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PubMed1980

Reduction of oxygen-pulsed cytochrome c oxidase by cytochrome c and other electron donors.

1. Stopped-flow experiments were performed in which solutions containing dithionite were mixed with air-saturated buffer. Cytochrome c oxidase present in the dithionite-containing syringe is fully oxidized within the mixing time and the oxygen-pulsed form of the oxidase is produced. 2. The reduction of this form by dithionite, by dithionite plus cytochrome c and by dithionite plus methyl viologen or benzyl viologen was followed and compared with the corresponding reduction reactions of the "resting" oxidized enzyme. Reduction by dithionite is relatively slow, but the rate of reduction is greatly increased by addition of cytochrome c or the viologens, which are even more effective than cytochrome c on a molar basis. 3. Profound differences between the transient kinetics of the reduction of the two oxidized oxidase derivatives were observed. The results are consistent with a direct reduction of cytochrome a followed by an intramolecular electron transfer to cytochrome a3 (k1obs = 7.5 s-1 for the oxygen-pulsed oxidase). 4. The spectrum of the oxygen-pulsed oxidase formed within 5 ms of the mixing closely resembles that of the "oxygenated" compound, but there were small differences between the two spectra.

Animals↗
PubMed1985

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.

Aerobiosis↗
PubMed1994

The use of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-labeled lipids in determining transmembrane lipid distribution.

Transbilayer lipid distribution of small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) was measured using 31P-nuclear magnetic resonance (NMR) spectroscopy, chemical modification with 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dithionite reduction of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-labeled lipid (NBD-lipid). The dithionite assay was the most reproducible of the three assays, with 1.2% error for SUVs and 3.9% error for LUVs. The dithionite assay also agreed best with theoretical inner:outer leaflet ratios, based on vesicle diameters determined by electron microscopy (Thomas et al. (1989) Biochem. Biophys. Acta 978, 85-90). Dithionite assay measurements were within 2.7% of theoretical ratios for SUVs and 2.3% for LUVs, while the NMR assay for SUVs was 14% lower than theoretical ratios and 23% lower for LUVs. The accuracy of NBD-lipids as markers for total transbilayer lipid was investigated. NBD-labeled phosphatidylserine, phosphatidylcholine and phosphatidylglycerol were accurate markers for total transbilayer lipid distribution, as their distributions were in close agreement with theoretical ratios. However, NBD-labeled phosphatidylethanolamine displayed a slight preference for the inner leaflet at low mole fractions of phosphatidylethanolamine, while native phosphatidylethanolamine showed a preference for the outer leaflet at the same concentration. NBD-labeled phosphatidic acid also showed a slight preference for the inner leaflet. We conclude that although dithionite-based assessment of NBD-labeled lipids across membrane bilayers can be a powerful analytical tool, caution must be used in the interpretation of results.

4-Chloro-7-nitrobenzofurazan↗
PubMed1974

The release of iron from horse spleen ferritin by reduced flavins.

Ferritin-Fe(III) was rapidly and quantitatively reduced and liberated as Fe(II) by FMNH(2), FADH(2) and reduced riboflavin. Dithionite also released Fe(II) from ferritin but at less than 1% of the rate with FMNH(2). Cysteine, glutathione and ascorbate gave a similar slower rate and yielded less than 20% of the total iron from ferritin within a few hours. The reduction of ferritin-Fe(III) by the three riboflavin compounds gave complex second-order kinetics with overlapping fast and slow reactions. The fast reaction appeared to be non-specific and may be due to a reduction of Fe(III) of a lower degree of polymerization, equilibrated with ferritin iron. The amount of this Fe(3+) ion initially reduced was small, less than 0.3% of the total iron. Addition of FMN to the ferritin-dithionite system enhanced the reduction; this is due to the reduction of FMN by dithionite to form FMNH(2) which then reduces ferritin-Fe(III). A comparison of the thermodynamic parameters of FMNH(2)-ferritin and dithionite-ferritin complex formation showed that FMNH(2) required a lower activation energy and a negative entropy change, whereas dithionite required 50% more activation energy and showed a positive entropy change in ferritin reduction. The effectiveness of FMNH(2) in ferritin-Fe(III) reduction may be due to a specific binding of the riboflavin moiety to the protein portion of the ferritin molecule.

