Formation of singlet molecular oxygen in illuminated chloroplasts. Effects on photoinactivation and lipid peroxidation

The formation of singlet molecular oxygen (¹O₂) in illuminatedchloroplasts and the effects of ¹O₂ on oxidation or destructionof components and functional integrity of chloroplasts werestudied. The rate of photoreduction of 2,6-dichloroindophenol(DCIP) and the extent of the 515-nm absorbance change were decreasedby light irradiation and by xanthine oxidase treatment. Malondialdehyde(MDA) formation, an indicator of lipid peroxidation, was observedin the light-irradiated chloroplast fragments, but not in thexanthine-xanthine oxidase-treated chloroplast fragments. MDAformation was absent under anaerobic conditions. MDA formation was stimulated when electron transfer on the oxidizingside of photosystem II (or I) was inhibited or inactivated bycarbonylcyanide m-chlorophenylhydrazone (CCCP), Tris-treatment,prolonged illumination, etc. MDA formation was also stimulatedby 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) when electrontransfer between water and the reaction center of photosystemII was intact. CCCPor DCMU-stimulated MDA formation was inhibitedby 1,4-diazabicyclo[2.2.2]octane, a quencher of singlet molecularoxygen (¹O₂). DCMU and electron donors for photosystem II, suchas ascorbate, hydroquinone and semicarbazide, inhibited MDAformation by illumination of the Tris-washed or CCCP-poisonedchloroplast fragments. Reduced DCIP, an electron donor for photosystemI, also inhibited MDA formation in the presence of DCMU. These results lead to the conclusion that MDA formation wasinitiated by ¹O₂ formed in illuminated chloroplasts. Of thethree mechanisms discussed for ¹O₂ generation in illuminatedchloroplasts, the formation by the electron transfer reactionbetween superoxide anion radical and the oxidant formed on theoxidizing side of photosystem II (or I) is mostimportant. (Received March 31, 1975; )     

Activation of Electron Donation from Hydrogen Peroxide by Manganese in Non-oxygen evolving Photosystem II Particles

Analysis of the activation of H₂O₂-supported 2,6-dichloroindophenol(DCIP) photoreduction by MnCl₂ showed two Mn²⁺-binding sitesin non-oxygen evolving PS II particles, with large (0.4IM) andsmall (0.04 µm) K_m values for Mn²⁺. Photoreduction throughthe high-affinity Mn²⁺.-binding site was inhibited by treatmentwith H₂O₂. (Received April 20, 1987; Accepted July 13, 1987)     

Specific inhibition of photosystem II activity in chloroplasts by fumigation of spinach leaves with SO2

The phytotoxic effects of sulfur dioxide (SO₂) were investigatedby fumigating spinach plants with SO₂. Inhibition of 2,6-dichloroindophenol(DCIP) photoreduction was observed in spinach chloroplasts isolatedfrom fumigated leaves. NADP and DCIP photoreductions were inhibitedto a similar extent by fumigation with 2.0 ppm SO₂ but electronflow from reduced DCIP to NADP was not affected. When electronflow from H₂O to NADP was inhibited by 36%, a 39% inhibitionof non-cyclic photophosphorylation was observed. However, phenazinemethosulfate(PMS)-catalyzed cyclic photophosphorylation wasas active as in the control chloroplasts. Moreover, in the presenceof PMS, no significant suppression was observed in the extentof light-induced H⁺ uptake or in the rate of H⁺ efflux in chloroplasts.From these results, it can be concluded that SO₂ inhibits theelectron flow driven by photosystem II when plants have beenfumigated with SO₂. In spinach leaves fumigated with SO₂, the rate of photosyntheticO₂ evolution was reduced under light-limited conditions, whilethe rate of respiratory O₂ uptake changed slightly. (Received February 8, 1979; )     

Requirement of Manganese for Electron Donation of Hydrogen Peroxide in Photosystem II Reaction Center Complex

Mn²⁺ was required for the electron donating reaction from H₂O₂,but not for that from diphenylcarbazide (DPC), in the PS IIreaction center complex which was prepared from spinach chloroplastsby Triton X-100 extraction. The reaction center complex showeda high activity of 2,6-dichloroindophenol (DCIP) photoreductionin the presence of DPC, but a low activity with H₂O₂. The H₂O₂-supportedDCIP photoreduction was suppressed by EDTA and enhanced by asmall amount of Mn²⁺. Ca²⁺ and Mg²⁺ could not replace Mn²⁺.The activation by Mn²⁺ and its binding showed two binding sitesof Mn²⁺ in the reaction center complex, with high (1.5?10⁷ M^–1)and low (1 ? 10⁶ M^–1) binding constants. (Received November 8, 1986; Accepted April 10, 1987)     

