reduction of cytochrome b(6) is initiated. Subsequent reduction of cytochrome f requires the additional movement of the ISP back to a site proximal to cytochromef. There is no discernible viscosity dependence for cytochrome b(6) reduction under oxidizing conditions, presumably because the oxidized ISP preferentially binds proximal to the quinone binding niche. The dependence of the cytochrome redox reaction on ambient viscosity implies that the tethered diffusional motion of the ISP is part of the rate limitation for charge transfer through the b(6)f complex.     

Thermal dissipation of light energy is regulated differently and by different mechanisms in lichens and higher plants

Modulated chlorophyll fluorescence was used to compare dissipation of light energy as heat in photosystem II of homoiohydric and poikilohydric photosynthetic organisms which were either hydrated or dehydrated. In hydrated chlorolichens with an alga as the photobiont, fluorescence quenching revealed a dominant mechanism of energy dissipation which was based on a protonation reaction when zeaxanthin was present. CO2 was effective as a weak protonating agent and actinic light was not necessary. In a hydrated cyanobacterial lichen, protonation by CO2 was ineffective to initiate energy dissipation. This was also true for leaves of higher plants. Thus, regulation of zeaxanthin-dependent energy dissipation by protonation was different in leaves and in chlorolichens. A mechanism of energy dissipation different from that based on zeaxanthin became apparent on dehydration of both lichens and leaves. Quenching of maximum or Fm fluorescence increased strongly during dehydration. In lichens, this was also true for so-called basal or Fo fluorescence. In contrast to zeaxanthin-dependent quenching, dehydration-induced quenching could not be inhibited by dithiothreitol. Both zeaxanthin-dependent and dehydration-induced quenching cooperated in chlorolichens to increase thermal dissipation of light energy if desiccation occurred in the light. In cyanolichens, which do not possess a zeaxanthin cycle, only desiccation-induced thermal energy dissipation was active in the dry state. Fluorescence emission spectra of chlorolichens revealed stronger desiccation-induced suppression of 685-nm fluorescence than of 720-nm fluorescence. In agreement with earlier reports of , fluorescence excitation data showed that desiccation reduced flow of excitation energy from chlorophyll b of the light harvesting complex II to emitting centres more than flow from chlorophyll a of core pigments. The data are discussed in relation to regulation and localization of thermal energy dissipation mechanisms. It is concluded that desiccation-induced fluorescence quenching of lichens results from the reversible conversion of energy-conserving to energy-dissipating photosystem II core complexes.     

The pgr1 mutation in the Rieske subunit of the cytochrome b6f complex does not affect PGR5-dependent cyclic electron transport around photosystem I

Although photosystem I (PSI) cyclic electron transport is essential for plants, our knowledge of the route taken by electrons is very limited. To assess whether ferredoxin (Fd) donates electrons directly to plastoquinone (PQ) or via a Q-cycle in the cytochrome (cyt) b(6)f complex in PSI cyclic electron transport, we characterized the activity of PSI cyclic electron transport in an Arabidopsis mutant, pgr1 (proton gradient regulation). In pgr1, Q-cycle activity was hypersensitive to acidification of the thylakoid lumen because of an amino acid alteration in the Rieske subunit of the cyt b(6)f complex, resulting in a conditional defect in Q-cycle activity. In vitro assays using ruptured chloroplasts did not show any difference in the activity of PGR5-dependent PQ reduction by Fd, which functions in PSI cyclic electron transport in vivo. In contrast to the pgr5 defect, the pgr1 defect did not show any synergistic effect on the quantum yield of photosystem II in crr2-2, a mutant in which NDH (NAD(P)H dehydrogenase) activity was impaired. Furthermore, the simultaneous determination of the quantum yields of both photosystems indicated that the ratio of linear and PSI cyclic electron transport was not significantly affected in pgr1. All the results indicated that the pgr1 mutation did not affect PGR5-dependent PQ reduction by Fd. The phenotypic differences between pgr1 and pgr5 indicate that maintenance of the proper balance of linear and PSI cyclic electron transport is essential for preventing over-reduction of the stroma.     

