Presence Of Dithionite Research Articles

A major integral protein of the plant plasma membrane binds flavin

Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor.

Energy and electron transfer in photosystem II of a chlorophyll b-containing Synechocystis sp. PCC 6803 mutant.

Using a Synechocystis sp. PCC 6803 mutant strain that lacks photosystem (PS) I and that synthesizes chlorophyll (Chl) b, a pigment that is not naturally present in the wild-type cyanobacterium, the functional consequences of incorporation of this pigment into the PS II core complex were investigated. Despite substitution of up to 75% of the Chl a in the PS II core complex by Chl b, the modified PS II centers remained essentially functional and were able to oxidize water and reduce Q(A), even upon selective excitation of Chl b at 460 nm. Time-resolved fluorescence decay measurements upon Chl excitation showed a significant reduction in the amplitude of the 60-70 ps component of fluorescence decay in open Chl b-containing PS II centers. This may indicate slower energy transfer from the PS II core antenna to the reaction center pigments or slower energy trapping. Chl b and pheophytin b were present in isolated PS II reaction centers. Pheophytin b can be reversibly photoreduced, as evidenced from the absorption bleaching at approximately 440 and 650 nm upon illumination in the presence of dithionite. Upon excitation at 685 nm, transient absorption measurements using PS II particles showed some bleaching at 650 nm together with a major decrease in absorption around 678 nm. The 650 nm bleaching that developed within approximately 10 ps after the flash and then remained virtually unchanged for up to 1 ns was attributed to formation of reduced pheophytin b and oxidized Chl b in some PS II reaction centers. Chl b-containing PS II had a lower rate of charge recombination of Q(A)(-) with the donor side and a significantly decreased yield of delayed luminescence in the presence of DCMU. Taken together, the data suggest that Chl b and pheophytin b participate in electron-transfer reactions in PS II reaction centers of Chl b-containing mutant of Synechocystis without significant impairment of PS II function.

Functional characterization of GcpE, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis

The gcpE gene product controls one of the terminal steps of isoprenoid biosynthesis via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. This pathway is utilized by a variety of eubacteria, the plastids of algae and higher plants, and the plastid-like organelle of malaria parasites. Recombinant GcpE protein from the hyperthermophilic bacterium Thermus thermophilus was produced in Escherichia coli and purified under dioxygen-free conditions. The protein was enzymatically active in converting 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the presence of dithionite as reductant. The maximal specific activity was 0.6 μmol min−1 mg−1 at pH 7.5 and 55°C. The kcat value was 0.4 s−1 and the Km value for HMBPP 0.42 mM.

Crystallization and preliminary X-ray analysis of the molybdenum-dependent pyrogallol-phloroglucinol transhydroxylase of Pelobacter acidigallici.

Crystals of the molybdo-/iron-sulfur protein pyrogallol:phloroglucinol hydroxyltransferase (transhydroxylase; EC 1.97.1.2) from Pelobacter acidigallici were grown by vapour diffusion in an N(2)/H(2) atmosphere using polyethylene glycol as a precipitant. In this microorganism, transhydroxylase converts pyrogallol to phloroglucinol in a unique reaction without oxygen transfer from water. Growth of crystals suitable for X-ray analysis was strongly dependent on the presence of dithionite as a reducing agent. The crystals belonged to space group P1 and MAD data were collected on the iron K edge to resolutions higher than 2.5 A.

Mobility of Sulfate Ester during Structural Determinations of Red Algal Galactans

The polysaccharide from Rhodophyllis membranacea (family Cystocloniaceae) was found to be approximately 80% L-carrageenan together with 20 % of a further precursor-like entity. Alkali treatment of the initial polymer gaveonly ι-carrageenan. A standard methylation of this polysaccharide in NaOH/DMSO gave 95 % of the product as permethylated β-carrageenan. This desulfation was avoided by removing reducing agents with bromine water, then carrying out a preliminary methylation with aqueous NaOH/dimethyl sulfate. Alkaline desulfation was investigated using L-carrageenan under non-aqueous alkaline conditions. Desulfation was achieved in the presence of strong reducing agents and ferric oxide, and quantitatively in the presence of dithionite or aluminium.

A new pathway of photoinactivation of photosystem II. Irreversible photoreduction of pheophytin causes loss of photochemical activity of isolated D1-D2-cytochrome b559 complex.

