Photooxidation Of Cytochrome Research Articles

Bipyridylium herbicide toxicity in an isolated lung cell culture system

An investigation has been made into the effect of low concentrations of 2,6-dichlorophenolindophenol (DCIP) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), in the presence of ascorbate, on light-induced absorbance changes in the α-band region of cytochrome f and b-type cytochromes. Strong photo-oxidation of cytochrome f occurred with DCIP, in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (2 μM), but not with TMPD. However, with TMPD a large light-induced absorbance increase occurred due to a broad band centred at 566 nm. With DCIP, the uncoupler, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), in conjunction with the electron acceptor 1,1′-ethylene-2,2′-bipyridylium dibromide (diquat), inhibited the cytochrome f response and an absorbance increase in the 560–575 nm region occurred similar to that with TMPD. Neither FCCP nor diquat had any great effect on the TMPD system. The results support a pathway of electron transport between the two photosystems in which (a) DCIP-ascorbate interacts with an intermediate on the Photosystem II side of a coupling site; (b) TMPD-ascorbate interacts after this site, and (c) cytochrome f is located on the Photosystem I side of the site.

Mechanism of the light state transition in photosynthesis. I. Analysis of the kinetics of cytochrome f oxidation in State 1 and State 2 in the red alga, Porphyridium cruentum

The kinetics of photooxidation and reduction of cytochrome f were examined spectrophotometrically in the red alga Porphyridium cruentum in light State 1 and light State 2. Experiments were performed on intact cells that had been chemically fixed and stabilized in the light states. The cytochrome f turnover was measured during conditions of linear electron transport driven by both photosystems and during several cyclic reactions mediated by the long-wavelength Photosystem (PS) I. The data show that the rate of photooxidation of cytochrome f increased in State 2 when the cells were activated by subsaturating intensities of green light absorbed primarily by the phycobilisome. No differences in kinetics were found between algae in State 1 or State 2 when they were activated by light absorbed primarily by the chlorophyll of PS I. The results confirm that changes in energy distribution between the two photosystems occur as a result of the light state transition and verify that the redistribution of excitation results in the predicted changes in electron transport.

Functional subunit structure of photosystem 1 reaction center in Synechococcus sp

Photochemical activities of six different P700-chlorophyll a-proteins (CP1-a, -b 1, -b 2, -c, -d, and -e) separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from digitonin particles of a thermophilic cyanobacterium Synechococcus sp. were examined. CP1-a, -b 1, -b 2, and -c contain the competent reaction center of photosystem 1: They were highly active in photooxidation of cytochrome c-553, the physiological electron donor to P700 in the organism, with methyl viologen as electron acceptor and showed flash-induced absorption changes indicating the charge separation between P700 and the secondary electron acceptors, P430 and A 2. The cytochrome photooxidation and P430 and A 2 photoresponses were significantly suppressed in CP1-d. CP1-e which lacks P430 and A 2 was least active in the cytochrome photooxidation. A 1, the primary electron acceptor of P700, is present in CP1-e as well as in other CP1 complexes. Comparison of the results with the polypeptide composition of CP1 complexes ( Y. Takahashi, H. Koike, and S. Katoh, 1982, Arch. Biochem. Biophys. 219, 209–218 ). indicates that CP1-c which contains four polypeptides with molecular weights of 62,000, 60,000, 14,000, and 10,000 represents the functional core of the photosystem 1 reaction center. P700, A 1, and antenna chlorophyll are associated with 62,000- and 60,000-dalton polypeptides, whereas 14,000- and 10,000-dalton polypeptides are assumed to carry P430 and A 2. The 13,000-dalton polypeptide which is associated with CP1-a, -b 1, and -b 2 is not required for the functioning of the reaction center.