Hydrogen peroxide inhibits cAMP-induced Cl- secretion across colonic epithelial cells

We examined the effects ofH₂O₂on Cl secretion acrosshuman colonic T84 cells grown on permeable supports and mounted in modified Ussing chambers. Forskolin-induced short-circuit current, ameasure of Cl secretion,was inhibited in a concentration-dependent fashion when monolayers werepretreated withH₂O₂for 30 min (30-100% inhibition between 500 µM and 5 mM).Moreover,H₂O₂inhibited 76% of the Clcurrent across monolayers when the basolateral membranes were permeabilized with nystatin (200 µg/ml). When the apical membrane waspermeabilized with amphotericin B,H₂O₂inhibited the Na⁺ current (ameasure ofNa⁺-K⁺-ATPaseactivity) by 68% but increased theK⁺ current more than threefold. Inaddition to its effects on ion transport pathways,H₂O₂also decreased intracellular ATP levels by 43%. We conclude that theprincipal effect ofH₂O₂on colonic Cl secretion isinhibitory. This may be due to a decrease in ATP levels followingH₂O₂treatment, which subsequently results in an inhibition of the apicalmembrane Cl conductance andbasolateral membraneNa⁺-K⁺-ATPaseactivity. Alternatively,H₂O₂may alter Cl secretion bydirect action on the transporters or alterations in signal transductionpathways.

     

Oxidation-reduction reactions between electron transfer components and hydrogen peroxide in photosystem II of chloroplasts

The light-induced oxygen evolution, photoreduction of 2,6-dichlorophenolindophenol (DPIP) and carotenoid photobleaching induced by carbonylcyanide m-chlorophenylhydrazone (CCCP) were investigated withspinach chloroplast fragments in the presence of H₂O₂. Oxygenevolution in the presence of H₂O₂ was not inhibited by CCCPand was only partially inhibited by 5 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) which completely inhibited the Hill reaction with DPIP.The degree of inhibition by DCMU was decreased by a simultaneousaddition of CCCP. Carotenoid photobleaching in the presenceof CCCP was stimulated by H₂O₂. The CCCP-induced carotenoidphotobleaching was completely inhibited by DCMU. However, itwas only partially inhibited by DCMU in the presence of H₂O₂.These data indicate that H₂O₂ donates electrons at a site betweenthe CCCP-sensitive site and the reaction center of photosystemII and is reduced at a site between the DCMU-blocked site andthe reaction center of photosystem II. ¹Present address: Department of Biology, Kyushu Dental College,Kitakyushu 803, Japan. (Received June 20, 1974; )     

Evidence for Involvement of the Superoxide Radical in the Conversion of 1-Aminocyclopropane-1-Carboxylic Acid to Ethylene by Pea Microsomal Membranes

Electron spin resonance (ESR) spectroscopy has provided evidencefor involvement of the superoxide anion (O₂^–) radicalin the conversion of l-aminocyclopropane-l carboxylic acid (ACC)to ethylene by microsomal membranes from etiolated pea seedlings.Formation of ethylene from ACC by the membrane system is oxygen-dependent,heat denaturable, inhibited by the radical scavenger n-propylgallate and sensitive to superoxide dismutase (SOD) and catalase.Addition of 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron)to the reaction mixture results in formation of the Tiron semiquinone(Tiron radical) ESR signal derived from O₂^–, and alsoinhibits ethylene production. The radical signal is oxygen-dependentand inhibited by SOD and catalase, but is formed both in thepresence and absence of ACC. Heat denaturation of the microsomalenzyme system completely blocks formation of the radical signal.The data collectively suggest that O₂^– generated by amembrane-bound enzyme facilitates the conversion of ACC to ethylene. (Received September 8, 1981; Accepted January 19, 1982)     

Effects of water stress on photochemical function and protein metabolism of photosystem II in wheat leaves

