Overcoming barriers to mental health care: multimodal trauma-focused treatment approach for unaccompanied refugee minors.

BACKGROUND: This study evaluated the feasibility of a short-term, multimodal trauma-focused treatment approach adapted specifically for unaccompanied refugee minors (URMs) in the Netherlands. This approach aims to overcome barriers to mental health care and to reduce symptoms of posttraumatic stress disorder (PTSD) and depression. METHODS: An uncontrolled study was conducted, evaluating the main request for help, treatment integrity and feasibility, and the course of symptoms of PTSD (Children's Revised Impact of Event Scale-13) and depression (Patient Health Questionnaire modified for Adolescents). RESULTS: In total, 41 minors were included in the study. Most participants were male (n = 27), predominately from Eritrea (75.6%) with a mean age of 16.5 (SD = 1.5). Minors mostly reported psychological problems, such as problems sleeping, and psychosocial problems, including worries about family reunification. Deviations from the approach were made to meet the current needs of the minors. Factors limiting the feasibility of the approach were often related to continuous stressors, such as news concerning asylum status. CONCLUSIONS: The results provide a first indication that this approach partly overcomes barriers to mental health care and emphasize the added value of collaborating with intercultural mediators and offering outreach care. TRIAL REGISTRATION: The study was registered in the Netherlands Trial Register (NL8585), 10 April 2020, Retrospectively registered, https://www.trialregister.nl/trial/8585 .

An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching.

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the ß-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

Orthopaedic evaluation in children with severe haemophilia A or B submitted to primary prophylaxis therapy in a coagulopathy treatment centre.

There is a lack of publications concerning the use of primary prophylaxis in developing countries. The aim of this study was to evaluate the effectiveness of primary prophylaxis therapy in preventing the development of arthropathy in children with severe haemophilia A or B. From January 1999 to April 2009, a prospective study was carried out involving 39 patients with severe haemophilia A or B. These haemophilia A and haemophilia B patients received 20-40 UI kg(-1) of factors VIII and IX, three and two times per week, respectively. The patients were followed up by a multidisciplinary team. The analysis was carried out in 23 patients who had been on prophylaxis therapy for at least 12 months. The orthopaedic evaluation was performed according to the recommendations of the Orthopedic Advisory Committee of the World Federation of Hemophilia, by evaluating pain and bleeding, and by conducting physical examination and radiological assessment (Pettersson's Joint Score and magnetic resonance): 82.6% of patients who had used the factor regularly did not present any clinical or radiographic changes in the studied joints; 17.4% used the factor irregularly at the beginning of the treatment and of those, most patients presented mild changes in the joints; and 4.3% presented transient knee and ankle pain in spite of regular factor use. The preliminary results of primary prophylaxis confirm its effectiveness in preventing haemophilic arthropathy. Socioeconomic factors did not play a significant role.

High satisfaction rates in women after DIEP flap breast reconstruction.

BACKGROUND: Breast reconstruction (BR) is aimed at improving quality of life (QoL) after mastectomy. Patient satisfaction is an important indicator to evaluate the success of BR. This study explored patient satisfaction and its determinants in women undergoing deep inferior epigastric artery perforator (DIEP) flap BR as well as the impact of the procedure on body image, sexuality and QoL. METHODS: Patient satisfaction and QoL were studied in 72 women who underwent DIEP flap BR using a study-specific questionnaire as well as the Short Form-36 (SF-36). RESULTS: Patient satisfaction was very high. Approximately 90% of the patients reported that they had been sufficiently informed about the procedure and its consequences, that their preoperative expectations had been met, that the reconstructed breast felt like their own and that they would choose the same procedure again and would recommend this procedure to a friend. Patient satisfaction was positively and significantly related to the reconstructed breast(s) feeling like their own. Women with secondary reconstructions were more positive about changes in sexuality and femininity than women with primary BRs. There were no clinically relevant differences in QoL between our study population and a random sample of Dutch females. CONCLUSIONS: Women with DIEP flap BRs reported high satisfaction rates. However, to compare these satisfaction rates with other forms of BR, prospective studies in comparable groups are necessary.

Clinical evaluation after 1 year of 153-samarium hydroxyapatite synovectomy in patients with haemophilic arthropathy.