A cytochrome f- b6 complex with plastoquinol-cytochrome c oxidoreductase activity from Anabaena variabilis

A cytochrome f- b 6 complex has been isolated from thylakoid membranes of Anabaena variabilis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of four major polypeptides - cytochrome f (molecular mass 31 or 38 kDa, depending on sample preparation for the electrophoresis), cytochrome b 6 (molecular mass 22.5 kDa), a band with slightly lower mobility than cytochrome b 6 resolved only in gels containing urea, and a 16.5 kDa polypeptide, - plus two small polypeptides below 10 kDa. The complex is devoid of chlorophyll and carotenoids, contains 2 cytochromes b 6 per cytochrome f, but no cytochrome b-559. The Rieske Fe-S center is present in substoichiometric amounts of cytochrome f. The complex is active in plastoquinol-cytochrome c oxidoreductase activity, which is specific for electron-accepting proteins with positive net charge at physiological pH. Cytochrome c from horse heart, cytochrome c-553 and plastocyanin, both from A. variabilis, are equally well reduced. The specificity for the electron-donating quinol is less stringent. The sensitivity of the oxidoreductase to various inhibitors is retained during the isolation procedure, 2,5-dibromo-3-methyl-isopropyl- p-benzoquinone being the most efficient. The complex exhibits oxidant-induced reduction of cytochrome b 6, and photooxidation of cytochrome f as well as transient photoreduction of cytochrome b 6 when illuminated in the presence of plastoquinol, plastocyanin and Photosystem I reaction centers.

In vivo Changes in Chloroplast ThylakoidMembrane Activity during Viable and Non-viable Dehydration of a Drought-tolerant Plant, Borya nitida

Expanded leaves of Borya nitida Labill. were detached and placed in atmospheres of 100, 96, 88, 53 or 13% relative humidity at 25°C. In vivo chlorophyll fluorescence induction kinetics and the photooxidation of cytochrome f were measured before and after dehydration and again after rehydration by floating on water. In contrast to slow dehydration at 96% R.H., which resulted in an initial stimulation of photosystem II activity, the slow development of a partial block on the photoreducing side of photosystem II and the eventual degreening of leaves to a yellow viable condition, fast dehydration at 13% R.H. induced rapid loss of photosystem I activity and a slower development of a block on the photooxidizing side of photosystem . Although the leaves remained green, photo- system activity did not recover upon rehydration. Charge separation in the photosystem II complex still took place in the dehydrated leaves but upon rehydration the photosystem II reaction centre appeared to be disassociated from the light harvesting system. Dehydration at intermediate relative humidities (88 and 53%) led to a partial block on the photoreducing side of photosystem II and, more slowly, inhibition of photosynthetic electron transfer on the photooxidizing side of photosystem II. Following rehydration there was partial recovery of reactions associated with photosystem I but the leaves were unable to return to their original condition.

Melittin: An inhibitor of chloroplast photochemical reactions

Melittin, a polypeptide component of bee venom, is an inhibitor of photochemical reactions in chloroplasts isolated from higher plants. At concentrations around 5 μM, melittin acts as an uncoupler of photophosphorylation and abolishes the 518 nm light induced absorbance changes. At higher concentrations (30–50 μM), melittin abolishes both the light-induced photooxidation of cytochrome f , and partially inhibits other reactions of photosynthetic electron transfer, without causing lysis of the membrane. The observed inhibitions appear to be due to changes in the properties of the membrane lipid bilayer, caused by penetration of melittin molecules.

The effect of some inhibitors of photosynthetic electron transport on the kinetics of redox changes of the reaction centre chlorophyll of photosystem I (P700) and of cytochrome f at sub-zero temperatures.

1 The effect of five inhibitory treatments on the extent of photooxidation of the reaction centre chlorophyll of photosystem I (P700) and cytochrome f in saturating continuous light and on the kinetics of their subsequent dark reduction was studied in spinach chloroplasts suspended at–17°C in a fluid medium containing 50 % (v/v) ethanediol: water. Under these conditions the turnover time of the uninhibited electron transport system is of the order of seconds. 2 Treatment with KCN or polylysine inhibited the photooxidation of cytochrome f and the reduction of P700, but not the reduction of the cytochrome or P700 oxidation. These observations are those expected if inhibition is at the level of plastocyanin and this component mediates electron flow between cytochrome f and P700. 3 Inhibition with bathophenanthroline or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone had no effect on the photooxidation of either cytochrome f or P700. In contrast the rate of dark reduction of both components was reduced in parallel by both inhibitors. This suggests a site of inhibition common to the reduction pathways of both cytochrome f and P700. 4 HgCl2 had the same inhibitory effects as polylysine and KCN but in addition the reduction of cytochrome f was inhibited. It is suggested that this inhibitor may act at more than one site. 5 In most cases cytochrome f reduction showed first-order kinetics and in no case was there any evidence for a lag in the reduction. Inhibitors preventing electron flow from plastocyanin to P700 caused no increase in the rate of cytochrome f reduction. These results do not fit the expected properties of a linear scheme of electron transfer from plastoquinol to P700 via cytochrome f and plastocyanin.