The effects of osmotic dehydration in wheat leaves ( Triticum aestivum L. cv. Longchun No. 10) on the photochemical function and protein metabolism of PSII were studied with isolated thylakoid and PSII membranes. The results indicated that PSII was rather resistant to water stress as mild water deficit in leaves did nut significantly affect its activity. However, extreme stress conditions such as 40% decrease in relative water content (RWC) or 1.8 MPa in water potential (Ψ) caused ca 50% reduction in O₂ evolution and ca 25% inhibition of DCIP (2.6-dichlorophenol indophenol) photoreduction of PSII. In addition, it was found that the inhibited DCIP photoreduction of PSII could not be reversed by DPC (2.2-diphenylcarbazide), a typical electron donor to PSII, suggesting that water stress did not affect electron donation to PSII. Urea-SDS-PAGE and western blot analysis showed that the steady slate levels of major PSII proteins, including the D1 and D2 proteins in the PSII reaction center, declined on a chlorophyll basis with increasing water stress, possibly as a result of increased degradation. In vitro translation experiments and quantitative analysis of chloroplast RNAs indicated that the potential synthesis of chloroplast proteins from their mRNAs was impaired by water stress. From the results it is concluded that the effects of water stress on PSII protein metabolism, especially on the reaction center proteins, may account for the damage to PSII photochemistry.     

Isolation and characterization of photosystem II from the filamentous sporophyte of Porphyra yezoensis

Thylakoid membranes were isolated and purified from diploid filamentous sporophytes of Porphyra yezoensis Ueda using sucrose density gradient ultracentrifugation (SDGUC). After thylakoid membranes were solubilized with SDS, the phtosystem II (PSII) particles with high 2, 6-dichloroindophenol (DCIP) photoreduction activity were isolated by SDGUC. The absorption and fluorescence spectra, DCIP photoreduction activity and oxygen evolution activity of the thylakoid membranes and PSII particles were determined. The polypeptide composition of purified PSII particles was distinguished by SDS-PAGE. Results showed that PSII particles of sporophytes differed from the gametophytes in spectral properties and polypeptide composition. Apart from 55 kDa D1-D2 heterodimer, CP47, CP43, 33 kDa protein, D1, D2, cyt b559 and 12 kDa protein were identified from PSII particles from sporophytes; a new 102 kDa protein was also detected. However, cyt c-550, 20 kDa, 14 kDa and 16 kDa proteins found in PSII particles from gametophytes were not detected in the sporophytes.     

Photoreduction of 18O2 and H218O2 with Concomitant Evolution of 16O2 in Intact Spinach Chloroplasts: Evidence for Scavenging of Hydrogen Peroxide by Peroxidase

Illuminated intact spinach chloroplasts decomposed one moleculeof H₂¹⁸O₂ which resulted in the evolution of a half moleculeof ¹⁶O₂, but little ¹⁸O₂. The chloroplasts showed the same rateof photoreduction of ¹⁸C₂ as that of the evolution of ¹⁶O₂ withoutaccumulation of H₂¹⁸O₂. These reactions were suppressed by DCMU,and also by several inhibitors of ascorbate peroxidase and dehydroascorbateand monodehydroascorbate reductases in chloroplasts. These observationsindicate that the hydrogen peroxide produced in chloroplastsis reduced to water by a peroxidase using a photoreductant asthe electron donor. The hydrogen peroxide scavenging systemof chloroplasts was inactivated if hydrogen peroxide was addedin the dark, but not if added during the light. (Received May 4, 1984; Accepted July 10, 1984)     

Inhibition of Hill Reaction and Photophosphorylation by l,l,l-Trichloro-2,2-bis(p-chlorophenyl)ethane

The effect of DDT on DCIP and Fe(CN)₆^3– photoreductions,and cyclic and non-cyclic photo-phosphorylations, in some 30varieties of barley from widely different parts of the worldis reported. Whereas resistant barleys were not affected byDDT treatment, chloroplasts from treated susceptible barleysshowed parallel inhibitions of all the investigated aspectsof photosynthesis. However, in a few susceptible varieties inhibitionsof Fe(CN)₆^3– photoreduction or non-cyclic photophosphorylationwere not so pronounced. Possible reasons for these anomaliesare discussed; in particular earlier reports that DDT had noeffect on these latter photosynthetic activities may have beendue to the use of hypotonic media during chloroplast isolation.     