Structural and functional analysis of the antiparallel strands in the lumenal loop of the major light-harvesting chlorophyll a/b complex of photosystem II (LHCIIb) by site-directed mutagenesis

The light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCIIb) fulfills multiple functions, such as light harvesting and energy dissipation under different illuminations. The crystal structure of LHCIIb at the near atomic resolution reveals an antiparallel strands structure in the lumenal loop between the transmembrane helices B/C. To study the structural and functional significances of this structure, three amino acids (Val-119, His-120, and Ser-123) in this region have been exchanged to Phe, Leu, and Gly, respectively, and the influence of the mutagenesis on the structure and function of LHCIIb has been investigated. The results are as follows. 1) Circular dichroism spectra of the mutations reveals that the antiparallel strands in the lumenal region are very important for adjusting pigment conformation in the neoxanthin domain of LHCIIb. Although the mutagenesis causes only a slight loss of the Neo binding in the complexes (V119F, 0.09; S123G, 0.19; and H120L, 0.27), it imparts remarkable changes to the pigment conformation. 2) Substituting Ser-123 with Gly results in a higher susceptibility to photodamage, an increased tendency to aggregate, and enhanced fluorescence quenching induced by the medium acidification. These results demonstrate that this antiparallel strands domain plays an important role in regulating the pigment conformation and in adjusting the aggregation and the fluorescence yield of LHCIIb.     

A few molecules of zeaxanthin per reaction centre of photosystem II permit effective thermal dissipation of light energy in photosystem II of a poikilohydric moss

The relationship between thermal dissipation of light energy (as indicated by the quenching of chlorophyll fluorescence), zeaxanthin availability and protonation reactions was investigated in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst. In the absence of zeaxanthin and actinic illumination, acidification by 20% CO₂ in air was incapable of quenching basal, so-called F ₀ fluorescence either in the moss or in spinach (Spinacia oleracea L.) leaves. However, 1-s light pulses given either every 40, 60 or 200 s increased thermal dissipation as indicated by F ₀ and F _m quenching in the presence of 20% CO₂ in air in the moss, but not in spinach while reaction centres of photosystem II (PSII) were photochemically open. In the moss, a few short light pulses, which were separated by prolonged dark times, were sufficient to raise zeaxanthin levels in the presence of 20% CO₂ in air. Simultaneously, quantum efficiency of charge separation in PSII was decreased. Increasing the CO₂ concentration beyond 20% further decreased quantum efficiency even in the absence of short light pulses. Under conditions optimal for fluorescence quenching, one molecule of zeaxanthin per reaction centre of PSII was sufficient to decrease quantum efficiency of charge separation in PSII by 50%. Thus, in combination with a protonation reaction, one molecule of zeaxanthin was as efficient at capturing excitation energy as a photochemically open reaction centre. The data are discussed in relation to the interaction between zeaxanthin and thylakoid protonation, which enables effective thermal dissipation of light energy in the antennae of PSII in the moss but not in higher plants when actinic illumination is absent. Received: 8 April 2000 / Accepted: 31 August 2000     

Activation of mechanisms of photoprotection by desiccation and by light: poikilohydric photoautotrophs

Mechanisms of protection against photo-oxidation in selected desiccation-tolerant lichens and mosses have been investigated by measuring loss of light absorption during desiccation and chlorophyll fluorescence as indicators of photoprotection. Apparent absorption (1-T) spectra measured in the reflectance mode revealed stronger absorption of photosynthetic pigments in hydrated than in desiccated organisms, but differences were pronounced only in a cyanolichen, less so in some chlorolichens, and even less in mosses. Since the amplitude of chlorophyll fluorescence is a product of (1-T) light absorption by chlorophyll and quantum yield of fluorescence, and since fluorescence is inversely related to thermal energy dissipation, when chemical fluorescence quenching is negligible, fluorescence measurements were used to measure changes in energy dissipation. Preincubation of the hydrated organisms and desiccation in darkness excluded the contribution of mechanisms of energy dissipation to photoprotection which are dependent on the presence of zeaxanthin or on the light-dependent formation of a quencher of fluorescence within the reaction centre of photosystem II. Fast drying in darkness or in very low light was less effective in decreasing chlorophyll fluorescence than slow drying. Heating the desiccated organisms increased fluorescence by inactivating the mechanism responsible for fluorescence quenching. Glutaraldehyde inhibited fluorescence quenching during desiccation. Prolonged exposure of a desiccated moss or a desiccated lichen to very strong light caused more photo-induced damage after fast drying than after slow drying. The photo-oxidative nature of damage was emphasized by the observation that irreversible loss of fluorescence was larger in air than in a nitrogen atmosphere. It is concluded from these observations that desiccation-induced conformational changes of a chlorophyll protein complex result in the fast radiationless dissipation of absorbed light energy. This mechanism of photoprotection is more effective in preventing photo-oxidative damage than other mechanisms of energy dissipation which require light for activation such as zeaxanthin-dependent energy dissipation or quencher formation within the reaction centre of photosystem II.     

