Probing the role of Valine 185 of the D1 protein in the Photosystem II oxygen evolution

In Photosystem II (PSII), the Mn₄CaO₅-cluster of the active site advances through five sequential oxidation states (S₀ to S₄) before water is oxidized and O₂ is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824–6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV–visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking Tyr_Z and the halide. The V185 contributes to the stabilization of S₂ into the low spin (LS), S?=?1/2, configuration. Indeed, in the V185T mutant a high proportion of S₂ exhibits a high spin (HS), S?=?5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S₁Tyr_Z?→?S₂^HSTyr_Z transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S₂^LSTyr_Z?→?S₃Tyr_Z transition was observed and we propose that it occurs in the S₂^LSTyr_Z?→?S₂^HSTyr_Z intermediate step before the S₂^HSTyr_Z?→?S₃Tyr_Z transition occurs. The dramatic slowdown of the S₃Tyr_Z?→?S₀Tyr_Z transition in the V185T mutant does not originate from a structural modification of the Mn₄CaO₅ cluster since the spin S?=?3?S₃ EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein.     

Some Photosystem II properties depending on the D1 protein variants in Thermosynechococcus elongatus

Cyanobacteria have multiple psbA genes encoding PsbA, the D1 reaction center protein of the Photosystem II complex which bears together with PsbD, the D2 protein, most of the cofactors involved in electron transfer reactions. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues constituting each of the 3 possible PsbA variants there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. In this review, we summarize the changes already identified in the properties of the redox cofactors depending on the D1 variant constituting Photosystem II in T. elongatus. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

The relative absorption cross-sections of Photosystem I and Photosystem II in chloroplasts from three types of Nicotiana tabacum

In the present study we used three types of Nicotiana tabacum, cv John William's Broad Leaf (the wild type and two mutants, the yellow-green Su/su and the yellow Su/su var. Aurea) in order to correlate functional properties of Photosystem II and Photosystem I with the structural organization of their chloroplasts. The effective absorption cross-section of Photosystem II and Photosystem I centers was measured by means of the rate constant of their photoconversion under light-limiting conditions. In agreement with earlier results (Okabe, K., Schmid, G.H. and Straub, J. (1977) Plant Physiol. 60, 150–156) the photosynthetic unit size for both System II and System I in the two mutants was considerably smaller as compared to the wild type. We observed biphasic kinetics in the photoconversion of System II in all three types of N. tabacum. However, the photoconversion of System I occurred with monophasic and exponential kinetics. Under our experimental conditions, the effective cross-section of Photosystem I was comparable to that of the fast System II component (α centers). The relative amplitude of the slow System II component (β centers) varied between 30% in the wild type to 70% in the Su/su var. Aurea mutant. The increased fraction of β centers is correlated with the decreased fraction of appressed photosynthetic membranes in the chloroplasts of the two mutants. As a working hypothesis, it is suggested that β centers are located on photosynthetic membranes directly exposed to the stroma medium.     

Differences in the Interactions between the Subunits of Photosystem II Dependent on D1 Protein Variants in the Thermophilic Cyanobacterium Thermosynechococcus elongatus

The main cofactors involved in the oxygen evolution activity of Photosystem II (PSII) are located in two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is encoded by either the psbA₁ or the psbA₃ gene, the expression of which is dependent on environmental conditions. It has been shown that the energetic properties of the PsbA1-PSII and those of the PsbA3-PSII differ significantly (Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Biochim. Biophys. Acta 1797, 1491–1499). In this work the structural stability of PSII upon a PsbA1/PsbA3 exchange was investigated. Two deletion mutants lacking another PSII subunit, PsbJ, were constructed in strains expressing either PsbA1 or PsbA3. The PsbJ subunit is a 4-kDa transmembrane polypeptide that is surrounded by D1 (i.e. PsbA1), PsbK, and cytochrome b₅₅₉ (Cyt b₅₅₉) in existing three-dimensional models. It is shown that the structural properties of the PsbA3/ΔPsbJ-PSII are not significantly affected. The polypeptide contents, the Cyt b₅₅₉ properties, and the proportion of PSII dimer were similar to those found for PsbA3-PSII. In contrast, in PsbA1/ΔPsbJ-PSII the stability of the dimer is greatly diminished, the EPR properties of the Cyt b₅₅₉ likely indicates a decrease in its redox potential, and many other PSII subunits are lacking. These results shows that the 21-amino acid substitutions between PsbA1 and PsbA3, which appear to be mainly conservative, must include side chains that are involved in a network of interactions between PsbA and the other PSII subunits.     