A new pathway of photoinactivation of photosystem II (PS II) connected with irreversible photoaccumulation of reduced pheophytin (Ph) in isolated D1-D2-cytochrome b559 complexes of reaction center (RC) of PS II was discovered. The inhibitory effects of white light illumination on photochemical activity of D1-D2-cytochrome b559 complexes of RCs of photosystem II, isolated from pea chloroplasts, have been compared under anaerobic conditions in the absence and in the presence of sodium dithionite, electron transfer from which to the oxidized primary electron donor P680+ results in the photoaccumulation of anion-radical of the primary electron acceptor, Ph(-.). In both cases, prolonged illumination (1-5 min, 120 W/m(2)) led to a pronounced loss of the photochemical activity as it was monitored by measuring the amplitude of the reversible photoinduced absorbance changes at 682 nm related to the photoreduction of Ph. The extent of the photoinactivation depended on the illumination time and pH of the medium. At pH 8.0, the presence of dithionite during photoinactivation brought about a protective effect compared to that in a control sample. In contrast, lowering pH to 6.0 increased the sensitivity to photoinactivation in the dithionite-containing samples. For 5 min irradiation, the photochemical activity in the absence and in the presence of dithionite decreased by 35 and 72%, respectively (this was accompanied by an irreversible bleaching of the pheophytin Q(x) absorption band at 542 nm). Degradation of the D1 and D2 proteins was not observed under these conditions. A subsequent addition of an electron acceptor, potassium ferricyanide, to the illuminated samples restored neither the amplitude of the signal at 682 nm nor absorption at 542 nm. It is suggested that at pH. 7.0 the photoaccumulated Ph(-.) is irreversibly converted into a secondary, most probably protonated form, that does not lead to destruction of the RCs but prevents the photoformation of the primary radical pair [P680+Ph(-.)]. A possible application of this effect to photoinactivation of PS II in vivo is discussed.

Appearance of nitrite reducing activity of cytochrome c upon heat denaturation.

The appearance of NO2- reducing activity of cytochrome c (Cyt c) upon heat denaturation was investigated with equine heart Cyt c. Denatured equine heart Cyt c (dCyt c), which was treated at 100 degrees C for 30 min, had NO2- reducing activity in the presence of dithionite and methylviologen in an aqueous solution under anaerobic conditions. In contrast, hemoglobin and myoglobin had no such activity under the same conditions. Using spectroscopic methods, we found that the appearance of this activity in the Cyt c was due to the following intramolecular changes: unfolding of the peptide chain, exposure of the heme, dissociation of the sixth ligand methionine sulfur, and appearance of autoxidizability. The dCyt c catalyzed NO2- reduction to NH4+ via ferrous-NO complexes, and this reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized dCyt c had activity similar strength to that in solution. The resin retained the activity after five uses and even after storage for 1 year. On the basis of these results, we concluded that Cyt c acquired a new catalytic activity upon heat treatment, unlike to other familiar biological molecules.

Mechanistic investigation of UDP-galactopyranose mutase from Escherichia coli using 2- and 3-fluorinated UDP-galactofuranose as probes.

The galactofuranose moiety found in many surface constituents of microorganisms is derived from UDP-D-galactopyranose (UDP-Galp) via a unique ring contraction reaction catalyzed by UDP-Galp mutase. This enzyme, which has been isolated from several bacterial sources, is a flavoprotein. To study this catalysis, the cloned Escherichia coli mutase was purified and two fluorinated analogues, UDP-[2-F]Galf (9) and UDP-[3-F]Galf (10), were chemically synthesized. These two compounds were found to be substrates for the reduced UDP-Galp mutase with the Km values determined to be 65 and 861 microM for 9 and 10, respectively, and the corresponding kcat values estimated to be 0.033 and 5.7 s(-1). Since the fluorine substituent is redox inert, a mechanism initiated by the oxidation of 2-OH or 3-OH on the galactose moiety can thus be firmly ruled out. Furthermore, both 9 and 10 are poorer substrates than UDP-Galf, and the rate reduction for 9 is especially significant. This finding may be ascribed to the inductive effect of the 2-F substituent that is immediately adjacent to the anomeric center, and is consistent with a mechanism involving formation of oxocarbenium intermediates or transition states during turnover. Interestingly, under nonreducing conditions, compounds 9 and 10 are not substrates, but instead are inhibitors for the mutase. The inactivation by 10 is time-dependent, active-site-directed, and irreversible with a K(I) of 270 microM and a k(inact) of 0.19 min(-1). Since the K(I) value is similar to Km, the observed inactivation is unlikely a result of tight binding. To our surprise, the inactivated enzyme could be regenerated in the presence of dithionite, and the reduced enzyme is resistant to inactivation by these fluorinated analogues. It is possible that reduction of the enzyme-bound FAD may induce a conformational change that facilitates the breakdown of the putative covalent enzyme-inhibitor adduct to reactivate the enzyme. It is also conceivable that the reduced flavin bears a higher electron density at N-1, which may play a role in preventing the formation of the covalent adduct or facilitating its breakdown by charge stabilization of the oxocarbenium intermediates/transition states. Clearly, this study has led to the identification of a potent inactivator (10) for this enzyme, and study of its inactivation has also shed light on the possible mechanism of this mutase.