Animals↗
PubMed1990

An analysis of the reaction kinetics of the hexahaem nitrite reductase of the anaerobic rumen bacterium Wolinella succinogenes.

The reduction kinetics of both the resting and redox-cycled forms of the nitrite reductase from the anaerobic rumen bacterium Wolinella succinogenes were studied by stopped-flow reaction techniques. Single-turnover reduction of the enzyme by dithionite occurs in two kinetic phases for both forms of the enzyme. When the resting form of the enzyme is subjected to a single-turnover reduction by dithionite, the slower of the two kinetic phases exhibits a hyperbolic dependence of the rate constant on the square root of the reductant concentration, the limiting value of which (approximately 4 s-1) is assigned to a slow internal electron-transfer process. In contrast, when the redox-cycled form of the enzyme is reduced by dithionite in a single-turnover experiment, both kinetic phases exhibit linear dependences of the rate on the square root of dithionite concentration, with associated rate constants of 150 M-1/2.s-1 and 6 M-1/2.s-1. Computer simulations of both the reduction processes shows that no unique set of rate constants can account for the kinetics of both forms, although the kinetics of the redox-cycled species is consistent with a much enhanced rate of internal electron transfer. Under turnover conditions the time course for reduction of the enzyme, in the presence of millimolar levels of nitrite and 100 mM-dithionite, is extremely complex. A working model for the mechanism of the turnover activity of the enzyme is proposed which very closely describes the reaction kinetics over a wide range of substrate concentrations, as shown by computer simulation. The similarity in the action of the nitrite reductase enzyme and mammalian cytochrome c oxidase is commented upon.

Anaerobiosis↗
PubMed1976

Cooperativity in the dissociation of nitric oxide from hemoglobin.

The dissociation of nitric oxide from hemoglobin, from isolated subunits of hemoglobin, and from myoglobin has been studied using dithionite to remove free nitric oxide. The reduction of nitric oxide by dithionite has a rate of 1.4 X 10(3) M-1 S-1 at 20 degrees in 0.05 M phosphate, pH 7.0, which is small compared with the rate of recombination of hemoglobin with nitric oxide (25 X 10(6) M-1 S-1 (Cassoly, R., and Gibson, Q. H. (1975) J. Mol. Biol. 91, 301-313). The rate of NO combination with chains and myoglobin was found to be 24 X 10(6) M-1 S-1 and 17 X 10(6) M-1 S-1, respectively. Hence, the observed progress curve of the dissociation of nitric oxide is dependent upon the dithionite concentration and the total heme concentration. Addition of excess carbon monoxide to the dissociation mixture reduces the free heme yielding a single exponential process for chains and for myoglobin which is dithionite and heme concentration independent over a wide range of concentrations. The rates of dissociation of nitric oxide from alpha chains, from beta chains, and from myoglobin are 4.6 X 10(-5) S-1, 2.2 X 10(-5) S-1, and 1.2 X 10(4) S-1, respectively, both in the presence and in the absence of carbon monoxide at 20 degrees in 0.05 M phosphate, pH 7.0. Analogous heme and dithionite concentration dependence is found for the dissociation of nitric oxide from tetrameric hemoglobin. The reaction is cooperative, the intrinsic rate constants for the dissociation of the 1st and 4th molecules of NO differing about 100-fold. With hemoglobin, replacement of NO by CO at neutral pH is biphasic in phosphate buffers. The rate of the slow phase is 1 X 10(-5) S-1 and is independent of pH. The amplitude of the fast phase increases with lowering of pH. By analogy with the treatment of the HbCO + NO reaction given by Salhany et al. (Salhany, J.M., Ogawa, S., and Shulman, R.G. (1975) Biochemistry 14, 2180-2190), the fast phase is attributed to the dissociation of NO from T state molecules and the slow phase to dissociation from R state molecules. Analysis of the data gives a pH-independent value of 0.01 for the allosteric constant c (c = Kr/Kt where Kr and Kt are the dissociation constants for NO from the R and T states, respectively) and pH-dependent values of L (2.5 X 10(7) at pH 7 in 0.05 M phosphate buffer). The value of c is considerably greater than that for O2 and CO. Studies of the difference spectrum induced in the Soret region by inositol hexaphosphate are also reported. This spectrum does not arise directly from the change of conformation between R and T states. The results show that if the equilibrium binding curve for NO could be determined experimentally, it would show cooperativity with Hill's n at 50% saturation of about 1.6.