Excitation trapping and charge separation in Photosystem II in the presence of an electrical field

To investigate the effects of a membrane potential on excitation trapping and charge separation in Photosystem II we have studied the chlorophyll fluorescence yield in osmotically swollen chloroplasts subjected to electrical field pulses. Significant effects were observed only in those membrane regions where a large membrane potential opposing the photochemical charge separation was built up. When the fluorescence yield was low, close to F₀, a much higher yield, up to F_max, was observed during the presence of the membrane potential. This is explained by an inhibition by the electrical field of electron transfer to the quinone acceptor Q, resulting in a decreased trapping of excitations. A field pulse applied when the fluorescence yield was high, Q and the donor side being in the reduced state, had the opposite effect: the fluorescence was quenched nearly to F₀. This field-induced fluorescence quenching is ascribed to reversed electron transfer from Q^? to the intermediate acceptor, pheophytin. Its field strength dependence suggests that the midpoint potential difference between pheophytin and Q is at most about 300 mV. Even then it must be assumed that electron transfer between pheophytin and Q spans 90% of the potential difference across the membrane.     

The D1-173 amino acid is a structural determinant of the critical interaction between D1-Tyr161 (TyrZ) and D1-His190 in Photosystem II

The main cofactors of Photosystem II (PSII) are borne by the D1 and D2 subunits. In the thermophilic cyanobacterium Thermosynechococcus elongatus, three psbA genes encoding D1 are found in the genome. Among the 344 residues constituting the mature form of D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. In a previous study (Sugiura et al., J. Biol. Chem. 287 (2012), 13336-13347) we found that the oxidation kinetics and spectroscopic properties of Tyr_Z were altered in PsbA2-PSII when compared to PsbA(1/3)-PSII. The comparison of the different amino acid sequences identified the residues Cys144 and Pro173 found in PsbA1 and PsbA3, as being substituted in PsbA2 by Pro144 and Met173, and thus possible candidates accounting for the changes in the geometry and/or the environment of the Tyr_Z/His190 phenol/imidizol motif. Indeed, these amino acids are located upstream of the α-helix bearing Tyr_Z and between the two α-helices bearing Tyr_Z and its hydrogen-bonded partner, D1/His190. Here, site-directed mutants of PSII, PsbA3/Pro173Met and PsbA2/Met173Pro, were analyzed using X- and W-band EPR and UV-visible time-resolved absorption spectroscopy. The Pro173Met substitution in PsbA2-PSII versus PsbA3-PSII is shown to be the main structural determinant of the previously described functional differences between PsbA2-PSII and PsbA3-PSII. In PsbA2-PSII and PsbA3/Pro173Met-PSII, we found that the oxidation of Tyr_Z by P₆₈₀^+● was specifically slowed during the transition between S-states associated with proton release. We thus propose that the increase of the electrostatic charge of the Mn₄CaO₅ cluster in the S₂ and S₃ states could weaken the strength of the H-bond interaction between Tyr_Z^● and D1/His190 in PsbA2 versus PsbA3 and/or induce structural modification(s) of the water molecules network around Tyr_Z.     

Water in Photosystem II: Structural,functional and mechanistic considerations

Water is clearly important for the functioning of Photosystem II (PSII). Apart from being the very substrate that needs to be transported in this water oxidation enzyme, water is also vital for the transport of protons to and from the catalytic center as well as other important co-factors and key residues in the enzyme. The latest crystal structural data of PSII have enabled detailed analyses of the location and possible function of water molecules in the enzyme. Significant progress has also been made recently in the investigation of channels and pathways through the protein complex. Through these studies, the mechanistic significance of water for PSII is becoming increasingly clear. An overview and discussion of key aspects of the current research on water in PSII is presented here. The role of water in three other systems (aquaporin, bacteriorhodopsin and cytochrome P450) is also outlined to illustrate further points concerning the central significance that water can have, and potential applications of these ideas for continued research on PSII. It is advocated that water be seen as an integral part of the protein and far from a mere solvent.     

