Isolation and properties of plastocyanin from Anabaena variabilis

Plastocyanin is a copper protein found in photosynethetic tissue and it exhibits the properties of a physiological redox reagent. This protein has been purified from the blue-green alga Anabaena variabilis. Plastocyanin is required for a number of partial reactions of the photosynthetic electron transfer chain. These reactions include the transfer of electrons from reduced 2,3′,6-trichlorophenolindophenol,N,N,N′,N′- tetramethyl-p-phenylenediamine and 2,3,5,6-tetramethyl-p-phenylenediamine to low potential oxidants. Reduced cytochrome c photooxidation does not appear to be dependent on plastocyanin. Cytochrome f, isolated from this alga, will partially replace plastocyanin in many of these reations. Inhibition of photosynthetic reactions by copper chelators appears to occur at some site other than the site of plastocyanin function.     

Observations on Photosystem II mutants of Scenedesmus: Pigments and proteinaceous components of the chloroplasts

Nine mutants of the green alga, Scenedesmus obliquus, which are blocked in the Photosystem II portion of photosynthesis were analyzed for possible deletion or alteration of (1) various components of the photosynthetic electron transport system, (2) of chloroplast lipids, (3) of total chlorophyll or of the chlorophyll a/chlorophyllb ratio, and (4) of their content of carotenes and carotenoids. No changes in content or activity of ferredoxin, ferredoxin-NADP⁺ reductase, plastocyanin, cytochrome c-552, and the membrane-bound b-type or c-type cytochromes were observed. The most consistent differences noted between the mutant strains and the wild-type strain were in the molar ratio of chlorophyll/plastoquinone A, the total chlorophyll content, and a decreased content of - and β-carotene with a concomitant increase of carotenoids. The loss of Photosystem II activity in these mutant strains, as observed either with whole cells or with isolated chloroplast fragments, may be accounted for by their decreased content of plastoquinone A. Their decreased chlorophyll content and altered carotene/xanthophyll ratio also suggests possible alteration of chloroplast membrances resulting in increased internal oxidation of the photosynthetic pigments.     

Cytochrome b and photosynthetic sulfur bacteria

Chromatophores isolated from the purple sulfur bacterium Chromatium and the green sulfur bacterium Chlorobium exhibit absorbance changes in the cytochrome -band region consistent with the presence of a b-type cytochrome. Cytochrome content determined by reduced minus oxidized difference spectra and by heme analysis suggests that each bacterium contains one cytochrome b per molecule of photochemically active bacteriochlorophyll (reaction-center bacteriochlorophyll).

The b-type cytochrome in Chromatium has an -band maximum at 560 nm and a midpoint oxidation-reduction potential of −5 mV at pH 8.0. The b-type cytochrome in Chlorobium has an -band maximum at 564 nm and an apparent midpoint oxidation-reduction potential near −90 mV.

Chromatophores isolated from both Chromatium and Chlorobium cells catalyze a photoreduction of cytochrome b that is enhanced in the presence of antimycin A. Antimycin A and 2-n-heptyl-4-hydroxyquinoline-N-oxide inhibit endogenous (but not phenazine methosulfate-mediated) cyclic photophosphorylation in Chromatium chromatophores and non-cyclic electron flow from Na₂S to NADP in Chlorobium chromatophores. These observations suggest that b-type cytochromes may function in electron transport reactions in photosynthetic sulfur bacteria.     

The role of a cytochrome c-552-cytochrome c complex in the oxidation of sulfide in Chromatium vinosum

The sulfide:cytochrome c oxidoreductase activity of the flavocytochrome c-522 from the purple sulfur bacterium Chromatium vinosum has been investigated. The oxidized sulfur product of the sulfide:cytochrome c reductase activity has been shown to be elemental sulfur. Cytochrome c-552 has been found to form a stable complex with horse heart cytochrome c that appears to be held together by electrostatic interactions. The stability of this complex and the sulfide:cytochrome c reductase activity of cytochrome c-552 are both ionic strength dependent, with maximal rates of cytochrome c reduction and extent of complex formation occurring over the same ionic strength range. Trifluoroacetylated cytochrome c is not reduced in the presence of cytochrome c-552 and sulfide, nor does it form a complex with cytochrome c-552. These results suggest the possible involvement of cytochrome c lysine residues in complex formation. Cytochrome c-552 migrates with an anomalously high apparent molecular weight on gel filtration columns equilibrated with low ionic strength buffers, suggesting the possibility of conformational changes or dimerization of the protein. However, complexation of cytochrome c-552 with cytochrome c still occurs at low ionic strength.     