Structural changes are induced in human neutrophil cytochrome b by NADPH oxidase activators,LDS, SDS,and arachidonate: intermolecular resonance energy transfer between trisulfopyrenyl-wheat germ agglutinin and cytochrome b(558)

Anionic amphiphiles such as sodium- and lithium dodecyl sulfate (SDS, LDS), or arachidonate (AA) initiate NADPH oxidase and proton channel activation in cell-free systems and intact neutrophils. To investigate whether these amphiphiles exert allosteric effects on cytochrome b, trisulfopyrenyl-labeled wheat germ agglutinin (Cascade Blue-wheat germ agglutinin, CCB-WGA) was used as an extrinsic fluorescence donor for resonance energy transfer (RET) to the intrinsic heme acceptors of detergent-solubilized cytochrome b. In solution, cytochrome b complexed with the CCB-WGA causing a rapid, saturable, carbohydrate-dependent quenching of up to approximately 55% of the steady-state fluorescence. Subsequent additions of SDS, LDS, or AA to typical cell-free oxidase assay concentrations completely relaxed the fluorescence quenching. The relaxation effects were specific, and not caused by dissociation of the CCB-WGA-cytochrome b complex or alterations in the spectral properties of the chromophores. In contrast, addition of the oxidase antagonist, arachidonate methyl ester, caused an opposite effect and was able to partially reverse the activator-induced relaxation. We conclude that the activators induce a cytochrome b conformation wherein the proximity or orientation between the hemes and the extrinsic CCB fluorescence donors has undergone a significant change. These events may be linked to NADPH oxidase assembly and activation or proton channel induction.     

A cytochrome c encoded by the nar operon is required for the synthesis of active respiratory nitrate reductase in Thermus thermophilus

The pgr1 mutant of Arabidopsis thaliana carries a single point mutation (P194L) in the Rieske subunit of the cytochrome b6/f (cyt b6/f) complex and is characterised by a reduced electron transport activity at saturating light intensities in vivo. We have investigated the electron transport in this mutant under in vitro conditions. Measurements of P700 reduction kinetics and of photosynthetic electron transport rates indicated that electron transfer from cyt b6/f to photosystem I is not generally reduced in the mutant, but that the pH dependence of this reaction is altered. The data imply that the pH-dependent inactivation of electron transport through cyt b6/f is shifted by about 1 pH unit to more alkaline pH values in pgr1 thylakoids in comparison with wild-type thylakoids. This interpretation was confirmed by determination of the transmembrane deltapH at different stromal pH values showing that the lumen pH in pgr1 mutant plants cannot drop below pH 6 reflecting most likely a shift of the pK and/or the redox potential of the oxidised Rieske protein.     