Binding Sites↗
PubMed1994

Investigations of ascorbate for direct labeling of antibodies with technetium-99m.

UNLABELLED: Recently, a method for the direct labeling of antibodies with 99mTc was described in which sulfhydryls were reportedly generated by reduction of antibody disulfides with ascorbic acid. Thereafter, these proteins may be labeled at high efficiency with 99mTc following reduction of pertechnetate with dithionite. This investigation was initially conducted to evaluate the mechanism of the increased stability towards cysteine challenge reported for the label and subsequently to determine the role of ascorbate in the labeling process. METHODS: It was possible to reproduce the reported high labeling efficiencies by increasing the dithionite concentration fivefold, presumably because of variabilities among lots of commercial sodium dithionite. RESULTS: Despite success in labeling, it was not possible to confirm that antibody reduction followed the treatment with ascorbate. Using both Ellman's reagent and 2,2' dithiodipyridine as indicators, we were unable to detect sulfhydryls on one IgG antibody treated at ten times the suggested ascorbate-to-antibody molar ratio. It was estimated that the number of sulfhydryls generated could not have been more than 1% (dithiodipyridine) to 2% (Ellman's). Furthermore, radiolabeling efficiencies for two IgG antibodies and stabilities of the label to cysteine challenge were unchanged when the ascorbate was eliminated. The number of sulfhydryls generated by treatment of the antibody with dithionite at 1-2 times the concentration required for adequate labeling was about 1% (dithiodipyridine) to 5% (Ellman's). CONCLUSION: For the conditions of this investigation and for the antibodies employed, ascorbate apparently played no more than a minor role at best in the labeling process. If antibody reduction occurred, this most likely was a result of residual dithionite presented to the protein along with the reduced 99mTc.

Antibodies↗
PubMed1993

Slow ('resting') forms of mitochondrial cytochrome c oxidase consist of two kinetically distinct conformations of the binuclear CuB/a3 centre--relevance to the mechanism of proton translocation.

We have purified slow ('resting') cytochrome oxidase from bovine heart, free of contamination with fast ('pulsed') enzyme. This form of the enzyme shows two kinetic phases of reduction of haem a3 by dithionite (k = 0.020 +/- 0.005 s-1 and k = 0.005 +/- 0.002 s-1). The presence of ligands that bind to the oxidized or reduced binuclear centre (formate or carbon monoxide respectively) has no effect on these rates. Varying the dithionite concentration also has no effect on either phase, although at low dithionite concentrations a lag phase is observed as the rate of haem a reduction is slower. The results are consistent with a model for reduction of the slow enzyme where the rate of electron transfer to the binuclear centre is the limiting step, rather than an equilibrium model where the haem a3 redox potential is low. Increasing the pH decreases the rate of the slower phase of dithionite reduction, but has no effect on the faster phase. EPR studies show that the slow phase (only) correlates with the disappearance of the g' = 12/g' = 2.95 signals, with the same pH dependence; again the presence of formate has no effect on these results. Deconvolution of the oxidized optical spectra shows that the enzyme reduced in the slow phase has a blue-shifted Soret band, relative to that reduced in the faster phase. Incubation of the oxidized enzyme at high pH causes a line-broadening of both the g' = 12 and g' = 2.95 EPR signals with no obvious effect on the amount of signal. The results are interpreted in a model where the presence of a carboxylate bridge between haem a3 and CuB defines the slow enzyme. It is suggested that the two rates of dithionite reduction are the result of different ligation to CuB--where water is the ligand the binuclear centre is FeIV/CuI (EPR-silent) and where hydroxide is the ligand the binuclear centre is FeIII/CuII (g' = 12/g' = 2.95 EPR signals).