Preparation and characterization of a chemically modified plastocyanin

The reaction of plastocyanin with tetranitromethane results in the nitration of only one of the three tyrosyl residues present in the protein. The modification does not affect the blue copper chromophore as both the characteristic visible spectrum of the chromophore and the redox potential of the protein are unchanged. Photochemical assays show that the modified plastocyanin is fully active in the reduction of photooxidized P700 and in the photooxidation of cytochrome f. The pK of the nitro-tyrosyl residue is about 7.3 indicating that the modified residue may be located in a negatively charged environment. Examination of the recently published X-ray structure of poplar plastocyanin suggests that Tyr-80 would be a likely candidate for the site of modification.

Characterization of synchronized cultures of Bumilleriopsis filiformis

Activity of the photosynthetic apparatus of synchronized cultures was studied with the xanthophycean alga Bumilleriopsis filiformis, following the kinetics of fluorescence induction and photooxidation of cytochrome f (= cytochrome c-553) of intact cells. During the beginning of the cell-division phase, minimum cellular photosynthetic activity is observed and a maximum after its completion, which is accompanied by corresponding changes in Hill reaction activity and re-reduction of cytochrome f by photosystem II light. At minimum activity, the level of steady state fluorescence was higher than at the maximum. This is due, at least in part, to the diminished electron flow between the two photosystems seemingly caused by decreased photosystem I activity. This explanation was suported by the kinetics of cytochrome-f photooxidation. Thus, electron transport activity of both photosystems appears to vary during the cell cycle.

Development of the Primary Photochemical Apparatus of Photosynthesis during Greening of Etiolated Bean Leaves

Seven-day-old dark-grown bean leaves were greened under continuous light. The amount of chlorophyll, the ratio of chlorophyll a to chlorophyll b, the O(2) evolving capacity and the primary photochemical activities of Photosystem I and Photosystem II were measured on the leaves after various times of greening. The primary photochemical activities were measured as the photo-oxidation of P(700), the photoreduction of C-550, and the photo-oxidation of cytochrome b(559) in intact leaves frozen to -196 C. The results indicate that the reaction centers of Photosystem I and Photosystem II begin to appear within the first few minutes and that Photosystem II reaction centers accumulate more rapidly than Photosystem I reaction centers during the first few hours of greening. The very early appearances of the primary photochemical activity of Photosystem II was also confirmed by light-induced fluorescence yield measurements at -196 C.

Concentration-dependent effects of salicylaldoxime on chloroplast reactions

Salicylaldoxime has been found to have a variety of concentration-dependent effects on chloroplast activities. At low concentrations (< 10 mM), salicylaldoxime reversibly inhibits all reactions which involve Photosystem II. Since the DCMU-insensitive silicomolybdate Hill reaction is also inhibited, one site of inhibition is definitely located before the DCMU-sensitive site, possibly before the photoact. The inhibition kinetics and the response of chloroplast fluorescence may indicate another site in the DCMU-sensitive region. At almost exactly the same concentrations (< 10 mM), salicylaldoxime uncouples phosphorylation reversibly, whether it is supported by Photosystem II or by Photosystem I. At higher concentrations (approx. 20 mM) salicylaldoxime inhibits Photosystem II irreversibly, uncouples irreversibly, and begins to cause changes in chloroplast light scattering which could be manifestations of membrane damage. At very high concentrations (approx. 45 mM) salicylaldoxime irreversibly inhibits Photosystem I activity in the region of plastocyanin. This is indicated by the ability of salicylaldoxime to inhibit the photooxidation of cytochrome f but not the photooxidation of P-700.

Light-induced absorbance changes in cell-free extracts of Neurospora crassa.