HCF164 Encodes a Thioredoxin-Like Protein Involved in the Biogenesis of the Cytochrome b6f Complex in Arabidopsis

To understand the biogenesis of the plastid cytochrome b(6)f complex and to identify the underlying auxiliary factors, we have characterized the nuclear mutant hcf164 of Arabidopsis and isolated the affected gene. The mutant shows a high chlorophyll fluorescence phenotype and is severely deficient in the accumulation of the cytochrome b(6)f complex subunits. In vivo protein labeling experiments indicated that the mutation acts post-translationally by interfering with the assembly of the complex. Because of its T-DNA tag, the corresponding gene was cloned and its identity confirmed by complementation of homozygous mutant plants. HCF164 encodes a thioredoxin-like protein that possesses disulfide reductase activity. The protein was found in the chloroplast, where it is anchored to the thylakoid membrane at its lumenal side. HCF164 is closely related to the thioredoxin-like protein TxlA of Synechocystis sp PCC6803, most probably reflecting its evolutionary origin. The protein also shows a limited similarity to the eubacterial CcsX and CcmG proteins, which are required for the maturation of periplasmic c-type cytochromes. The putative roles of HCF164 for the assembly of the cytochrome b(6)f complex are discussed.     

Characterization of a nonphotochemical quenching-deficient Arabidopsis mutant possessing an intact PsbS protein, xanthophyll cycle and lumen acidification

Arabidopsis thaliana plants grown from ethyl methane sulfonate-treated seeds were screened for so-called que mutants, which are affected in non-photochemical energy quenching. Based on video imaging of chlorophyll fluorescence an energy dissipation mutant, que1, was identified, isolated and characterized. Similar to the npq mutants, the que1 mutant showed a drastically reduced capacity for pH-dependent energy dissipation, qE, but without affecting the Δ pH-dependent conformational changes at 535 nm (ΔA ₅₃₅), which have been supposed to be obligatorily correlated with qE and to reflect pH-regulated binding of zeaxanthin to the PsbS protein. Western blot and DNA sequence analysis revealed that neither a reduced expression of the PsbS protein nor a mutation in the PsbS gene was responsible for the missing qE in que1. Measurements of 9-aminoacridine fluorescence quenching showed that the acidification of the thylakoid lumen was also not affected in the mutant. Furthermore, que1 was able to convert violaxanthin to zeaxanthin. However, unusual characteristics of zeaxanthin formation in the mutant pointed at an altered availability of violaxanthin for de-epoxidation. This was further accompanied by a decrease of the photochemical quenching of chlorophyll fluorescence (qP), an increase of the portion of oxidized P700 and a reduction of the electron transport rate. These characteristics indicate changes in the organization of the thylakoid membrane that affect linear electron transport (but not lumen acidification) and the formation of energy dissipation in photosystem II. Preliminary genetic analysis revealed that the phenotype of que1 is related to two different mutations, mapped to the lower arms of chromosomes 1 and 4.     

细胞色素C和含心磷脂的人工脂膜相互作用

 本文报告以芘为荧光探剂,研究细胞色素C和含心磷脂的人工脂膜的相互作用。1.由于芘和细胞色素C的血红素团之间的能量转移,细胞色素C与心磷脂结合引起芘的单体荧光发射峰(395nm)强度下降。这种淬灭效应受脂膜的相行为影响,在液晶相时淬灭效应小于凝胶相;2.氧化态细胞色素C与还原态相比,对心磷脂结合的视和度稍高;3.在以芘的激发二聚体荧光峰(475nm)强度与单体荧光峰强度之比做为脂膜流动性的指标,发现还原态细胞色素C与含心磷脂脂膜结合后引起流动性增加的效应高于氧化态的结合。     

Thermostability and photostability of photosystem II of the resurrection plant Haberlea rhodopensis studied by chlorophyll fluorescence

The stability of PSII in leaves of the resurrection plant Haberlea rhodopensis to high temperature and high light intensities was studied by means of chlorophyll fluorescence measurements. The photochemical efficiency of PSII in well-hydrated Haberlea leaves was not significantly influenced by temperatures up to 40 degrees C. Fo reached a maximum at 50 degrees C, which is connected with blocking of electron transport in reaction center II. The intrinsic efficiency of PSII photochemistry, monitored as Fv/Fm was less vulnerable to heat stress than the quantum yield of PSII electron transport under illumination (phiPSII). The reduction of phiPSII values was mainly due to a decrease in the proportion of open PSII centers (qP). Haberlea rhodopensis was very sensitive to photoinhibition. The light intensity of 120 micromol m(-2) s(-1) sharply decreased the quantum yield of PSII photochemistry and it was almost fully inhibited at 350 micromol m(-2) s(-1). As could be expected decreased photochemical efficiency of PSII was accompanied by increased proportion of thermal energy dissipation, which is considered as a protective effect regulating the light energy distribution in PSII. When differentiating between the three components of qN it was evident that the energy-dependent quenching, qE, was prevailing over photoinhibitory quenching, qI, and the quenching related to state 1-state 2 transitions, qT, at all light intensities at 25 degrees C. However, the qE values declined with increasing temperature and light intensities. The qI was higher than qE at 40 degrees C and it was the major part of qN at 45 degrees C, indicating a progressing photoinhibition of the photosynthetic apparatus.     