Animals↗
PubMed1995

Reactivity of carbon monoxide dehydrogenase from Rhodospirillum rubrum with carbon dioxide, carbonyl sulfide, and carbon disulfide.

The reactivities of CO2 and the related compounds COS and CS2 with the nickel- and iron- sulfur-containing carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum have been investigated. Both CO2 and COS were substrates for CODH in a reductant-dependent reaction resulting in the formation of CO. CO2 was reduced to CO and H2O, while COS was reduced to CO and H2S. CO was a potent inhibitor of CO2 reduction at dissolved concentrations as low as 1 microM, but this inhibition could be prevented by quantitatively trapping CO as it was formed by including reduced hemoglobin in the assays. The addition of hemoglobin to the assays also allowed the formation of CO to be monitored in real time by following the decrease in absorbance at 433 nm resulting from carboxyhemoglobin formation. A variety of low-potential reductants, including dithionite, titanium(III) citrate, and dithionite-reduced viologens (methyl and benzyl), were suitable electron donors for the reduction of CO2 and COS. Dithionite-reduced methyl viologen supported the highest rates of CO2 and COS reduction, and the stimulation of CO2 reduction (170-fold increased rate over dithionite alone) was much more dramatic than the stimulation of COS reduction (2.6-fold increased rate over dithionite alone). CO2 was reduced to CO with a Km for CO2 of 190 microM and a Vmax of 44 mumol of CO formed min-1 (mg of protein)-1, while COS was reduced with a Km for COS of 2.2 microM and a Vmax of 0.51 mumol of CO formed min-1 (mg of protein)-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Oxidoreductases↗
PubMed1971

Properties of nonheme iron in a cell envelope fraction from Escherichia coli.

The reaction with o-phenanthroline of nonheme iron found in cell envelope fractions from Escherichia coli has been investigated. About 20% of the total nonheme iron reacts directly with o-phenanthroline. This iron appears to be in the ferric state but is reducible by protein sulfhydryl groups in the presence of the chelating agent. A further 20% of the nonheme iron will react with o-phenanthroline only in the presence of dithionite. Succinate can replace dithionite but only produces about 35% of the reaction given by dithionite. The reduction of cytochrome b(1) of the respiratory chain by succinate shows similar behavior to the reaction of iron with o-phenanthroline in the presence of succinate. Both of these components react completely only in the presence of 2-heptyl-4-hydroxyquinoline-N-oxide. The remaining 60% of the nonheme iron of the cell envelope will not react with o-phenanthroline even in the presence of dithionite or 6 m urea. Triton X-100 with dithionite will permit a small part (10%) of this iron to react with o-phenanthroline. The iron which does not react with o-phenanthroline is not associated with succinate, reduced nicotinamide adenine dinucleotide, or d-lactate dehydrogenases.

Cell Wall↗
PubMed1988

The measurement of cytochrome b559 in polymorphonuclear leukocytes and macrophages in the presence of hemoglobin or mitochondrial cytochromes.

A cyanide-insensitive b-type cytochrome (cytochrome b559) is involved in the respiratory burst of phagocytes. Spectroscopic measurement of its content in polymorphonuclear leukocytes or macrophages is complicated by signals arising from contaminating Hb or mitochondrial cytochromes upon reduction by dithionite. These interferences can be overcome by reducing the sample with dithionite, aerating it, and reducing it a second time with dithionite, in the presence of 10 microM cyanide. Cyanide suppresses the response of the interfering proteins to the second dithionite reduction. This modified method allows an accurate determination of the content of cytochrome b559 using smaller numbers of cells than previously feasible.