Effect of Oxygen on Photosynthetic 14CO2 Fixation in Chroomonas sp. (Cryptophyta) II. Effects of Inhibitors, Uncouplers and an Artificial Electron Mediator on the Inhibition of 14CO2 Fixation by Anaerobiosis

In "air-grown" Chroomonas sp. cells, low concentrations of DCMU(less than 0.1 µM) could prevent the inhibition of ¹⁴CO₂fixation by anaerobiosis under light-saturating conditions (morethan 40 W.m^–2), with phenazine methosulfate showing asimilar effect. Antimycin A, carbonyl cyanide m-chlorophenylhydrazone(CCCP), and N,N'-dicyclohexylcarbodiimide strongly inhibitedanaerobic photosynthesis at concentrations which did not significantlyinhibit the rate under 2% O₂ at high light intensity (200 W.m^–2),although 0.2 µM CCCP stimulated the rate under 2% O₂ tosome extent. On the other hand, KCN inhibited the rate muchmore strongly under 2% O₂ than N₂, although it inhibited therate very strongly at concentrations above 5 µM both underN₂ and 2% O₂. These results suggest that the inhibition of photosynthetic¹⁴CO₂ fixation by anaerobiosis in this alga result from ATPdeficiency caused by over-reduction of electron carriers ofthe cyclic electron flow and that oxygen can prevent the over-reduction.Cyclic electron flow seems to be necessary to provide additionalATP for CO₂ reduction under anaerobic conditions, although itseems to be less necessary under aerobic conditions. (Received July 21, 1983; Accepted January 23, 1984)     

Photosynthesis by Guard Cell Chloroplasts of Vicia faba L.: Effects of Factors Associated with Stomatal Movement

The properties of photosynthetic O₂ evolution by mesophyll cellchloroplasts (MCC) and guard cell chloroplasts (GCC) isolatedfrom protoplasts of Vicia faba L. have been studied and effectson O₂ evolution of factors known to regulate stomatal movementshave been compared. The O₂ evolution of GCC was CO₂-dependent.The saturating light intensity for O₂ evolution was between150 and 200 µmol m^–2s^–1 for MCC and was between400 and 1,000µmol m^–2s^–1 for GCC. Light quality(red vs. blue) had no significant effect on O₂ evolution byeither MCC or GCC. The O₂ evolution rate of MCC was stronglydependent on external K⁺ concentration, but GCC did not respondsignificantly to variations in external K⁺ concentration between0 and 250 mM. The optimal external pH for O₂ evolution by MCCwas approximately 7.5, and either higher or lower external pHsignificantly inhibited O₂ evolution. However, O₂ evolutionby GCC was only slightly enhanced when external pH was increasedfrom 6.0 to 8.0. Our observation of differential sensitivityof MCC and GCC to light intensity and to variations of cytoplasmicK⁺ and pH may indicate differential regulation of photosynthesisin MCC and GCC. ¹Current address: Biology Department, Pennsylvania State University,208 Mueller Laboratory, University Park, PA 16802, U.S.A.     

Effects of lanthanum and calcium on photoelectron transport activity and the related protein complexes in chloroplast of cucumber leaves

The effects of lanthanum and calcium ions on electron transport, dichlorephenol indophenol (DCIP) photoreduction, and oxygen evolution activities in chloroplast from cucumber (Cucumis satives L.) were determined. The lanthanum inhibited the whole electron chain-transport activity of chloroplast. DCIP photoreduction and oxygen evolution activities of the photosystem I (PSII) also decrease after treatment with La³⁺. But the diminished activities of PSII and chloroplast caused by La³⁺ could be reversed by Ca²⁺ and even became higher than the control level. The concentration analysis of related protein complexes to photoelectron transport in chloroplast included that La³⁺ induced the concentration of chlorophyll protein complexes increasing but caused some nonchlorophyll protein complexes to decompose partially. This increasing effect of La³⁺ on chlorophyll protein complexes results in the improvement of chlorophyll content, which will improve the absorption of photoelectron and energy transport in the process of photosynthesis.     