Effects of light intensity on cyclic electron flow around PSI and its relationship to non-photochemical quenching of Chl fluorescence in tobacco leaves

We tested the hypothesis that plants grown under high light intensity (HL-plants) had a large activity of cyclic electron flow around PSI (CEF-PSI) compared with plants grown under low light (LL-plants). To evaluate the activity of CEF-PSI, the relationships between photosynthesis rate, quantum yields of both PSII and PSI, and Chl fluorescence parameters were analyzed simultaneously in intact leaves of tobacco plants which had been grown under different light intensities (150 and 1,100 micromol photons m(-2) s(-1), respectively) and with different amounts of nutrients supplied. HL-plants showed a larger value of non-photochemical quenching (NPQ) of Chl fluorescence at the limited activity of photosynthetic linear electron flow. Furthermore, HL-plants had a larger activity of CEF-PSI than LL-plants. These results suggested that HL-plants dissipated the excess photon energy through NPQ by enhancing the ability of CEF-PSI to induce acidification of the thylakoid lumen.     

The xanthophyll cycle of Mantoniella squamata converts violaxanthin into antheraxanthin but not to zeaxanthin: consequences for the mechanism of enhanced non-photochemical energy dissipation

The prasinophycean alga Mantoniella squamata uses in vivo an incomplete violaxanthin cycle. Although the violaxanthin cycle in Mantoniella is capable of converting violaxanthin to zeaxanthin, in intact cells only antheraxanthin accumulates during periods of strong illumination. Antheraxanthin enhances non-photochemical quenching of chlorophyll fluorescence. Inhibition of antheraxanthin synthesis by the de-epoxidase inhibitor dithiothreitol abolishes increased thermal energy dissipation. Antheraxanthin-dependent non-photochemical quenching, like zeaxanthin-mediated non-photochemical quenching in higher plants, is uncoupler-sensitive. Mantoniella squamata cells cultivated at high light intensities contain higher amounts of violaxanthin than cells grown at low light. The increased violaxanthin-cycle pool size in high-light-grown Mantoniella cells is accompanied by higher de-epoxidation rates in the light and by a greater capacity to quench chlorophyll fluorescence non-photochemically. Antheraxanthin-dependent amplification of non-photochemical quenching is discussed in the light of recent models developed for zeaxanthin- and diatoxanthin-mediated enhanced heat dissipation. Received: 4 September 1997 / Accepted: 22 December 1997     

A new subunit of cytochrome b6f complex undergoes reversible phosphorylation upon state transition

A 15.2-kDa polypeptide, encoded by the nuclear gene PETO, was identified as a novel cytochrome b(6)f subunit in Chlamydomonas reinhardtii. The PETO gene product is a bona fide subunit, subunit V, of the cytochrome b(6)f complex, because (i) it copurifies with the other cytochrome b(6)f subunits in the early stages of the purification procedure, (ii) it is deficient in cytochrome b(6)f mutants accumulating little of the complex, and (iii) it colocalizes with cytochrome f, which migrates between stacked and unstacked membrane regions upon state transition. Sequence analysis and biochemical characterization of subunit V shows that it has a one transmembrane alpha-helix topology with two large hydrophilic domains extending on the stromal and lumenal side of the thylakoid membranes, with a lumenal location of the N terminus. Subunit V is reversibly phosphorylated upon state transition, a unique feature that, together with its topological organization, points to the possible role of subunit V in signal transduction during redox-controlled short term and long term adaptation of the photosynthetic apparatus in eukaryotes.     