The H2 Sensor of Ralstonia eutropha: BIOCHEMICAL CHARACTERISTICS, SPECTROSCOPIC PROPERTIES, AND ITS INTERACTION WITH A HISTIDINE PROTEIN KINASE

Previous genetic studies have revealed a multicomponent signal transduction chain, consisting of an H(2) sensor, a histidine protein kinase, and a response regulator, which controls hydrogenase gene transcription in the proteobacterium Ralstonia eutropha. In this study, we isolated the H(2) sensor and demonstrated that the purified protein forms a complex with the histidine protein kinase. Biochemical and spectroscopic analysis revealed that the H(2) sensor is a cytoplasmic [NiFe]-hydrogenase with unique features. The H(2)-oxidizing activity was 2 orders of magnitude lower than that of standard hydrogenases and insensitive to oxygen, carbon monoxide, and acetylene. Interestingly, only H(2) production but no HD formation was detected in the D(2)/H(+) exchange assay. Fourier transform infrared data showed an active site similar to that of standard [NiFe]-hydrogenases. It is suggested that the protein environment accounts for a restricted gas diffusion and for the typical kinetic parameters of the H(2) sensor. EPR analysis demonstrated that the [4Fe-4S] clusters within the small subunit were not reduced under hydrogen even in the presence of dithionite. Optical spectra revealed the presence of a novel, redox-active, n = 2 chromophore that is reduced by H(2). The possible involvement of this chromophore in signal transduction is discussed.

Direct FeS cluster involvement in generation of a radical in lysine 2,3-aminomutase.

Lysine 2,3-aminomutase (KAM) belongs to a class of enzymes that use FeS clusters and S-adenosyl-L-methionine to initiate radical-dependent chemistry. Selenium K-edge X-ray absorption spectroscopic analysis of KAM poised at various stages of catalysis, in the presence of selenomethionine or Se-adenosyl-L-selenomethionine, reveals that the cofactor is cleaved only in the presence of dithionite and the substrate analogue trans-4,5-dehydrolysine. A new Fourier transform peak at 2.7 A, assigned as a Se-Fe interaction, appears concomitant with this cleavage. This is the first demonstration of a direct interaction of S-adenosyl-L-methionine, or its cleavage products, with the FeS cluster in this class of enzymes.

Studies with Substrate and Cofactor Analogues Provide Evidence for a Radical Mechanism in the Chorismate Synthase Reaction

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The strict requirement for a reduced FMN cofactor and a trans-1,4-elimination are unusual. (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites in the presence of dithionite. This conversion was associated with the oxidation of FMN to give a stable flavin semiquinone. The IC(50) of the fluorinated substrate analogue was 0.5 and 250 microm with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not. The lack of dissociation of the flavin semiquinone and chorismate from the enzyme appears to be the basis of the essentially irreversible inhibition by this analogue. A dithionite-dependent transient formation of flavin semiquinone during turnover of (6S)-6-fluoro-EPSP has been observed. These reactions are best rationalized by radical chemistry that is strongly supportive of a radical mechanism occurring during normal turnover. The lack of activity with 5-deaza-FMN provides additional evidence for the role of flavin in catalysis by the E. coli enzyme.

Effects of hypoxia and dithionite on catecholamine release from isolated type I cells of the rat carotid body.