Inhibition of photosystem I and photosystem II in chloroplasts by UV radiation

The effects of UV radiation on the low temperature fluorescenceand primary photochemistry of PSII and PSI of spinach chloroplastswere studied. Fluorescence induction curves at –196°Cwere measured at 695 nm for PSII fluorescence and at 730 nmfor PSI fluorescence to determine both the initial F_o and finalF_M levels. The primary photochemistry of PSII was measured asthe rate of photoreduction of C-550 at – 196°C, thatof PSI as the rate of photooxidation of P₇₀₀ at –196°C.The results were analyzed in terms of a model for the photosyntheticapparatus which accounts for the yields of fluorescence andprimary photochemistry. According to this analysis UV radiationincreases nonradiative decay processes at the reaction centerchlorophyll of PSII. However, the effect of UV radiation isnot uniform throughout the sample during irradiation so thataccount must be taken of the fraction of PSII reaction centerswhich have been irradiated at any given time. UV radiation alsoinactivates P700 and causes a slight increase in nonradiativedecay in the antenna chlorophyll of PSI. All fluorescence ofvariable yield, F_V = F_M–F_o, at 730 nm is due to energytransfer from PSII to PSI so that the sensitivity of Fv to UVradiation is the same at 730 and 695 nm. ¹Present address: Department of Biology, Faculty of Science,Toho University, Narashino, Chiba 275, Japan. ²Present address: Central Research Laboratories, Fuji PhotoFilm Co., Ltd., 105 Mizonuma, Asaka-Shi, Saitama 351, Japan. (Received September 10, 1975; )     

Weak Light-Induced Oxygen Consumption Observed during Photoreactivation Is Coupled to the Recovery of Oxygen Evolving Activity

During photoreactivation of the O₂-evolving center in Tris-inactivated/Mn-depletedthylakoids, a slow O₂-consumption occurred. This O₂-consumptionbecame detectable when the O₂-evolving activity of thylakoidswas inactivated by Tris-treatment and decreased as photoreactivationproceeded. The O₂-consumption and photoreactivation similarlyrequired Mn²⁺ at µM levels in addition to PSII electrondonors and shared severa common characteristics. Stimulationof O₂-consumption and photoreactivation by these cofactors werealways accompanied by enhancement in chlorophyll fluorescenceinduction, suggesting the involvement of a Mehler-type reactionin photoreactivation. Although the electron transport due tothis O₂-consumption was rapid enough to oxidize 4 Mn²⁺ ionsto reconstitute the tetranuclear Mn-cluster in each O₂-evolvingcenter in a few seconds, actual recovery of O₂-evolving activityoccurred more slowly in a few minutes. It was inferred thatphotoreactivation in Tris-inactivated thylakoids is not a simplephotooxidation of Mn2²⁺ but involves more complicated processeswhich are coupled to the Mehlertype electron transport fromPSII to oxygen via PSI. (Received July 11, 1994; Accepted August 23, 1996)     

Photoswelling and light-inactivation of isolated chloroplasts II. Functional and structural changes in isolated chloroplasts under the influence of lysolecithin

Lysolecithin was added to spinach chloroplasts in suspension,and its effect as a detergent on structure and electron transportactivities was examined. At low concentrations of lysolecithin,the activities of FeCN photoreduction and O₂-linked DCPIPH₂photo-oxidation were stimulated but the photoreduction of NADPwas not enhanced and decreased with an increase in concentration.Absorption bands over the whole visible region were intensifiedwithout any shifts. At high concentrations, the activities ofFeCN photoreduction and O₂-linked DCPIPH₂ photo-oxidation decreasedfrom a maximum to 10–50% of the control activity, andNADP photoreduction was completely inhibited. Absorption bandswere further intensified and the red band was slightly shifted.Results indicate that lysolecithin treatment is useful in chloroplastbiochemistry. Lysolecithin formation and the deterioration ofchloroplasts during light-aging is also discussed. (Received August 15, 1974; )     

Photoproduction of the Azidyl Radical from the Azide Anion on the Oxidizing Side of Photosystem II and Suppression of Photooxidation of Tyrosine Z by the Azidyl Radical