Examination of chlorophyll fluorescence in sulfur-deprived cells of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Pulse amplitude modulation fluorimetry was used to assess chlorophyll fluorescence parameters in Chlamydomonas reinhardtii cells during sulfur deprivation. A significant (fourfold) increase in the chlorophyll fluorescence yield (parameters F ₀ and F _m) normalized to the chlorophyll concentration was shown for deprived cells. The chlorophyll content did not change during the deprivation experiments. An analysis of nonphotochemical quenching of chlorophyll fluorescence indicated a considerable modification of the energy deactivation pathways in photosystem II (PSII) of sulfur-deprived cells. For example, starved cells exhibited a less pronounced pH-dependent quenching of excited states and a higher thermal dissipation of excess light energy in the reaction centers of PSII. It was also shown that the photosynthetic apparatus of starved cells is primarily in state 2 and that back transition to state 1 is suppressed. However, these changes cannot cause the discovered elevation of chlorophyll fluorescence intensity (F ₀ and F _m) in the cells under sulfur limitation. The observed increase in the chlorophyll fluorescence intensity under sulfur deprivation may be due to partial dissociation of peripheral light-harvesting complexes from the reaction centers of PSII or a malfunction of the dissipative cycle in PSII, involving cytochrome b ₅₅₉.     

Chlorophyll a fluorescence measurements of isolated spinach thylakoids obtained by using single-laser-based flow cytometry

Flow cytometry data of spinach thylakoid membrane preparations indicate the presence of a homogeneous thylakoid population. Fluorescence data from a flow cytometer and comparison with data from two other fluorometers show that chlorophyll a fluorescence detected with a flow cytometer has the character of maximum fluorescence (Fmax), not of the constant component (Fo). This conclusion is important since Fo measures fluorescence that is affected mostly by changes in excitation energy transfer and Fmax-Fo (the variable fluorescence) by changes in photochemistry. This was demonstrated by: 1) The light intensity as well as diffusion rate dependence of the quenching effect of various quinones (p-benzoquinone, phenyl-benzoquinone, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on fluorescence yield; quenching for the same concentration of these quinones was lower at the higher than at the lower light intensities. 2) Temperature dependence of the fluorescence yield; increasing the temperature from 20 to 70 degrees C did not show an increase in fluorescence yield using a flow cytometer in contrast to measurements with weak excitation light, but similar to those obtained for Fmax. 3) Addition of an inhibitor diuron up to 100 microM did not change the fluorescence intensity. A comparison of quenching of fluorescence by various quinones obtained by flow cytometry with those by other fluorometers suggests that the high intensity used in the cytometry produces unique results: the rate of reduction of quinones in much larger than the rate of equilibration with the bulk quinones.     

Modulation of gene expression of carotene biosynthesis-related protein by photosynthetic electron transport for the acclimation of intertidal macroalga Ulva fasciata to hypersalinity and excess light

A gene (UfCBR) encoding carotene biosynthesis-related (CBR) protein that potentially functions for the dissipation of excessive energy has been cloned from the intertidal green macroalga Ulva fasciata Delile. Hypersalinity and high light ≥300 μmol m(-2) s(-1) increased both UfCBR mRNA level and non-photochemical quenching (NPQ). The increase of UfCBR mRNA level and NPQ by high light was inhibited by treatment of photosynthetic electron transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, but not by stigmatellin, an inhibitor that blocks electron transfer from quinol oxidase to iron-sulfur protein in cytochrome b(6) f complex. Treatment of dimethylthiourea, an H(2) O(2) scavenger, under 1200 μmol m(-2) s(-1) condition inhibited H(2) O(2) accumulation but did not affect UfCBR mRNA level, while treatment of H(2) O(2) in 150 μmol m(-2) s(-1) condition decreased UfCBR mRNA level. Thus, an reactive oxygen species-independent redox control via a more reduced state downstream the cytochrome b(6) f complex is involved in high light up-regulation of UfCBR expression in U. fasciata. The expression of UfCBR in U. fasciata against oxidative stress occurring in high light or high salinity in relation to NPQ is discussed.     

Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase

The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS)