Addition Of Dithionite Research Articles

Spectra properties of rat liver mitochondrial choline dehydrogenase.

Choline dehydrogenase contains the prosthetic group FAD, non-haem iron and acid labile sulfur. However, the absorption spectra of the purified enzyme do not change after adding substrate. The reduced absorption spectra of choline dehydrogenase can only be determined after the addition of dithionite. Those choline dehydrogenases situated in the mitochondrial inner membrane can be reduced by substrate and exist in the reduced state. When cholate was used to solubilize the substrate-reduced choline dehydrogenase, the reduced spectra will gradually disappear. However, if solubilization is carried out under anaerobic conditions, the reduced spectra can be retained, suggesting that the solubilized choline dehydrogenase can use oxygen as an acceptor.

Dithionite‐Induced Fluorescence Quenching does not Reflect Reductive Activation in Spinach Chloroplasts*

AbstractDithionite is found to cause substantial quenching of chlorophyll fluorescence in intact spinach chloroplasts. In a light‐on induction curve, the I‐P fluorescence rise is selectively suppressed, with a half‐maximal effect at 1.7 × 10−5 M dithionite. Quenching analysis by the saturation pulse method reveals that the dithionite‐induced quenching is photochemical, in analogy to the effect of a Hill reagent. The quenching is suppressed by catalase, KCN and by O2 removal before dithionite addition. These observations, in combination with recent findings on H2O2‐induced photochemical quenching (Neubauer and Schreiber, 1989), lead to the conclusion that H2O2 is formed from reduction products of O2 by dithionite and that it is this H2O2 which, via the action of the ascorbate peroxidase, sustains Hill activity. The results are discussed with reference to the concept of reductive activation of the ferredoxin: NADP oxidoreductase put forward by Satoh (Satoh, 1982). It is concluded that such activation is unlikely to play an essential role in spinach chloroplasts. This conclusion is supported by the observation of suppression of the I‐P fluorescence rise by oxidized NADP in dark‐adapted spinach chloroplasts.

Effect of redox potential on rat liver F 1-ATPase

Redox titration of F 1-ATPase from rat liver mitochondria referred to the modification of the hydrolytic activity on Mg-ATP has resulted in a three-step pattern, with three distinct jumps of activity separated by clear plateaus. The measured potentials ranged from −400 mV to +400 mV and were obtained by the addition of dithionite and ferricyanide. Electron exchange was facilitated with a mixture of different redox mediators. At pH 7.4 the midpoint potentials were +210 mV, +40 mV and −230 mV. These three midpoint potentials were displaced towards more negative values by 2,4-dinitrophenol or by an increase of the pH of the medium. The titration curves were described by n=2 Nernst equations.

Activation by reduction of the resting form of cytochrome c oxidase: Tests of different models and evidence for the involvement of Cu B

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2 · 10 4 M −1 · s −1. In the absence of cyanide, ferrocytochrome a 3 appears at a rate ( k obs) of 0.016 s −1. Ferricytochrome a 3 maintains its 418 nm Soret maximum until reduced. The rate of a 3 reduction is independent of dithionite concentration over a range 0.9 mM–131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a 3/cyanide complex occurs at the same rate as a 3 reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a 3 with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a 3 shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a 3. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow ( k obs ≈ 0.02 s) entry of a third electron. Above +200 mV, the number of electrons taken up in the initial fast phase drops as a redox center (presumably Cu A) titrates with an apparent mid-point potential of +240 mV. The slow phase of reduction remains at the more positive redox values. (4) The results are interpreted in terms of an initial fast reduction of cytochrome a (and Cu A at redox values more negative than +240 mV) followed by a slow reduction of Cu B. Cu B reduction is proposed to spin-uncouple cytochrome a 3 to form a cyanide sensitive center, and trigger a conformational change to an activated form of the enzyme with faster intramolecular electron transfer.

Purification and properties of an enzyme oxidizing nitrite to nitrate form Candida rugosa.

A nitrite-oxidizing enzyme was isolated (1300-fold purification, 15% yield) from Candida rugosa IFO 0591 using a reaction mixture containing glucose oxidase and glucose. The enzyme (molecular weight 220,000) consisted of four identical subunits with molecular weight of 58,000. It showed absorption maxima at 277 and 405 nm with small peaks at 492, 535, and 625 nm. The spectrum was not altered by the addition of dithionite. These and other properties suggested that the enzyme was catalase and that the nitrite-oxidizing reaction was dependent on its peroxidase activity. Some aspects of the nitrite oxidation by the purified enzyme and various preparations of C. rugosa are described.

Extraction of vitamin K-1 from Photosystem I particles by treatment with diethyl ether and its effects on the A −1 EPR signal and System I photochemistry

Treatment of Photosystem I particles, prepared by digitonin treatment of spinach chloroplasts, with dry diethyl ether extracted all the vitamin K-1 (2.2 molecules/P-700 in the original Photosystem I particles) and carotenoids as well as 60% of antenna chlorophylls. P-700 and FeS centers (F X , F A and F B) were not extracted, and were photochemically oxidized and reduced, respectively, upon continuous illumination. This preparation showed the A − 0 EPR signal but no A − 1 signal even after long illumination at 210 or 230 K, in which condition A − 1 was accumulated in the untreated Photosystem I particles. Laser flash excitation induced only a small absorption change, corresponding to 20% of the total P-700, at 429 nm in the extracted preparation, suggesting that charge recombination took place on a submicrosecond time scale. Addition of vitamin K-1 or K-3 but not that of benzyl viologen increased the extent of the slow decay phase of P-700 +. The extraction also induced a new fluorescence band peaking at 692 nm, the intensity of which increased upon addition of dithionite. This fluroescence band is possibly related to the rapid charge recombination between A − 0 and P-700 +. These results strongly suggest that A 1 is vitamin K-1 and that it functions to mediate electron transfer between A 0 and FeS centers. Similar results were also obtained with the ‘P-700 enriched particles’ with 7–16 antenna chlorophyll a P-700 which can be obtained by extraction of Photosystem I particles with diethyl ether containing (80% saturated) water.

Primary radical pair in the Photosystem II reaction centre

Primary Photosystem II (PS II) reactions have been studied by flash absorption spectroscopy with nanosecond resolution. In isolated reaction centers which are devoid of the 47- and 43-kDa chlorophyll-binding polypeptides, an absorption change (ΔA) is induced immediately by a flash. The difference spectrum (450–560 nm, 820 nm) indicates the formation of a radical pair (P-680 +, Pheo −) decaying with t 1 2 = 40 ns at 120 K. At 140 ns after the flash the ΔA can be attributed to the triplet state of P-680 ( 3P-680) and of a carotenoid ( 3Car, λ max = 545 nm). 3Car is formed with a yield of ≈3% and it rises with t 1 2 ≈ 12 ns . At 276 K, the radical pair decays, with t 1 2 ≈ 32 ns . One radical pair is formed per 20 chlorophylls. The data are best interpreted if 3P-680 is formed as a product of radical pair recombination with a yield of 23% at 276 K (80% at 10 K) and if 3Car is formed only in a minority of pigment complexes. The ΔA at 820 nm disappear under conditions (addition of dithionite and methyl viologen, plus continuous illumination) designed to reduce pheophytin. The signals reappear after turning off the continuous light. More intact PS II particles (core complex) were also studied. At 820 nm, the data show that P-680 + is formed under oxidizing conditions and is re-reduced in the microsecond time range. Under reducing conditions the primary biradical decays with t 1 2 = 25 ns . 3P-680 is formed, with properties analogous to those in isolated reaction centers. 3Car is also formed ( λ max = 535 nm). The results show that the PS II reaction center has strong functional analogies with the reaction center of purple bacteria. The behavior of the carotenoid is a remarkable exception. The primary biradical decays much more slowly than expected on the basis of fluorescence measurements.

Redox reactions of plasma membranes isolated from soybean hypocotyls by phase partition

Ninety-five percent pure plasma membranes were isolated from the microsomal fraction of soybean hypocotyls ( Glycine max L. Merr. cv. Wayne) by phase partition in an aqueous polymer system. It was found that isolated plasma membranes could reduce ferricyanide, cytochrome c or various iron complexes with NADH as an electron donor. The rates of these reactions varied from 300 nmol (mg protein) −1 min −1 for NADH-ferricyanide reductase to 21 nmol (mg protein) −1 min −1 for NADH-cytochrome c reductase. The flavin content on these membranes was 2.4 nmol flavin/mg protein. The membranes also showed evidence of a b- type cytochrome in the oxidized minus reduced difference spectrum ( λ max 561 nm) upon addition of dithionite. The Soret band peak for this b- type cytochrome was at 428 nm in the difference spectrum. The cytochrome content of the isolated plasma membranes was 0.5 nmol/mg protein. The reductase activity of plasma membranes isolated from soybean hypocotyls by two-phase partition was stimulated from 88 to 175% by low concentrations of Triton X-100. The reductase reactions in these membranes were inhibited from 33 to 83% by several nucleotides, manganese and zinc ions, p- chloromercuribenzene sulfonate (PCMBS) and 4-nitrophenyl acetate. These inhibitions were affected differently by Triton X-100 in the assay.

Autotrophic growth of nitrogen-fixingAzospirillum species and partial characterization of hydrogenase from strain CC

Out of 15 strains ofAzospirillum spp. isolated from the roots of different plants, only 4 (CY, M, CC, and AM) were able to grow autotrophically with H2 and CO2. All of them showed H2 uptake in the presence of oxygen or methylene blue and ribulose-1,5-bisphosphate carboxylase activity. Among the four strains, strain CC isolated from the roots ofCenchrus cilliaris showed maximum H2+O2 uptake (32.5 μl/min. mg protein) as well as H2 uptake in the presence of methylene blue (41.4 μl/min·mg protein) and also the maximum activity of ribulose-1,5-bisphosphate carboxylase (17 units [U]/g protein). The doubling time of this strain under autotrophic growth conditions and at low oxygen concentration (2.5%, vol/vol) was 10 h. At the same O2 concentration the maximal rates of H2+O2 uptake were reached. The distribution of hydrogenase activity among soluble and particulate protein fractions revealed that the hydrogenase ofAzospirillum strain CC is a membrane-bound enzyme. It showed cross-reaction with antibodies raised against the membrane-bound hydrogenase ofAlcaligenes eutrophus. The hydrogenase in intact cells and crude extracts reacted with methylene blue, phenazine methosulfate, and ferricyanide, but not with NAD or FMN. The specific hydrogenase activity, with methylene blue as an acceptor, was 5.71 U/mg protein in crude extract at 9.38 U/mg protein in the membrane suspension. Hydrogen evolution from reduced viologen dyes could not be demonstrated. The hydrogenase is oxygen sensitive and can be optimally stabilized by addition of dithionite to H2-gased samples.

Fumarate reductase and other mitochondrial activities in Trypanosoma cruzi

Subcellular fractions obtained from Trypanosoma cruzi epimastigotes broken by freezing and thawing were assayed for fumarate reductase activity with reduced methyl viologen as electron donor and fumarate as electron acceptor under anaerobic conditions. Two distinct activities were detected: one in the mitochondrial membranes, 115 mU(mg protein) −1, accounting for 96% of the total and the other in the cytosol, 3 mU(mg protein) −1, accounting for 3% of the total. The activity of membrane-bound fumarate reductase correlated statistically with either the activity or the amount of mitochondrial markers such as succinate and NADH dehydrogenases, cytochromes b + c 558, cytochrome a 611 and 5,7-diene sterols in the obtained subcellular fractions (580 × g, 12 000 × g, and 105 000 × g sediments and supernatant). Mitochondrial fumarate reductase was inhibited by succinate, malonate, cyanide, and 2-thenoyltrifluoroacetone (TTFA); whereas the soluble enzyme was inhibited by succinate and not by TTFA. The 12 000 × g sediment (mitochondrial membranes) showed after dithionite addition, absorption maxima at 611, 560 and 530 nm accounting for the presence of cytochromes b 560, c 558 and a 611. A CO-binding cytochrome o was also detected. A scheme of the T. cruzi mitochondrial respiratory chain is presented.

Comparison of ligninase-I and peroxidase-M2 from the white-rot fungus Phanerochaete chrysosporium

Ligninase-I ( M r 42,000–43,000; carbohydrate, 21%) and peroxidase-M2 ( M r 45,000–47,000; carbohydrate, 17%), two representative, hydrogen peroxide-dependent extracellular enzymes produced by ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium BKM-F-1767, were purified and their properties compared. Spectroscopic studies showed that both native enzymes are heme proteins containing protoporphyrin IX. EPR spectroscopy indicated that iron ions are coordinated with the enzymes' prosthetic groups as high-spin ferriheme complexes. We confirmed reports of others that the ligninase-hydrogen peroxide complex (activated enzyme) reverts to its native state on addition of dithionite or one of the enzyme's substrates (e.g., veratryl alcohol); however, we found that the peroxidase-M2-hydrogen peroxide complex required Mn 2+ ions to accomplish a similar cycle. The peroxidase oxidized Mn 2+ to a higher oxidation state, and the oxidized Mn acted as a diffusible catalyst able to oxidize numerous organic substrates. Unlike ligninase-I which is found free extracellularly, peroxidase-M2 appears to be associated closely with the fungal mycelium. In its peroxidatic reactions, ligninase-I oxidizes a variety of nonphenolic and phenolic lignin model compounds. In the presence of Mn 2+, peroxidase-M2 oxidizes numerous phenolic compounds, especially syringyl (3,5-dimethoxy-4-hydroxyphenyl) and vinyl side-chain substituted substrates. Also, the peroxidase-Mn 2+ system (without hydrogen peroxide) expresses oxidase activity against NADPH, GSH, dithiothreitol, and dihydroxymaleic acid, forming hydrogen peroxide at the expense of oxygen. Both enzymes were believed to play roles in lignin degradation, and these are discussed.

Kinetic studies on the nitrite reductase of Wolinella succinogenes.

The six haem groups of the nitrite reductase enzyme isolated from Wolinella succinogenes are rapidly reduced by the addition of dithionite (S2O4(2-)). The reduction, however, is not homogeneous. Two of the haem groups, namely those that show spectral characteristics typical of five-co-ordinated haem groups, are reduced in a dithionite-concentration-dependent fashion with a rate limit of 1.5 S-1. The other four haem groups, which show spectral characteristics very similar to those of normal six-co-ordinate c-haem groups, reduce in a linear dithionite-concentration-dependent manner with a second-order rate constant of 150 M-1/2 X S-1. The ratio of the amplitudes of the two reduction phases observed in stopped-flow studies is found to be dependent on the concentration of dithionite used. A model is proposed to account for these observations, and computer simulations show that the model represents a good fit to the experimental data. The two haem groups with five-co-ordinate spectral characteristics bind CO. Flash photolysis of the CO complex exhibits one major recombination process with a linear dependence in rate on CO concentration with a second-order rate constant of 2 X 10(6) M-1 X S-1. By contrast, stopped-flow mixing of the reduced protein with CO shows a very complex pattern of combination, with most of the observed absorbance change associated with a concentration-independent step. These findings are rationalized in terms of structural changes in the protein consequent to ligand binding.

Effect of free-radical and oxygen scavengers on photochemically generated oxygen toxicity and on the aerotolerance of Campylobacter jejuni.