Abstract— The mycelium of Neurospora crassa was ground and extracted with buffer and separated into a soluble supernatant fraction and a particulate fraction by centrifugation. Both fractions were examined for light‐induced absorbance changes. Irradiation of the supernatant fraction caused a reversible photooxidation of cytochrome c which was inhibited by sodium azide or by dialysis. The action spectrum for the photooxidation showed that the response was mediated by an endogenous flavin. The photooxidation of cytochrome c, lost by dialysis, could be restored by adding flavin mononucleotide. Irradiation of the particulate fraction with blue light caused a reversible photoreduction of cytochrome c and cytochrome oxidase and, in some samples, of cytochrome b as well. The supernatant fraction showed photooxidation of cytochrome c rather than the more usual photoreductive changes because of the presence of super‐oxide dismutase activity.

Further investigation of the light-induced cytochrome b-559 reaction in membrane fragments of the blue-green alga Anabaena variabilis

Oxidation-reduction reactions of the low redox potential cytochrome b-559 were studied for membrane fragments of the blue-green alga Anabaena variabilis. Cytochrome photooxidation was observable at room temperature when the membrane fragments had been preincubated at room temperature in the dark. A CCCP addition (10−4M) strongly enhanced the reaction. Oxidation consisted of a DCMU-sensitive and an insensitive reaction. The former depended on actinic illumination of short wavelength. The latter showed a dependency on longer wavelength light. The former was assumed to be induced by the action of photosystem II and the latter by that of photosystem I. The photosystem II oxidation was small before preincubation, and was enhanced by added DPIP or Ferro. This was interpreted as photosystem II action inducing oxidation as well as reduction; reduction being inactivated during dark incubation or being suppressed by added redox reagents which compete for electron acceptance from photosystem II so that oxidation was apparently enhanced. The oxidationreduction reactions of this cytochrome with low redox potential were assumed to be almost identical with those of the high redox potential form, at least in the membrane fragments of Anabaena variabilis.

Nature of photochemical reactions in chromatophores of Chromatium D III. Heterogeneity of the photosynthetic units

The effect of isooctane extraction on photooxidation of c-type cytochromes was investigated in Chromatium chromatophores. Photooxidation of cytochrome c-555 was not affected by isooctane-extraction except that the dark recovery was accelerated. Photooxidation of cytochrome c-552 was abolished by thorough extraction of ubiquinone-7, but the quantum yield of the cytochrome photooxidation remained unchanged until 90|X% of the total ubiquinone was extracted. The photooxidation of cytochrome c-552 was recovered by the addition of ubiquinone-7 but not by menaquinone. A dark incubation of sufficient length was needed for maximal quantum yield of cytochrome c-555 photooxidation in the presence of 30 mM ascorbate. It is proposed that there are two types of photosynthetic units (or associations of molecules involved in the primary redox reactions) in Chromatium chromatophores. The combinations of primary electron donor-reaction center chlorophyll-primary electron acceptor may be cytochrome c-552-P890-ubiquinone in one type and cytochrome c-555-P890-X in another.

Light-induced absorbance changes due to Photosystems 1 and 2 in spinach chloroplasts at −50 °C

Absorbance changes in the region 500–565 nm and at 702 nm, brought about by excitation of Photosystems 1 and 2, respectively, were measured in spinach chloroplasts at −50 °C. Either dark-adapted chloroplasts were used or chloroplasts preilluminated with a number of short saturating flashes just before cooling. Both photosystems were found to cause a light-induced increase of absorbance at 518 nm (due to “P518”). The System 1-induced change was not affected by preillumination. It decayed within 1 s in the dark and showed similar kinetics as P700. Experiments in the presence of external electron acceptors (methylviologen or Fe(CN) 6 3−) suggested that P518 was not affected by the redox state of the primary electron acceptor of System 1. The absorbance increase at 518 nm due to System 2 decayed in the dark with a half-time of several min. The kinetics were similar to those of C-550, the presumed indicator of the primary electron acceptor of System 2. After two flashes preillumination the changes due to P518 and C-550 were reduced by about 40%, and a relatively slow, System 2-induced oxidation of cytochrome b 559 occurred which proceeded at a similar rate as the increase in yield of chlorophyll a fluorescence. The results indicate that at −50°C two different photoreactions of System 2 occur. One consists of a photoreduction of the primary electron acceptor associated with C-550, accompanied by the oxidation of an unknown electron donor; the other is less efficient and results in the photooxidation of cytochrome b 559.