Polypeptide composition of a Photosystem II core complex. Presence of a herbicide-binding protein

The polypeptide composition of a Photosystem II (PS II) core complex from higher plant chloroplasts has been characterized by subjecting the isolated complex to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides in the 40–50 kDa size class, attributed to the chlorophyll a-binding apoproteins of PS II, were resolved when the urea concentration in the SDS-polyacrylamide gel electrophoresis was greater than 1 M. The two chlorophyll a-binding proteins were dissimilar in their primary structure based upon their different hydrolysis products on SDS-polyacrylamide gel electrophoresis following papain treatment. The core complex contained three additional polypeptides. Two polypeptides in the 30–34 kDa size class were resolved when the urea concentration in the gel system was increased to greater than 4 M. One of the polypeptides in this size class was identified as the herbicide-binding protein from azido[¹⁴C]atrazine labeling studies. The herbicide-binding protein displayed an anomalous electrophoretic migration behavior in SDS-polyacrylamide gel electrophoresis in the presence or absence of urea; its apparent molecular weight decreased when the urea concentration increased. The fifth protein component of the core complex was attributed to cytochrome b-559 which was found to consist of the ascorbate- and dithionite-reducible forms in the samples prior to SDS solubilization.     

Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA1 or psbA3 gene

The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA₃ gene compared to that deduced from the psbA₁ gene points a difference of 21 residues. In this work, PSII isolated from a wild type T. elongatus strain expressing PsbA1 or from a strain in which both the psbA₁ and psbA₂ genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of Pheo_D1 is increased by 17 mV from −522 mV in PsbA1-PSII to −505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S₂Q_A^−• charge recombination and the C ≡ N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the Q_B pocket. The temperature dependences of the S₂Q_A^−• charge recombination show that the strength of the H-bond to Pheo_D1 is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of Q_A (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P₆₈₀^+• and Y_Z is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P₆₈₀/P₆₈₀^+• couple (and hence that of ¹P₆₈₀^*/P₆₈₀^+•) is tuned as well when shifting from PsbA1 to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes.     

Proteolytic activity against the light-harvesting complex and the D1/D2 core proteins of Photosystem II in close association to the light-harvesting complex II trimer

Light-harvesting complex II (LHCII) prepared from isolated thylakoids of either broken or intact chloroplasts by three independent methods, exhibits proteolytic activity against LHCII. This activity is readily detectable upon incubation of these preparations at 37 °C (without addition of any chemicals or prior pre-treatment), and can be monitored either by the LHCII immunostain reduction on Western blots or by the Coomassie blue stain reduction in substrate-containing “activity gels”. Upon SDS-sucrose density gradient ultracentrifugation of SDS-solubilized thylakoids, a method which succeeds in the separation of the pigment-protein complexes in their trimeric and monomeric forms, the protease activity copurifies with the LHCII trimer, its monomer exhibiting no activity. This LHCII trimer, apart from being “self-digested”, also degrades the Photosystem II (PSII) core proteins (D1, D2) when added to an isolated PSII core protein preparation containing the D1/D2 heterodimer. Under our experimental conditions, 50% of LHCII or the D1, D2 proteins are degraded by the LHCII-protease complex within 30 min at 37 °C and specific degradation products are observed. The protease is light-inducible during chloroplast biogenesis, stable in low concentrations of SDS, activated by Mg²⁺, and inhibited by Zn²⁺, Cd²⁺, EDTA and p-hydroxy-mercury benzoate (pOHMB), suggesting that it may belong to the cysteine family of proteases. Upon electrophoresis of the LHCII trimer on substrate-containing “activity gels” or normal Laemmli gels, the protease is released from the complex and runs in the upper part of the gel, above the LHCII trimer. A polypeptide of 140 kDa that exhibits proteolytic activity against LHCII, D1 and D2 has been identified as the protease. We believe that this membrane-bound protease is closely associated to the LHCII complex in vivo, as an LHCII-protease complex, its function being the regulation of the PSII unit assembly and/or adaptation.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

Light-Induced Damage of Histidine Residues in Photosystem II Reaction Center D1/D2/cyt b559 Complex

Histidine residue content of photosystem Ⅱ reaction center D1/D2/cytochrome b559 complex decreased by about 26% after illumination. The result suggests that some histidine residues are damaged by illumination. The damage of histidine residues may be related to the changes of the spectra properties during the incubation in the dark following preillumination of the reaction center complex.     