Azide ions inhibited O₂ evolution in PSII membranes from spinachin a time-dependent manner in the light until all activity disappeared.Illumination in the presence of azide (azide-phototreatment)irreversibly inhibited the following processes: (1) both theoxidation of water and the electron transport between the redox-activetyrosine 161 of the D1 protein (Y_Z) and the secondary quinoneelectron acceptor (Q_B) site, to the same extent; (2) the donationof electrons to the primary quinone electron acceptor (Q_A),as measured by monitoring the maximum variable fluorescenceof Chl; and (3) the photoproduction of the Y_Z radical (Y). Thus,the primary site of inhibition appeared to lie between Y_Z andQ_A. On illumination of Tris-treated PSII membranes in the presenceof azide, production of the azidyl radical was observed by spin-trappingESR. Yield of Y in Tris-treated membranes on illumination wassuppressed by azide. Electron transport from Y_Z to Q_B in Tris-treatedmembranes was inhibited only when the azidyl radical was photoproduced,and it was inhibited more rapidly than it was in the oxygenicPSII membranes. These results indicate that the azidyl radicalwas produced via a univalent oxidation of azide by Y and thatit irreversibly inhibited the electron transport from Y_Z toQ_A in Tris-treated membranes. Although the azidyl radical wasundetectable in the oxygenic PSII membranes, probably due tosteric interference by the peripheral proteins of water-oxidizingcomplex with the access of the spin-trapping reagent to theproduction site of the radical, the participation of the azidylradical in the inhibition of the oxygenic PSII membranes issuggested since simultaneous occurrence of both electron transportand azide was required for the inhibition. Possible inhibitorymechanisms and the target sites of azidyl radical are discussed. (Received April 21, 1995; Accepted July 3, 1995)     

The Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase is Fragmented into 37-kDa and 16-kDa Polypeptides by Active Oxygen in the Lysates of Chloroplasts from Primary Leaves of Wheat

Lysates of chloroplasts isolated from wheat (Triticum aestivumL. cv. Aoba) leaves were incubated on ice (pH 5.7) for 0 to60 min in light (15 µmol quanta m^–2 s^–1),and degradation of the large subunit (LSU) of ribulose-l,5-bis-phosphatecarboxylase/oxygenase (Rubisco: EC 4.1.1.39 [EC] ) was analyzed byapplying immunoblotting with site-specific antibodies againstthe N-terminal, internal, and C-terminal amino acid sequencesof the LSU of wheat Rubisco. The most dominant product of thebreakdown of the LSU and that which was first to appear wasan apparent molecular mass of 37-kDa fragment containing theN-terminal region of the LSU. A 16-kDa fragment containing theC-terminal region of the LSU was concomitantly seen. This fragmentationof the LSU was inhibited in the presence of EDTA or 1,10-phenanthroline.The addition of active oxygen scavengers, catalase (for H₂O₂)and n-propyl gallate (for hydroxyl radical) to the lysates alsoinhibited the fragmentation. When the purified Rubisco fromwheat leaves was exposed to a hydroxyl radical-generating systemcomprising H₂O₂, FeSO₄ and ascorbic acid, the LSU was degradedin the same manner as observed in the chloroplast lysates. Theresults suggest that the large subunit of Rubisco was directlydegraded to the 37-kDa fragment containing the N-terminal regionand the 16-kDa fragment containing the C-terminal region ofthe LSU by active oxygen, probably the hydroxyl radical, generatedin the lysates of chloroplasts. (Received October 28, 1996; Accepted February 7, 1997)     

Effects of Light Stress on Redox Potential Forms of Cyt b-559 in Photosystem II Membranes Depleted of Water-Oxidizing Complex

Effects of photoinhibition on the redox properties of Cyt b-559were studied with NH₂OH treated PSII membranes, which are depletedof the water-oxidizing complex. The membranes contained threeredox forms (HP-, IP- and LP-forms) of Cyt b-559, with E_m valuesof +435, +237 and +45 mV, respectively. A novel intermediate-potentialform of Cyt b-559 was generated during photoinhibition on thedonor side of PSII: photoinhibitory illumination (7,000 µEm^–2 s^–1) for 1 min induced a 30% decrease in thelevel of the HP-form, with concomitant generation of the intermediate-potential(IP-) form whose E_m value was about +350mV. Prolonged illumination(10 min) resulted in complete loss of the HP-form and an apparentincrease in the level of the IPform. After further photoinhibitorytreatment (60 min), complete loss of the IP'-form was observedand levels of the IP- and LP-forms each increased to about 50%of the total amount of Cyt b-559. Kinetic analysis of thesedata led to the conclusion that the HP-form is converted tothe LP-form via two intermediate-potential forms (IP' and IP),and that IP'-form appears only at the early phase of photoinhibition. (Received March 30, 1994; Accepted February 27, 1995)