Reduction of nitrite to nitrous oxide by a cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus

A cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus reduced nitrite to nitrous oxide in a stoichiometric reaction without nitric oxide as free intermediate. The membrane system had a specific requirement for FMN with NAD(P)H as electron donors. Other electron donors were ascorbate-reduced cytochrome c-551 or phenazine methosulfate. The membrane fraction contained tightly bound cytochrome cd which represented only a small portion of the total cytochrome cd of the cell. As further terminal oxidase cytochrome o was identified. The membrane fraction produced also nitrous oxide from nitric oxide, however, at a substantially lower rate than from nitrite when using ascorbate-reduced phenazine methosulfate as electron donor.     

Lipophilicity and catalysis of photophosphorylation. II. Quinoid compounds as artificial carriers in cyclic photophosphorylation and photoreductions by Photosystem I

The topography of the chloroplast membrane has been studied using the following pairs of quinoid compounds with similar structure and chemical properties, but with different lipid solubility: phenazine/sulfophenazine, naphthoquinone/naphthoquinone sulfonate, indophenol/sulfoindophenol and lumiflavin/FMN.

All these compounds in the oxidized form are able to accept electrons from the photosynthetic electron transport chain in Hill reactions. However, only the lipophilic compounds in the reduced form can donate electrons to Photosystem I, when electron flow from Photosystem II is blocked by inhibitors. This is in agreement with the notation that the oxidizing site of Photosystem I (P₇₀₀⁺) and the electron donors for Photosystem I (cytochrome f and plastocyanin) are located inside the lipid barrier of the inner chloroplast membrane. The reducing sites in the Hill reactions must be located on the outer surface, accessible from the suspending medium.

It has been known for a long time that N,N′-tetramethyl-p-phenylenediamine can donate electrons to Photosystem I, but contrary to diaminodurene (2,3,5,6-tetramethyl phenylenediamine) it does not induce ATP formation. Both compounds are lipophilic and have similar redox potentials, but only the latter carries hydrogens which are involved in the redox reaction. For energy conservation, coupled to electon flow in Photosystem I, it therefore seems necessary that the lipophilic redox compound in the reduced form can carry hydrogens through the chloroplast membrane.     

Effects of dibromothymoquinone on oxidation-reduction reactions and the midpoint potential of the Rieske iron-sulfur center in photosynthetic electron transport of Synechococcus Sp.

The effects of 2,5-dibromo-3-methyl-p-benzoquinone (DBMIB) on the reduction kinetics of flash-oxidized P-700 and cytochrome c-553 were studied in the thermophilic cyanobacterium Synechococcus sp. (1) The reduction kinetics of P-700 showed two exponential phases with half-times of 0.2 and 2 ms at the recording time used (Nanba, M. and Katoh, S. (1983) Biochim. Biophys. Acta 725, 272–279). DBMIB strongly slowed down the 2 ms reduction phase but not the 0.2 ms phase. (2) The content of an electron donor which transfers its electrons to P-700 with the half time of 0.2 ms was estimated to be comparable to that of cytochrome f. (3) The magnitudes of the 0.2 ms reduction phase and cytochrome c-553 oxidation decreased as the flash interval was shortened below 2 s in the poisoned cells. Assuming a rapid equilibrium of electrons in the electron donor pool of Photosystem I, the midpoint potential of the 0.2 ms donor was estimated as 280 mV by comparing its percent reduction with that of cytochrome c-553 at three different flash intervals. (4) A similar value was obtained for the midpoint potential of the 0.2 ms donor in the cells in which the plastoquinone pool had been oxidized by dark starvation. It is concluded that the 0.2 ms reduction phase of P-700 is due to the electron donation from the Rieske iron-sulfur center and that DBMIB inhibits strongly but incompletely the reduction of the iron-sulfur center with electrons from the plastoquinone pool, whereas the inhibitor has no effect on the midpoint potential and Photosystem-I-dependent oxidation of the iron-sulfur center.     

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates.