Measurement of the relative electron-transport capacity of Photosystem I and Photosystem II in spinach chloroplasts

The ratio of Photosystem (PS) II to PS I electron-transport capacity in spinach chloroplasts was compared from reaction-center and steady-state rate measurements. The reaction-center electron-transport capacity was based upon both the relative concentrations of the PS II_α, PS II_β and PS I centers, and the number of chlorophyll molecules associated with each type of center. The reaction-center ratio of total PS II to PS I electron-transport capacity was about 1.8:1. Steady-state electron-transport capacity data were obtained from the rate of light-induced absorbance-change measurements in the presence of ferredoxin-NADP⁺, potassium ferricyanide and 2,5-dimethylbenzoquinone (DMQ). A new method was developed for determining the partition of reduced DMQ between the thylakoid membrane and the surrounding aqueous phase. The ratio of membrane-bound to aqueous DMQH₂ was experimentally determined to be 1.3:1. When used at low concentrations (200 μM), potassium ferricyanide is shown to be strictly a PS I electron acceptor. At concentrations higher than 200 μM, ferricyanide intercepted electrons from the reducing side of PS II as well. The experimental rates of electron flow through PS II and PS I defined a PS II/PS I electron-transport capacity ratio of 1.6:1.     

Inhibition of CO2-fixation and its effect on the activity of Photosystem II,on D1-protein synthesis and phosphorylation

To study the effects of limitations in the Calvin-cycle on Photosystem (PS) II function and on its repair by D1-protein turnover, glycerinaldehyde (DLGA) was applied to 1 h dark-adapted pea leaves via the petiole. The application resulted in a 90% inhibition of photosynthetic oxygen evolution after 90 min illumination at either 120 or 500 µmol m^–2 s^–1. In the control leaves an increase of light-dependent oxygen production to 147 and 171% was observed after 90 min illumination. According to chlorophyll fluorescence quenching analysis the inhibition of photosynthetic electron transport by DLGA led to a substantial increase in the reduction state of the primary quinone acceptor of PS II, QA, and to a rise in membrane energetisation. However, PS II functionality was hardly affected by DLGA at the low light intensity as indicated by the constant high yield of variable fluorescence, Fv/Fm. Only at 500 µmol m^–2 s^–1 a 15% loss of Fv/Fm was observed in the presence of DLGA indicating that inactivated PS II centres had accumulated. The control leaves also showed a slight loss of Fv/Fm which did not affect photosynthetic electron transport due to a faster reoxidation of QA. The relative stability of PS II function in the presence of DLGA could not be ascribed to an increased repair by the rapid turnover of the D1-protein. Radioactive pulse-labelling studies with [14C] leucine in combination with immunological determination of the protein content revealed that both synthesis and degradation of the protein were inhibited in DLGA-treated leaves whereas in the control leaves a stimulation of D1-protein turnover was observed. The changes of D1-protein turnover could be explained by differences in the occupancy state of the QB-binding niche. A relation between the phosphorylation status of the PS II polypeptides and the turnover of the D1-protein could not be established. As shown by radioactive labelling with [32P]i, addition of DLGA led to an increase in the phosphorylation level of the PS II polypeptides D1 and D2 at the low light intensity when compared to the non-treated control. At the higher light intensity the phosphorylation level of the PS II polypeptides in control and DLGA-treated leaves were identical in spite of the substantial differences in D1-protein turnover.     

Psb30 contributes to structurally stabilise the Photosystem II complex in the thermophilic cyanobacterium Thermosynechococcus elongatus

A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA₃ gene (WT*). The ΔPsb30 mutant appears more susceptible to photodamage, has a cytochrome b₅₅₉ that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the ?Psb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b₅₅₉ into the low potential form. Structural reasons for such effects are discussed.     

Site-directed mutations at D1-His198 and D1-Thr179 of photosystem II in Synechocystis sp. PCC 6803: deciphering the spectral properties of the PSII reaction centre

Site-directed mutations were constructed in photosystem II of Synechocystis sp. PCC6803 in which the axial ligand, D1-His198, of special pair chlorophyll PD1 was replaced with Gln and where D1-Thr179, which overlies monomeric chlorophyll ChlD1, was replaced with His. The D1-His198Gln mutation produces a 3nm displacement to the blue of the bleaching minimum in the Soret and in the Qy region of the (P+QA--PQA) absorbance difference spectrum. To a first approximation, the bleaching can be assigned to the low-energy exciton transition of the special pair chlorophylls PD1/PD2. The D1-Thr179His mutation produces a 2nm displacement to the red of the bleaching minimum in the Qy region of the (3P-1P) absorbance difference spectrum. Analysis of the flash-induced (P+QA--PQA) and (3P-1P) absorbance difference spectra of both mutants compared with wild-type at 80K indicate that the cation of the oxidized donor P+ is predominantly localized on the chlorophyll PD1 of the special pair and that the reaction centre triplet state, produced upon charge recombination from 3[P+Pheo-], when the primary quinone electron acceptor QA is doubly reduced, is primarily localized on ChlD1.     