1. Amperometric recordings were conducted to investigate the ability of hypoxia and anoxia to evoke quantal catecholamine secretion from isolated type I cells of the rat carotid body. 2. Hypoxia (PO2 8-14 mmHg) consistently failed to evoke catecholamine secretion from type I cells, when cells were perfused either at room temperature (21-24 C) or at 35-37 C, and regardless of whether Hepes- or HCO3-/CO2-buffered solutions were used. 3. Elevating extracellular [K+] caused concentration-dependent secretion from individual type I cells, with a threshold concentration of approximately 25 mM. In the presence of this level of extracellular K+, hypoxia (PO2 8-14 mmHg) caused a marked enhancement of secretion which was fully blocked by 200 microM Cd2+, a non-specific blocker of voltage-gated Ca2+ channels. 4. Anoxia (N2-equilibrated solution containing 0.5 mM dithionite) evoked exocytosis from type I cells when extracellular [K+] was 5 mM. This secretion was completely inhibited by removal of extracellular Ca2+, but was not significantly affected by Cd2+ (200 microM), Ni2+ (2 mM), Zn2+ (1 mM) or nifedipine (2 microM). Secretion was also observed when 0.5 mM dithionite was added to air-equilibrated solutions. 5. Anoxia also evoked secretion from chemoreceptive phaeochromocytoma (PC12) cells, which was wholly Ca2+ dependent, but unaffected by Cd2+ (200 microM). 6. Our results suggest that hypoxia can evoke catecholamine secretion from isolated type I cells, but only in the presence of elevated extracellular [K+]. This may be due to the cells being relatively hyperpolarized following dissociation. In addition, we have shown that dithionite evokes catecholamine release regardless of PO2 levels, and this release is due mainly to an artefactual Ca2+ influx pathway activated in the presence of dithionite.

Anoxic decrease in potassium outward currents of hippocampal cultured neurons in absence and presence of dithionite

The effect of brief anoxia on voltage dependent K +-currents of hippocampal cultured neurons was studied. The oxygen scavenger dithionite (hydrosulphite) was previously used for creating zero oxygen pressure. However, dithionite consumes O 2 in parallel with generation of superoxide radicals and is a strongly reducing agent. In this study anoxia was produced by perfusion of the neurons with a solution bubbled with nitrogen for 1 h using a chamber with an argon layer isolating the anoxic bath flow from atmospheric oxygen in presence and absence of dithionite. Oxygen partial pressure of dithionite-free solution was determined by oxygen dependent quenching of the phosphorescence of Pd-coproporphyrin to be 0.15±0.02 Torr (values are given as mean±S.D., n=6). Slow ( I K)- and fast ( I A)-inactivating K +-currents were measured with the patch clamp technique in the whole cell configuration. Exposure of the neurons to anoxia reversibly decreased the amplitude of I K at a test pulse of 0 mV to 77±12% ( n=7) in absence and to 83±7% ( n=6) in presence of 2 mM dithionite; the amplitude of I A decreased to 78±11% in absence and to 82±9% in presence of 2 mM dithionite. Voltage dependence of activation and inactivation shifted 5 min after exposure to anoxia reversibly by about 6 mV in depolarizing direction. The decay times of inactivation were insensitive to anoxia. Dithionite had no significant effects on K +-currents. In 15 of 21 neurons not employed for analysis on K +-currents, a reversible increase in holding current under dithionite was observed. In absence of dithionite in 4 of 19 neurons the holding current reversibly increased during anoxia. Although dithionite does not affect K +-currents, changes in holding current show that the dithionite may affect neurons independently of oxygen deprivation.

MgATP-independent hydrogen evolution catalysed by nitrogenase: an explanation for the missing electron(s) in the MgADP–AlF4 transition-state complex

When the MoFe (Kp1) and Fe (Kp2) component proteins of Klebsiella pneumoniae nitrogenase are incubated with MgADP and AlF4(-) in the presence of dithionite as a reducing agent, a stable putative transition-state complex is produced [Yousafzai and Eady (1997) Biochem. J. 326, 637-640]. Surprisingly, the EPR signal associated with reduced Kp2 is not detectable, but Kp1 retains the S=3/2 EPR signal arising from the dithionite reduced state of the MoFe cofactor centre of the protein. This is consistent with the [Fe4S4] centre of the Fe protein in the complex being oxidized, and similar observations have been made with the complex of Azotobacter vinelandii [Spee, Arendsen, Wassink, Marritt, Hagen and Haaker (1998) FEBS Lett. 432, 55-58]. No satisfactory explanation for the fate of the electrons lost by Kp2 has been forthcoming. However, we report here that during the preparation of the MgADP-AlF4 K. pneumoniae complex under argon, H2 was evolved in amounts corresponding to one half of the FeMoco content of the Kp1 (FeMoco is the likely catalytic site of nitrogenase with a composition Mo:Fe7:S9:homocitrate). This is surprising, since activity is observed during incubation in the absence of MgATP, normally regarded as being essential for nitrogenase function, and in the presence of MgADP, a strong competitive inhibitor of nitrogenase. The formation of H2 by nitrogenase in the absence of AlF4(-) was also observed in reaction mixtures containing MgADP but not MgATP. The reaction showed saturation kinetics when Kp1 was titrated with increasing amounts of Kp2 and, at saturation, the amount of H2 formed was stoichiometric with the FeMoco content of Kp1. The dependence of the rate of formation of H2 on [MgADP] was inconsistent with the activity arising from MgATP contamination. We conclude that MgATP is not obligatory for H+ reduction by nitrogenase since MgADP supports a very low rate of hydrogen evolution.