Animals↗
PubMed1978

Variable chlorophyll a fluorescence from P-700 enriched photosystem I particles dependent on the redox state of the reaction centre.

1. Photosystem I particles enriched in P-700 prepared by Triton X-100 treatment of chloroplasts show a light-induced increase in fluorescence yield of more than 100% in the presence of dithionite but not in its absence. 2. Steady state light maintains the P-700, of these particles, in the oxidised state when ascorbate is present but in the presence of dithionite only a transient oxidation occurs. 3 EPR data show that, in these particles, the primary electron acceptor (X) is maintained in the reduced state by light at room temperature only when the dithionite is also present. In contrast, the secondary electron acceptors are reduced in the dark by dithionite. 4. Fluorescence emission and excitation spectra and fluorescence lifetime measurements for the constant and variable fluorescence indicate a heterogeneity of the chlorophyll in these particles. 5. It is concluded that the variable fluorescence comes from those chlorophylls which can transfer their energy to the reaction centre and that the states PX and P+X are more effective quenchers of chlorophyll fluorescence than PX-, where P is P-700.

Chlorophyll↗
PubMed1988

Activation by reduction of the resting form of cytochrome c oxidase: tests of different models and evidence for the involvement of CuB.

(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 a3 appears at a rate (kobs) of 0.016 s-1. Ferricytochrome a3 maintains its 418 nm Soret maximum until reduced. The rate of a3 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 a3/cyanide complex occurs at the same rate as a3 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 a3 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 a3 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 a3. 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 (kobs approximately 0.02 s) entry of a third electron.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

A comparison of iron extraction methods for the determination of degree of pyritisation and the recognition of iron-limited pyrite formation.

Measurements of degree of pyritisation require an estimate of sediment iron which is capable of reaction with dissolved sulphide to form pyrite, either directly or indirectly via iron monosulphide precursors. Three dissolution techniques (buffered dithionite, cold 1 M HCl, boiling 12 M HCl) were examined for their capacity to extract iron from a variety of iron minerals, and iron-bearing sediments, as a function of different extraction times and different grain sizes. All the iron oxides studied are quantitatively extracted by dithionite and boiling HCl (but not by cold HCl). Both HCl techniques extract more iron from silicates than does dithionite but probably about the same amounts as are potentially capable of sulphidation. Modern sediment studies indicate that most sedimentary pyrite is formed rapidly from iron oxides, with smaller amounts formed more slowly from iron silicates (if sufficient geologic time is available). It is therefore recommended that the degree of pyritisation be defined with respect to the dithionite-extractable (mainly iron oxide) pool and/or the boiling HCl-extractable pool (which includes some silicate iron) for the recognition of iron-limited pyritisation.

Dithionite↗
PubMed1995

An improved assay for measuring the transverse redistribution of fluorescent phospholipids in plasma membranes.

The internalization of fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-phospholipids from the plasma membrane can be assessed by the irreversible quenching of analogues in the outer leaflet by dithionite. Here we have utilized this assay to follow the redistribution of short-chain C6-NBD-sphingomyelin and C6-NBD-phosphatidylserine from the cell membrane of human gingiva fibroblasts. The significant uptake of dithionite across the plasma membrane and the subsequent reduction of NBD-analogues exposed to the cytoplasmic lumen does not allow an accurate measurement of the amount of internalized lipid probes even at low temperature. We could show that a precise determination can be achieved by extraction of analogues remaining in the exoplasmic half by a short pretreatment with bovine serum albumin prior to addition of dithionite. The fluorescence of those analogues localized to the cytoplasmic lumen was slowly destroyed by permeating dithionite. The fluorescence of those NBD-probes which are localized in the inner layer of intracellular vesicles remained almost unaffected in the time course of the assay. Thus, this approach allows to distinguish between different routes of internalization of NBD-phospholipids from the plasma membrane.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

Organization of clusters and internal electron pathways in CO dehydrogenase from Clostridium thermoaceticum: relevance to the mechanism of catalysis and cyanide inhibition.