Purification and characterization of a stable oxygen-evolving Photosystem II complex from a marine centric diatom, Chaetoceros gracilis

Oxygen-evolving Photosystem II particles (crude PSII) retaining a high oxygen-evolving activity have been prepared from a marine centric diatom, Chaetoceros gracilis (Nagao et al., 2007). The crude PSII, however, contained a large amount of fucoxanthin chlorophyll a/c-binding proteins (FCP). In this study, a purified PSII complex which was deprived of major components of FCP was isolated by one step of anion exchange chromatography from the crude PSII treated with Triton X-100. The purified PSII was still associated with the five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31 and PsbU, and showed a high oxygen-evolving activity of 2135 μmol O₂ (mg Chl a)^− 1 h^− 1 in the presence of phenyl-p-benzoquinone which was virtually independent of the addition of CaCl₂. This activity is more than 2.5-fold higher than the activity of the crude PSII. The activity was completely inhibited by 3-(3,4)-dichlorophenyl-(1,1)-dimethylurea (DCMU). The purified PSII contained 42 molecules of Chl a, 2 molecules of diadinoxanthin and 2 molecules of Chl c on the basis of two molecules of pheophytin a, and showed typical absorption and fluorescence spectra similar to those of purified PSIIs from the other organisms. In this study, we also found that the crude PSII was significantly labile, as a significant inactivation of oxygen evolution, chlorophyll bleaching and degradation of PSII subunits were observed during incubation at 25 °C in the dark. In contrast, these inactivation, bleaching and degradation were scarcely detected in the purified PSII. Thus, we succeeded for the first time in preparation of a stable PSII from diatom cells.     

Gel electrophoresis of chloroplast membranes of mutants of Chlamydomonas reinhardii which have impaired Photosystem II function and lack photosynthetic cytochromes

Photosystem (PS) II-enriched particles or chloroplast fragments of the wild type and of three nonphotosynthetic mutants of Chlamydomonas reinhardii, which lack chloroplast cytochromes, were analyzed by lithium dodecyl sulfate polyacrylamide gel electrophoresis at 4°C to locate which chlorophyll complexes and which proteins are associated with cytochrome b-559. Two mutants, Fl 39 and Fl 50, have previously been shown to contain, respectively, 3.6- and 2.7-times less hydroquinone-reducible high-potential cytochrome b-559 than the wild type. They have impaired PS II functions. In the presence of ADRY agents: carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p) or 2-(3,4,5-trichloro)-anilino-3,5-dinitrothiophene (ANT 2s), Fl 50 carried out photo-oxidation of cytochrome b-559 with half the amplitude of that of the wild type. No photo-oxidation was observed with Fl 39. We show here that in both these mutants chlorophyll-protein complexes CP III, CP IV and CP V were missing. There were traces of the corresponding apoproteins (45 000, 42 000 and 33 000 daltons, respectively) in Fl 50 but none in Fl 39. In addition, a 19 000 dalton protein was missing in Fl 39 and present in a very small amount in Fl 50. In another mutant, Fl 9, previously characterized as lacking both cytochromes b-563 and c-553 with a normal cytochrome b-559 content, CP III-CP V and the 19 000 dalton protein were detected. CP I (110 000 daltons) and CP II (24 000 daltons) were present in all strains. These observations confirmed the close relationship between deficiencies in cytochrome b-559, lack of CP III and CP IV and anomalies in the photochemistry of PS II. They provided additional evidence that CP V and a 19 000 dalton protein are also involved in this PS II photochemistry. Staining of the gels with 3,3′,5,5′-tetramethylbenzidine and H₂O₂ allowed us to distinguish clearly four heme protein bands having peroxidase activity. Three of these bands (45 000, 42 000 and 19 000 daltons), which were shown in wild-type, Fl 39 and Fl 50 preparations but not in Fl 9, appeared related to cytochromes b-563 and c-553. The fourth heme protein (14 000 daltons) occurred in wild type and Fl 9 but was missing in Fl 39 and Fl 50; it appeared related to cytochrome b-559.