It is suggested that besides the reaction sequence cytochrome 553 → plastocyanin → Photosystem I reaction center, a second pathway cytochrome 553 → Photosystem I reaction center may operate additionally.     

Localization of the reaction site of cytochrome 552 in chloroplasts from Euglena gracilis. Cytochrome content and photooxidation in different chloroplast preparations

Isolated Euglena chloroplasts retain up to 50% of cytochrome 552 on a chlorophyll basis compared to the content of cells. Cytochrome 563 is found in equal amount in chloroplasts and cells. The amount of cytochrome 552 retained depends on the isolation procedure of chloroplasts.Cytochrome 552 can be further liberated from chloroplasts by mechanical treatment or incubation with detergent. It is concluded that cytochrome 552 is not tightly bound in the membrane but rather trapped in the thylakoids of the chloroplasts.In photosynthetic electron flow, cytochrome 552 is functioning as donor for photosystem I, mediating electron flow from cytochrome 558 to P₇₀₀ under our conditions.Antimycin A stimulates the photooxidation of cytochrome 552 and of cytochrome 558.The rates of electron flow from water to NADP⁺ and of cyclic photophosphorylation mediated by phenazine methosulfate correlate with the content of endogenous cytochrome 552 in the chloroplasts. External readdition of cytochrome 552 to deficient chloroplasts causes reconstitution of NADP⁺ reduction but not of cyclic photophosphorylation. Mechanical treatment or other means of fragmentation of chloroplasts results in the exposure of originally buried reaction sites for external cytochrome 552.     

The role of plastidic cytochrome c in algal electron transport and photophosphorylation.

By an improved isolation procedure chloroplasts could be obtained from the alga Bumilleriopsis filiformis (Xanthophyceae) which exhibited high electron transport rates tightly coupled to ATP formation. Uncouplers both stimulate electron transport and inhibit photophosphorylation. These chloroplasts retain almost all soluble cytochrome c-553 besides a membrane-bound cytochrome c-554.5 (=f-554.5). Sonification or iron deficiency removed the soluble cytochrome only with a concurrent decrease of electron transport from water to methyl viologen or to NADP and decreased non-cyclic and cyclic photophosphorylation. However, photosynthetic control and the P/2e ratios remain unaltered. In Bumilleriopsis, which apparently has no plastocyanin, the soluble cytochrome c-553 seemingly links electron transport between the bound cytochrome c and P-700.     

The photochemical activities and electron carriers of developing barley leaves

The development of photochemical activities in isolated barley plastids during illumination of dark-grown plants has been studied and compared with the behaviour of plastocyanin, cytochromes f, b-559_LP, b-563 and b-559_HP and pigments P546 (C550) and P700. Electron-transport activity dependent on Photosystem 1 and cyclic photophosphorylation dependent on N-methylphenazonium methosulphate (phenazine methosulphate) were very active relative to the chlorophyll content after only a few minutes of illumination of etiolated leaves, and then rapidly declined during the first few hours of greening. By contrast, Photosystem 2 activity (measured with ferricyanide as electron acceptor) and non-cyclic photophosphorylation were not detectable during the first 2½h of greening, but then increased in total amount in parallel with chlorophyll. The behaviour of the electron carriers suggested their association with either Photosystem 1 or 2 respectively. In the first group were plastocyanin, cytochrome f and cytochrome b-563, whose concentrations in the leaf did not change during greening, and cytochrome b-559_LP whose concentration fell to one-half its original value, and in the second group were cytochrome b-559_HP and pigment P546, the concentrations of which closely followed the activities of Photosystem 2. Pigment P700 could not be detected during the first hour, during which time some other form of chlorophyll may take its place in the reaction centre of Photosystem 1. The plastids started to develop grana at about the time that Photosystem 2 activity became detectable.     

Reactions of b-cytochromes in the red alga Porphyridium aerugineum

1. 1. The kinetics of light-induced absorbance changes due to oxidation and reduction of cytochromes were measured in a suspension of intact cells of the unicellular red alga Porphyridium aerugineum. Absorbance changes in the region 540–570 nm upon alternating far-red light and darkness indicated the oxidation of cytochrome ƒ and reduction of cytochrome b₅₆₃ upon illumination. The relative efficiencies of far-red and orange light indicated that both reactions were driven by Photosystem I.

2. 2. Experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), with anaerobic cells and in alternating far-red and orange light indicated that cytochrome b₅₆₃ reacts in a cyclic chain around Photosystem I, and that the reduced cytochrome does not react with oxygen or with another oxidized product of Photosystem II. The quantum requirement for the photoreduction was about 6 quanta/equiv at 700 nm. A low concentration of N-methylphenazonium methosulphate (PMS) enhanced the rate of reoxidation of cytochrome b₅₆₃ in the dark. In the presence of higher concentrations of PMS a photooxidation, driven by Photosystem I, instead of reduction was observed. These observations suggest that PMS enhances the rate of reactions between reduced cytochrome b₅₆₃ and oxidized products of Photosystem I.

3. 3. In the presence of carbonylcyanide m-chlorophenylhydrazone (CCCP) a light-induced decrease of absorption at 560 nm occurred. Spectral evidence suggested the photooxidation of cytochrome b₅₅₉ under these conditions. Inhibition by DCMU and a relatively efficient action of orange light suggested that this photooxidation is driven by Photosystem II.

Iron protein content of Thiocapsa pfennigii, a purple sulfur bacterium of atypical chlorophyll composition

Four out of five soluble electron transport iron proteins of Thiocapsa pfennigii plus the particulate cytochromes have been found to be analogous to those of Chromatium vinosum strain D. In addition to ferredoxin, high potential iron-sulfur protein. cytochrome c′, and cytochrome c-552(550), T. pfennigii contains a cytochrome c-552(545) not previously isolated from photosynthetic bacteria. It is concluded that T. pfennigii is more closely related to C. vinosum than to Rhodopseudomonas viridis, the only other known bacterial species having bacteriochlorophyll b.     

Photochemical properties of a Photosystem II subchloroplast fragment

1. The Photosystem II fraction (D-10) obtained by incubation of spinach chloroplasts with digitonin was further purified by incubation with Triton X-100. The resulting Photosystem II subchloroplast fragment (DT-10) contained 1 mole of cytochrome b-559 per 170 moles of chlorophyll. It lacked cytochrome f and cytochrome b₆ and its content of P700 was low.

2. The DT-10 fragment showed only traces of photochemical activity with water as electron donor, but it was active in a Photosystem II reaction with 2,6-dichlorophenolindophenol as electron acceptor and diphenyl carbazide as donor. Photoreduction of NADP⁺ with diphenyl carbazide as donor was negligible. There was some photoreduction of NADP⁺ with ascorbate plus 2,6 dichlorophenolindophenol as donor but this activity could be accounted for by contamination with Photosystem I. These results are consistent with the Z-scheme of photosynthesis with Photosystems I and II operating in series for the reduction of NADP⁺ from water. DT-10 subchloroplast fragments showed a light-induced rise in fluorescence yield at 20 °C in the presence of diphenyl carbazide. A light-induced fluorescence increase also was observed at 77 °K.

3. During the preparation of the DT-10 fragment, the high potential form of cytochrome b-559 was largely converted to a form of lower potential and C-550 was converted to the reduced state. A photoreduction of C-550 was observed at liquidnitrogen temperature, provided the C-550 was oxidised with ferricyanide prior to cooling. Some photooxidation of cytochrome b-559 was obtained at 77 °K if the preparation was reduced prior to cooling, but the degree of photooxidation was variable with different preparations. C-550 does not appear to be identical with the primary fluorescence quencher, Q.

4. Photosystem I subchloroplast fragments (D-144) released by the action of digitonin were compared with Photosystem I fragments (DT-144) released from D-10 fragments by Triton X-100. There were no significant differences between D-144 and DT-144 fragments either in chlorophyll a/b ratio or in P700 content.     