A T14C Variant of Azotobacter vinelandii Ferredoxin I Undergoes Facile [3Fe-4S]0 to [4Fe-4S]2+Conversion in Vitro but Not in Vivo

[4Fe-4S]2+/+ clusters that are ligated by Cys-X-X-Cys-X-X-Cys sequence motifs share the general feature of being hard to convert to [3Fe-4S]+/0 clusters, whereas those that contain a Cys-X-X-Asp-X-X-Cys motif undergo facile and reversible cluster interconversion. Little is known about the factors that control the in vivo assembly and conversion of these clusters. In this study we have designed and constructed a 3Fe to 4Fe cluster conversion variant of Azotobacter vinelandii ferredoxin I (FdI) in which the sequence that ligates the [3Fe-4S] cluster in native FdI was altered by converting a nearby residue, Thr-14, to Cys. Spectroscopic and electrochemical characterization shows that when purified in the presence of dithionite, T14C FdI is an O2-sensitive 8Fe protein. Both the new and the indigenous clusters have reduction potentials that are significantly shifted compared with those in native FdI, strongly suggesting a significantly altered environment around the clusters. Interestingly, whole cell EPR have revealed that T14C FdI exists as a 7Fe protein in vivo. This 7Fe form of T14C FdI is extremely similar to native FdI in its spectroscopic, electrochemical, and structural features. However, unlike native FdI which does not undergo facile cluster conversion, the 7Fe form T14C FdI quickly converts to the 8Fe form with a high efficiency under reducing conditions.

New Catalytic Properties of Iron Porphyrins: Model Systems for Cytochrome P450-Catalyzed Dehydration of Aldoximes

Various iron porphyrin systems were found to catalyze the dehydration of aldoximes, such as heptanaldoxime or phenylacetaldoxime, into the corresponding nitriles under mild conditions (t = 20 °C, neutral or slightly acidic pH). In all these systems, the presence of both the iron porphyrin catalyst and a reducing agent is required, indicating that the active species is the iron(II) porphyrin. The most efficient systems used either an organosoluble iron porphyrin, such as Fe(OEP), in the presence of a carboxylic acid and zinc amalgam as reducing agent, or a water-soluble heme fragment of cytochrome c (microperoxidase MP-11) in the presence of dithionite. The catalytic activity of the systems was greatly increased when using electron-rich iron porphyrins bearing an electron-donating axial ligand, such as imidazole, and a carboxylic acid cocatalyst in close proximity to the iron center. The activity of the best systems was comparable to that of microsomal cytochromes P450 (between 1 and 10 turnovers per min)....

Secondary Pair Charge Recombination in Photosystem I under Strongly Reducing Conditions: Temperature Dependence and Suggested Mechanism

Photoinduced electron transfer in photosystem I (PS I) proceeds from the excited primary electron donor P700 (a chlorophyll a dimer) via the primary acceptor A 0 (chlorophyll a) and the secondary acceptor A 1 (phylloquinone) to three [4Fe-4S] clusters, F X, F A, and F B. Prereduction of the iron-sulfur clusters blocks electron transfer beyond A 1. It has been shown previously that, under such conditions, the secondary pair P700 +A 1 − decays by charge recombination with t 1/2 ≈ 250 ns at room temperature, forming the P700 triplet state ( 3P700) with a yield exceeding 85%. This reaction is unusual, as the secondary pair in other photosynthetic reaction centers recombines much slower and forms directly the singlet ground state rather than the triplet state of the primary donor. Here we studied the temperature dependence of secondary pair recombination in PS I from the cyanobacterium Synechococcus sp. PCC6803, which had been illuminated in the presence of dithionite at pH 10 to reduce all three iron-sulfur clusters. The reaction P700 +A 1 − → 3P700 was monitored by flash absorption spectroscopy. With decreasing temperature, the recombination slowed down and the yield of 3P700 decreased. In the range between 303 K and 240 K, the recombination rates could be described by the Arrhenius law with an activation energy of ∼170 meV. Below 240 K, the temperature dependence became much weaker, and recombination to the singlet ground state became the dominating process. To explain the fast activated recombination to the P700 triplet state, we suggest a mechanism involving efficient singlet to triplet spin evolution in the secondary pair, thermally activated repopulation of the more closely spaced primary pair P700 +A 0 − in a triplet spin configuration, and subsequent fast recombination (intrinsic rate on the order of 10 9 s −1) forming 3P700.

Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands.

A cDNA encoding barley hemoglobin (Hb) has been cloned into pUC 19 and expressed in Escherichia coli. The resulting fusion protein has five extra amino acids at the N terminus compared with the native protein, resulting in a protein of 168 amino acids (18.5 kDa). The recombinant Hb is expressed constitutively. Extracts made from the bacteria containing the recombinant fusion construct contain a protein with a subunit molecular mass of approximately 18.5 kDa comprising approximately 5% total soluble protein. Recombinant Hb was purified to homogeneity according to SDS-polyacrylamide gel electrophoresis by sequential polyethylene glycol precipitation and fast protein liquid chromatography. Its native molecular mass as assessed by fast protein liquid chromatography-size exclusion was 40 kDa suggesting that it is a dimer. Ligand binding experiments demonstrate that 1) barley Hb has a very slow oxygen dissociation rate constant (0.0272 s-1) relative to other Hbs, and 2) the heme of ferrous and ferric forms of the barley Hb is low spin six-coordinate. The subunit structure, optical spectrum, and oxygen dissociation rate of native barley hemoglobin are indistinguishable from those obtained for the recombinant protein. The implications of these kinetic data on the in vivo function of barley Hb are discussed.

Electron transport and triplet formation in membrane fragments of the green sulfur bacterium Prosthecochloris aestuarii

Under conditions where normal electron transport is blocked, at high pH in the presence of dithionite, three triplets could be observed upon flash illumination of membrane fragments of the green sulfur bacterium Prosthecochloris aestuarii. The first triplet decayed in 7.5 μs and is assigned to carotenoid. The second triplet decays in 67 μs and is assigned to bacteriochlorophyll (BChl) a of the Fenna-Matthews-Olson (FMO) complex, since a triplet with the same spectrum and lifetime was also formed in the isolated FMO complex. The third triplet decayed in 165 μs and is assigned to BChl a of the reaction center core complex, based on its main bleaching at 837 nm. There is insufficient evidence to decide whether this triplet is located on the primary electron donor P840 or on a long-wavelength antenna BChl a of the core. A multiple-flash experiment indicated the presence of two photo-oxidizable hemes per reaction center (RC), both having a difference spectrum centered around 553 nm. The oxidation time was 25 μs for both cytochromes. However, a 75- μs delay was observed for the oxidation of the second heme, indicating that another process must take place before this reaction can occur. This result, together with the observed low efficiency of oxidation of the second heme, suggests the presence of a four-heme cytochrome (as observed in some other species of green sulfur bacteria), with only one heme in direct contact with the RC, rather than a model with two cytochromes symmetrically attached to the RC, as proposed by others. The observed delay can then be explained by a relatively slow heme-to-heme electron transfer. The cytochrome oxidation time of both hemes increased with viscosity, suggesting that some molecular motion is involved in the oxidation process.

Increases in the fluorescence F o level and reversible inhibition of Photosystem II reaction center by high-temperature treatments in higher plants

Effects of high temperatures on the fluorescence Fm (maximum fluorescence) and Fo (dark level fluorescence) levels were studied and compared with those of the photochemical reactions of PS II. These comparisons were performed during and after the high temperature treatments. The following results were obtained; (1) increases in the Fo level at high temperatures were partly reversible, (2) the Fm level in the presence of dithionite in spinach chloroplasts decreased at high temperatures and also showed a partial reversibility, (3) photoreductions of pheophytin a and Qa were reversibly inhibited at high temperatures parallel to the decrease in the difference between the Fm and Fo levels, and (4) the decrease in the fluorescence Fm level seemed to be related to denaturation of chlorophyll-proteins. All the data suggested that, as well as the separation of light-harvesting chlorophyll a/t b protein complexes of PS II from the PS II core complexes, partly reversible inactivation of the PS II reaction center at high temperatures is the cause of the increase in the Fo level.