Exposure of a nutrient agar medium to the combined action of fluorescent light and air produced toxic factors in the medium which affected the growth of Campylobacter jejuni. Sodium dithionite (5-10 mM), a powerful reducing agent, and catalase were effective in counteracting the injurious action of light and air. Among the quenchers of singlet oxygen tested, only histidine had a beneficial effect on the recovery of C. jejuni in the photo-oxidized medium, while the addition of superoxide dismutase, a hydroxyl radical scavenger such as cysteamine, or the free radical antioxidants tocopherol and butylated hydroxy toluene, did not increase the recovery rate of photochemically injured cells. Histidine (40 mM) and dithionite (5-10 mM) also assisted recovery of C. jejuni inoculated on nutrient agar stored in air in the dark. Cysteamine and dithionite were toxic to Campylobacter when added at concentrations of greater than or equal to 10 mM and greater than or equal to 20 mM, respectively. A high inoculum of C. jejuni could not be recovered in unsupplemented nutrient agar incubated in air but was recovered in atmospheres containing 17 or 21% oxygen plus 10% carbon dioxide. The addition of dithionite, catalase or histidine resulted some colony formation on nutrient agar incubated in air. Among the scavengers tested, only dithionite was consistently able to maintain the viability of C. jejuni on nutrient agar stored in air for longer than 4 weeks. In view of the ability of catalase, dithionite and histidine to enhance the aerotolerance of C. jejuni, it is concluded that various oxygen species might be involved in the toxicity of high levels of oxygen.

Low fluorescence yield in anaerobic chloroplasts and stimulation of chlorophyll a fluorescence by oxygen and inhibitors that block electron flow between photosystems II and I

Intact or broken chloroplasts exhibited strong reversible quenching of chlorophyll fluorescence when illuminated with red light in the absence of oxygen and other electron acceptors. Fluorescence quenching was not accompanied by formation of a significant proton gradient. It was caused by excitation of photosystem II, not photosystem I. The extent of quenching and its quantum yield was increased by carbonyl cyanide- m -chlorophenylhydrazone (CCCP), which protected chloroplasts against photobleaching of pigments in the absence of oxygen. CCCP also increased reversibility of fluorescence quenching, perhaps by facilitating cyclic electron transfer around photosystem II. Addition of oxygen or, in intact chloroplasts, of nitrite or oxaloacetate, increased fluorescence. A similar increase was brought about by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) or other inhibitors that interrupt electron flow between the photosystems. In the presence of these inhibitors, fluorescence quenching could no longer be observed but it was restored by the addition of dithionite. It is proposed that a main part of anerobic fluorescence quenching is caused by accumulation of reduced phaeophytin, and that this accumulation is prevented by electron transport inhibitors acting between photosystem II and I.

Variable fluorescence of photosystem I particles and its application to the study of the structure and function of photosystem I

Chlorophyll a fluorescence in Photosystem I (PSI) particles isolated according to the method of Bengis and Nelson [ J. Biol. Chem. 252, 4564–4569 (1977)]was found to be dependent on the redox state of both P700 and X (an acceptor on the reducing side of PSI). Addition of dithionite plus neutral red to PSI caused an increase in fluorescence intensity and a shift of the main fluorescence peak from 689 to 674 nm. Addition of electron acceptors such as ferredoxin and methyl viologen decreased the fluorescence yield when added to PSI incubated under anaerobic conditions in the presence of excess dichlorophenol indophenol (DCIPH 2). The K m for ferredoxin agreed with that determined from direct measurements of ferredoxin reduction, showing that X is a quencher of fluorescence. P700 was also found to be a quencher of fluorescence, since electron donors such as DCIPH 2, TMPD, and plastocyanin decreased fluorescence with K m 's nearly identical to those observed for P700 + reduction. Chemical modification of PSI (with ethylene diamine + a water-soluble carbodiimide) to make it positively charged increased the fluorescence yield and shifted the 689-nm peak to 674 nm. The K m 's for DCIPH 2 and ferredoxin were decreased. In contrast, modification of PSI with succinic anhydride, which increased the net negative charge, increased the K m for ferredoxin. Salts affected the interaction of methyl viologen with PSI. Both anion and cation selectivity were observed. Limited proteolysis increased the K m for both methyl viologen and ferredoxin, indicating that their binding site on PSI was altered. These results suggest that the binding site for ferredoxin is on either the 70- or the 20-kDa subunit of PSI.