An NAD(P) reductase derived from Chlorobium thiosulfa-tophilum: Purification and some properties

A highly purified preparation of an NAD(P) reductase was obtained from Chlorobium thiosulfatophilum and some of its properties were studied. The enzyme possesses FAD as the prosthetic group, and reduces benzyl viologen, 2,6-dichloro-phenolindophenol and cytochromes c, including cytochrome c-555 (C. thiosulfato-philum), with NADPH or NADH as the electron donor. It reduces NADP⁺ or NAD⁺ photosynthetically with spinach chloroplasts in the presence of added spinach ferredoxin. It reduces the pyridine nucleotides with reduced benzyl viologen. The enzyme also shows a pyridine nucleotide transhydrogenase activity. In these reactions, the type of pyridine nucleotide (NADP or NAD) which functions more efficiently with the enzyme varies with the concentration of the nucleotide used; at concentrations lower than approx. 1.0 mM, NADPH (or NADP⁺) is better electron donor (or acceptor), while NADH (or NAD⁺) is a better electron donor (or acceptor) at concentrations higher than approx. 1.0 mM. Reduction of dyes or cytochromes c catalysed by the enzyme is strongly inhibited by NADP⁺, 2′-AMP and and atebrin.     

Localization of the reaction site of cytochrome 552 in chloroplasts from Euglena gracilis. Effects of a specific antibody on endogenous, external and reincorporated cytochrome 552 in chloroplast membranes

Euglena chloroplasts, isolated by Yeda press treatment contain endogenous cytochrome 552. Antibodies against cytochrome 552 from Euglena gracilis do not agglutinate chloroplasts and do not inhibit photosynthetic electron flow from water to NADP⁺. There is also no influence on cyclic photophosphorylation with phenazine methosulfate as mediator and on photooxidation of endogenous cytochrome 552. However, in the presence of cholate the photooxidation of the cytochrome is inhibited by antibodies.Cyclic photophosphorylation is not restored by addition of cytochrome 552 to the assay mixture but is stimulated by trapping the cytochrome in the thylakoid vesicles during sonication.Trapped cytochrome 552 is not accessible to antibodies. It is concluded that the original site of action for endogenous cytochrome 552 is inside the thylakoids. This site can be dislocated to the outside during fragmentation of chloroplasts.     

On the site of action of plastocyanin in isolated chloroplasts

Adding plastocyanin to plastocyanin-depleted chloroplast particles, restored both their ability to catalyse the photoinduced electron transfer from ascorbate-DCIP to NADP, and to induce the photooxidation of cytochrome f. It is concluded, therefore, that plastocyanin mediates the photoinduced oxidation of cytochrome f, as previously suggested.     

The interaction of mucidin with anaerobically grown cells of Paracoccus denitrificans

Mucidin similar to antimycin inhibits the electron flow to cytochrome c and the enzyme activities dependent on cytochrome c reduction in the cells of Paracoccus denitrificans, but it does not inhibit the electron flow to nitrate reductase and cytochrome o. Unlike antimycin mucidin does not permit a residual electron flow through the cytochrome bc₁ region. In the presence of antimycin the electron flow to nitrate is lower than in using mucidin in contrast with a higher extent of cytochrome b reduction. This result is in contradiction to the participation of the constitutive cytochrome b as an electron donor in the nitrate reduction.     

Single and multiple turnover reactions in the ubiquinone-cytochrome b-c2 oxidoreductase of Rhodopseudomonas sphaeroides. The physical chemistry of the major electron donor to cytochrome c2, and its coupled reactions

We have examined the thermodynamic properties of the physiological electron donor to ferricytochrome c₂ in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides. This donor (Z), which is capable of reducing the ferri-cytochrome with a halftime of 1–2 ms under optimal conditions, has an oxidation-reduction midpoint potential of close to 150 mV at pH 7.0, and apparently requires two electrons and two protons for its equilibrium reduction.

The state of reduction of Z, which may be a quinone · protein complex near the inner (cytochrome c₂) side of the membrane, appears to govern the rate at which the cyclic photosynthetic electron transport system can operate. If Z is oxidized prior to the flash-oxidation of cytochrome c₂, the re-reduction of the cytochrome takes hundreds of milliseconds and no third phase of the carotenoid bandshift occurs. In contrast if Z is reduced before flash activation, the cytochrome is rereduced within milliseconds and the third phase of the carotenoid bandshift occurs. The prior reduction of Z also has a dramatic effect on the uncoupler sensitivity of the rate of electron flow; if it is oxidized prior to activation, uncoupler can stimulate the cytochrome re-reduction after several turnovers by less than tenfold, but if it is reduced prior to activation, the stimulation after several turnovers can be as dramatic as a thousandfold. The results suggest that Z plays a central role in controlling electron and proton movements in the ubiquinone cytochrome b-c₂ oxido-reductase.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.