Nature of photochemical reactions in chromatophores of Chromatium D. II. Quantum yield of photooxidation of cytochromes in Chromatium chromatophores

Initial rates of the light-induced absorption decrease in Chromatium chromatophores due to the oxidation of cytochromes were measured under various experimental conditions. The initial rate in the presence of 10 mM potassium ferrocyanide and 50 μM potassium ferricyanide was about one-half to two-thirds of that in the presence of 30 mM ascorbate or in a medium with a redox potential ( E h) of − 78 mV. Light-minus-dark difference spectrum indicated that, in the presence of 10 mM ferrocyanide and 50 μM ferricyanide, only cytochrome c-555 was photooxidized. In the presence of 30 mM ascorbate or at E h values lower than about 0 mV, both cytochrome c-555 and cytochrome c-552 were photooxidized. The quantum yield of cytochrome c-555 photooxidation was calculated to be about 0.4. The results obtained in the present study are compared with other investigators' and the possibility of the presence of two types of associations between the cytochromes and reaction-center bacteriochlorophyll is discussed.

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.

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 P 700 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 effect of temperature on the primary reaction of chloroplast Photosystem II Evidence for a temperature-dependent back reaction

The primary reaction of Photosystem II has been studied over the temperature range from −196 to −20 °C. The photooxidation of the reaction-center chlorophyll (P680) was followed by the free-radical electron paramagnetic resonance signal of P680 +, and the photoreduction of the Photosystem II primary electron acceptor was monitored by the C-550 absorbance change. At temperatures below −100 °C, the primary reaction of Photosystem II is irreversible. However, at temperatures between −100 and −20 °C a back reaction that is insensitive to 3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) occurs between P680 + and the reduced acceptor. The amount of reduced acceptor and P680 + present under steady-state illumination at temperatures between −100 and −20 °C is small unless high light intensity is used to overcome the competing back reaction. The amount of reduced acceptor present at low light intensity can be increased by adjusting the oxidation-reduction potential so that P680 + is reduced by a secondary electron donor (cytochrome b 559) before P680 + can reoxidize the reduced primary acceptor. The photooxidation of cytochrome b 559 and the accompanying photoreduction of C-550 are inhibited by DCMU. The inhibition of C-550 photoreduction by DCMU, the dependence of P680 photooxidation and C-550 photoreduction on light intensity, and the effect of the availability of reduced cytochrome b 559 on C-550 photoreduction are unique to the temperature range where the Photosystem II primary reaction is reversible and are not observed at lower temperatures.

The functions of plastoquinone and β-carotene in Photosystem II of chloroplasts

1. 1. We have investigated the role of plastoquinone and β-carotene in Photosystem II by examining their effects when added back to freeze-dried chloroplasts that had been extracted with heptane. 2. 2. Under our conditions, extraction removed about 99.5% of the β-carotene and 90% of the plastoquinone. Photochemical activities associated with Photosystem II were reduced to a low level and 85–90% of the cytochrome b-559 HP ( E′ o = +0.37 V) was converted into a low-potential form. 3. 3. Plastoquinone alone restored the potential of the cytochrome to the original high value. The amount of cytochrome restored by plastoquinone was as great as by the heptane extract, and β-carotene was either ineffective or slightly inhibitory. Plastoquinone allowed a substantial rate of 2,6-dichlorophenolindophenol (DCIP) photoreduction, but a greater rate was observed when β-carotene was also present (in agreement with previous work). β-Carotene improved the efficiency of light utilisation and protected the chloroplasts from photoinactivation. 4. 4. The Photosystem II-linked oxidation of cytochrome b-559 HP and reduction of C550 (P546) at 77 °K could only be observed when both β-carotene and plastoquinone were present. A small effect with β-carotene alone could be attributed to unextracted plastoquinone. 5. 5. The results support a model in which C550 (depending on β-carotene) indicates the redox state of the primary acceptor but is not essential for electron flow; β-carotene is needed to maintain the optimum conformation of membrane components for light utilisation and cytochrome photooxidation.