Molybdenum EXAFS of the desulfovibrio gigas MO(2Fe-2S) protein—structural similarity to “desulfo” xanthine dehydrogenase

The molybdenum EXAFS of the Mo(2Fe-2S) protein from Desulfovibrio gigas has been examined using fluorescence detection and synchrotron radiation. In the oxidized form the molybdenum environment is found to contain two terminal oxo groups and two long (2.47 Å) Mo-S bonds. Evidence was also found for an oxygen or nitrogen donor ligand a 1.90 Å. Addition of dithionite to the oxidized enzyme results in loss of a terminal oxo group, perhaps due to protonation. In addition, a 0.1 Å contraction in the Mo-S bond lengths is observed. The behavior of both oxidized and dithionite-treated forms is similar to that observed previously with “desulfo” xanthine oxidase.

Spectrally distinct cytochrome b -563 components in a chloroplast cytochrome b — f complex: Interaction with a hydroxyquinoline N -oxide

The two heme equivalents of cytochrome b-563 in the photosynthetic cytochrome b-f complex can be distinguished by their rate of reduction with dithionite at 25 degrees C and by their optical absorption spectra at 77 K. The cytochrome b component that is rapidly reduced after addition of dithionite or reduced ferredoxin possesses an alpha band that splits at 77 K into two peaks, at 557 and 561 nm. Prolonged incubation with reductant reveals a second, approximately equimolar cytochrome b component that has at 77 K an unsplit alpha-band maximum at 561 nm. The designations cytochrome b-563(H) and cytochrome b-563(L), respectively, are proposed for the rapidly and more slowly reduced cytochrome b-563 components. Potentiometric titration establishes a midpoint potential, E(m), of -30 mV (electron change n approximately 2) for cytochrome b-563(H) and -150 mV (n = 1) for cytochrome b-563(L) at pH 7.5. The reduction potential of these components is raised by 2-heptyl-4-hydroxyquinoline N-oxide, giving E(m) values of +57 and -34 mV, respectively, with each titration slope approximating n = 2.

Reactions of hydrogen and oxygen with Photosystem I of isolated heterocysts from Anabaena variabilis (ATCC 29413)

Isolated heterocysts from Anabaena variabilis show high rates of light- and hydrogen-dependent acetylene reduction. This heterocyst preparation also shows light-induced redox reactions of cytochromes f-556 and b-564. Both cytochromes are reversibly oxidized by light or by oxygen (in the dark). Oxygen-oxidized cytochromes assume their initially reduced state by addition of dithionite or flushing the reaction vessel with hydrogen or argon. All three agents remove oxygen from solutions more or less efficiently, thereby creating reducing conditions; hydrogen-induced reduction is slow and generally completed after about 10 min. No evidence has been obtained for a direct electron donation of hydrogen to either of the cytochromes measured. Photoreduction of nitrogen in the presence of hydrogen is explained by a combined operation of properly poised cyclic photophosphorylation providing ATP, and a light-independent hydrogenase reaction providing reductant.

Characterization of a new type of dissimilatory sulfite reductase present in Thermodesulfobacterium commune.