Cyanide inhibits the CO oxidation activity of carbon monoxide dehydrogenase from Clostridium thermoaceticum by binding tightly to the form of the C-cluster yielding the gav = 1.82 signal (the C1.82 form). CN- dissociates and the enzyme reactivates upon addition of CO, CO2 plus dithionite, or CS2 plus dithionite. Dithionite slows the inhibition of the enzyme by CN-, but it cannot reactivate the enzyme. This behavior is explained by assuming that binding of CO, CO2, or CS2 at a modulator site accelerates the dissociation of CN- from the C-cluster. With CN- bound at the C-cluster, dithionite, but not CO, can reduce those Fe-S clusters in the enzyme whose redox status can be monitored at 420 nm. The electron pathway used for CO oxidation appears to be as follows: C-cluster-->Fe-S Clusters-->external electron acceptors. The electron used to reduce the NiFe complex originates predominantly from the C-cluster, and this reduction is inhibited when CN- is bound at the C-cluster. The NiFe complex is reduced more slowly (in the absence of CN-) than CO is catalytically oxidized, indicating that this reduction is not part of the catalytic mechanism for CO oxidation. The form of the C-cluster yielding the g(av) = 1.86 signal (C1.86) is proposed to be two electrons more reduced than C1.82 and able to bind and reduce CO2. CO is proposed to be oxidized by C1.82. Neither CO or CN- appears to bind C1.86.

Aldehyde Oxidoreductases↗
PubMed2006

Mössbauer and EPR study of recombinant acetyl-CoA synthase from Moorella thermoacetica.

Mössbauer and EPR spectroscopies were used to study the electronic structure of the A-cluster from recombinant acetyl-CoA synthase (the alpha subunit of the alpha2beta2 acetyl-CoA synthase/CO dehydrogenase). Once activated with Ni, these subunits have properties mimicking those associated with the alpha2beta2 tetramer, including structural heterogeneities. The Fe4S4 portion of the A-cluster in oxidized, methylated, and acetylated states was in the 2+ core oxidation state. Upon reduction with dithionite or Ti3+ citrate, samples of Ni-activated alpha developed the ability to accept a methyl group. Corresponding Mössbauer spectra exhibited two populations of A-clusters; roughly, 70% contained [Fe4S4]1+ cubanes, while approximately 30% contained [Fe4S4]2+ cubanes, suggesting an extremely low [Fe4S4](1+/2+) reduction potential for the 30% portion (perhaps <-800 mV vs NHE). The same population ratio was observed when Ni-free unactivated alpha was used. The 70% fraction exhibited paramagnetic hyperfine structure in the absence of an applied magnetic field, excluding the possibility that it represents an [Fe4S4]1+ cluster coupled to a (proximal) Ni(p)1+. EPR spectra of dithionite-reduced, Ni-activated alpha exhibited features at g = 5.8 and g(ave) approximately 1.93, consistent with a physical mixture of {S = 3/2; S = 1/2} spin-states for A-clusters containing [Fe4S4]1+ clusters. Incubation of Ni-activated alpha with dithionite and CO converted 25% of alpha subunits into the S = 1/2 A(red)-CO state. Previous correlation of this state to functional A-clusters suggests that only the 30% fraction not reduced by dithionite or Ti3+ citrate represents functional A-clusters. Comparison of spin states in oxidized and methylated states suggests that two electrons are required for reductive activation, starting from the oxidized state containing Ni(p)2+. Refitting published activity-vs-potential data supports an n = 2 reductive activation. Enzyme starting in the methylated state exhibited catalytic activity in the absence of an external reductant, suggesting that the two electrons used in reductive activation are retained by the enzyme after each catalytic cycle and that the enzyme does not have to pass through the A(red)-CO state during catalysis. Taken together, our results suggest that a Ni(p)0 state may form upon reductive activation and reform after each catalytic cycle.

Acetate-CoA Ligase↗
PubMed2002

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.

Chloroplasts↗