A new type of dissimilatory bisulfite reductase, desulfofuscidin, was isolated from the nonsporeforming thermophilic sulfate-reducing microorganism Thermodesulfobacterium commune. The molecular weight of the enzyme was estimated at 167,000 by sedimentation equilibrium, and the protein was pure by both disc electrophoresis and ultracentrifugation. The bisulfite reductase was a tetramer and had two types of subunits with an alpha(2)beta(2) structure and an individual molecular weight of 47,000. The enzyme exhibited absorption maxima at 576, 389, and 279 nm, with a weak band at 693 nm. Upon the addition of dithionite, the absorption maxima at 576 and 693 nm were weakened, and a new band appeared at 605 nm. The protein reacted with CO in the presence of dithionite to give a complex with absorption peaks at 593, 548, and 395 nm. The extinction coefficients of the purified enzyme at 576, 389, and 279 nm were 89,000, 310,000, and 663,000 M(-1) cm(-1), respectively. Siroheme was detected as the prosthetic group. The protein contains 20 to 21 nonheme iron atoms and 16 to 17 acid-labile sulfur groups per molecule. The data suggest the presence of four sirohemes and probably four (4Fe-4S) centers per molecule by comparison with desulfoviridin, the dissimilatory sulfite reductase from Desulfovibrio species. The protein contains 36 cysteine residues and is high in acidic and aromatic amino acids. The N-terminal amino acids of the alpha and beta subunits were threonine and serine, respectively. With reduced methyl viologen as electron donor, the major product of sulfite reduction was trithionate, and the pH optimum for activity was 6.0. The enzyme was stable to 70 degrees C and denatured rapidly above this temperature. The dependence of T. commune bisulfite reductase activity on temperature was linear between 35 and 65 degrees C, and the Q(10) values observed were above 3. The presence of this new type of dissimilatory bisulfite reductase in T. commune is discussed in terms of taxonomic significance.

Optical and EPR characterizations of oxygen-evolving Photosystem II subchloroplast fragments isolated from the thermophilic blue-green alga Phormidium laminosum

An oxygen-evolving, Photosystem II particle was isolated from the thermophilic, blue-green alga, Phormidium laminosum, according to the procedure of Stewart and Bendall (Stewart, A.C. and Bendall, D. (1979) FEBS Lett. 107, 308–312). Our particle has an oxygen-evolution activity of 1500–1600 μmol O 2/mg chlorophyll per h. The oxygen-evolution activity has a pH optimum at 5–6, and is abolished at pH 9. Maximum oxygen evolution occurs at approx. 47°C in whole cells, but at 29°C in the particles. The activity decreases to 50% when the cells are heated for 30 min at 55°C; with the particles, 50% inactivation occurred at 47°C for the same heating time of 30 min. Flash excitation of the particle at 100 K produced absorbance changes whose difference spectrum in the ultraviolet-to-near infrared region shows photochemical charge separation and recombination of P-680 + and Q − in the dark with t 1 2 of 1.75 ms. An EPR spectrum for the P-680 + free radical, with g 2.0027 and ΔH pp = 8 G, was constructed from flash-induced EPR changes under conditions identical to those used for obtaining P-680 absorbance changes. The actinic light-induced variable fluorescence yield is 5-fold that induced by the weak probing beam alone. Addition of dithionite to the particle brings the fluorescence to the same maximum level. Under the reducing condition, strong actinic light caused the fluorescence to decrease. This observation is consistent with the notion that variable fluorescence yield in Photosystem II originates, as in green-plant chloroplasts, from recombination luminescence, the attenuation of which corresponds to photoaccumulation of reduced pheophytin under these conditions. Broad segments (300 nm) of the difference spectrum for pheophytin photoreduction were recorded by an intensified photodiode array in conjunction with a phosphoroscopic photometer. Kinetic spectrophotometric assays together with chemical analysis showed a rather clean and simple stoichiometry in these particles, namely, 1 P-680:1 Ph:1 Q:4 Mn:44 Chl. Initial investigation failed to reveal the doublet EPR spectrum previously observed for Ph −·Q − Fe in spinach subchloroplast particles (Klimov, V.V., Dolan, E. and Ke, B. (1980). FEBS Lett. 118, 97–100). A hyperfine EPR spectrum consisting of 16–20 lines and presumably associated with the manganese clusters in the oxygen-evolving protein has been confirmed in these particles. Tris washing but not washing with EDTA eliminates this signal. Active oxygen-evolving particles also yield the II vf signal with a t 1 2 of approx. 800 μs. Upon Tris washing, the II f signal appears which decays in 23.5 ms.