The aim is to evaluate the efficiency of the treatment with 153-samarium hydroxyapatite (153-Sm-HA) in haemophilic arthropathy. Thirty-one patients (30 males) with ages ranging from 8 to 34 years (average age = 20.6 years) were treated with fixed intra-articular dose of 185 MBq (5 mCi) and divided into two groups: infantile-juvenile (13 patients with up to 18 years of age, an average age of 12.7 years and arthropathy evolution of 7.8 years), and adult (18 patients older than 18 years, an average age of 24 years and arthropathy evolution of 18.7 years). The clinical evaluation before and after 1 year of synovectomy used the following criteria: subjective (pain through visual scale, articulation inspection), objective (articular movement through flexion level, sensitivity to palpation and leakage through joint circumference), reduction on the use of the coagulation factor, number of haemarthrosis, and the occurrence of adverse effects. The results were classified as: 1, good (remission from 70% to 100% of manifestations); 2, moderate (remission from 40% to 69%); and 3, poor (remission from 0% to 39%). Seventy-eight joints were tested: 15 knees, 36 elbows, 24 ankles, 1 shoulder and 2 hips. Early scintigraphic (1-2 h) and late scintigraphic (24-72 h) studies were performed after synoviorthesis. The cost of the procedure per joint was also estimated. No significant difference in the synoviorthesis result between groups was observed. The results were good for 75% of elbows, 87.5% of ankles and 40% of knees; the reduction in haemarthrosis and use of the coagulation factor was respectively 78% and 80% for elbows, 82% and 85% for ankles and 30% and 35% for knees. Four cases of reactional synovitis were observed in the 31 patients. The scintigraphic control showed homogeneous distribution of the material with no articular escape. The use of 153 Sm-HA in the treatment of the haemophilic arthropathy is effective for intermediate-size joints (elbows and ankles), but less effective for knees. Moreover, this treatment presents an excellent safety profile and accessible cost.

Efficacy of synovectomy in haemophilic patients with 153Sm-hydroxyapatite

Introduction - We preconized the use of 153Sm-Hydroxyapatite (HYP) in the synoviorthesis of haemophilic patients (pts) by physical, chemical and biological characteristics. The real efficacy is beeing confirmed by the actual experience over the previous results. These are the first results with 153Sm-HYP in our literature review. Material and Methods - Fiftheen pts. were treated, all males, with ages between 15 and 31 years (average = 22,8 years old), with an intraarticular injection of 185MBq (5mCi) of 153Sm-HYP, totalizing 29 joints: 12 knees, 11 elbows, 4 ankles and 2 shoulders. The 153Sm, produced by IPEN/CNEN - São Paulo, targeted hydroxyapatite particles with a diameter between 1-10µm, radiochemical purity superior to 95 percent and stability of 96,6 percent 1440min after the labeling process. The intraarticular punction was made after local antiseptic, aspirating synovial fluid, injecting 0,5 ml of radioactive solution and flushing the tract with a saline with a total volume no greater than 1,5 ml. The images were obtained 2 and 24h after injection in all pts in a large field of view gamma-camera. The clinical evaluation was made before and one year after it, using objective (range of motion, joint tenderness, degree of joint effusion) and subjective criteria (joint pain by visual scale and joint aspect). The response were graded: 1 - Excellent (E); 2 - Good (G); 3 - Mild (M); 4 - Bad (B); 5 - Worse (W). The reduction in clotting factor use and heamarthroses were others aspects evaluated. Results - The scintigrafies showed homogeneous distribution of the material in joints (2h) and no articular scape (24h). The was a reduction of 34 percent in the clotting factor use and of 51,4 percent in haemarthroses including the poor responses in knees. The results by patients were: 53,3 percent E, 20 percent G, 13,3 percent M, 13,3 percent B, 0 percent W and by joints were 47,75 with excellent and good responses and 84,75 percent when included the mild group. The...

Satisfaction with prophylactic mastectomy and breast reconstruction in genetically predisposed women.

BACKGROUND: Prophylactic mastectomy with breast reconstruction is a risk-reducing strategy for women at increased risk of breast cancer. It remains a very radical intervention, and long-term data on satisfaction are insufficiently available. In the present follow-up study, the authors assess satisfaction with prophylactic mastectomy and breast reconstruction and its impact on sexual relationships. METHODS: The authors conducted a retrospective study using a short self-report questionnaire administered to 114 genetically predisposed women who underwent prophylactic mastectomy and breast reconstruction mainly by subpectorally implanted silicone prostheses performed at one institution. RESULTS: The median follow-up time between prophylactic mastectomy/breast reconstruction and completion of the questionnaire was 3 years. Sixty percent of all participants were satisfied with the result of prophylactic mastectomy/breast reconstruction. Satisfaction was significantly and negatively correlated with perceived lack of information, experienced complications, ongoing complaints, whether or not the reconstructed breasts feel "like your own," and not choosing this type of breast reconstruction again. Adverse effects in the patient's sexual relationship were strongly correlated with perceived lack of information, discrepant expectations, ongoing complaints and limitations, whether or not the reconstructed breasts feel "like your own," altered feelings of femininity, partner's negative perception on femininity and sexuality, and not choosing this type of breast reconstruction again. CONCLUSIONS: A majority of women would choose the procedure again, but adverse effects and untoward changes in the perception of the sexual relationship need to be addressed in the counselling of women at high risk, to optimize an informed choice and enable adequate adjustment postoperatively.

Factors that determine the unusually low reduction potential of cytochrome c550 in cyanobacterial photosystem II.

A new purification protocol for cytochrome c550 (cyt c550) from His-tagged SYnechocYstis PCC 6803 photosystem II (PSII) was developed which allows the protein to be isolated in high yield and purity. Electron paramagnetic resonance spectroscopy of cyt c550, both free in solution and in intact PSII preparations, yields identical spectra with g values at 1.50, 2.23, and 2.87, which are characteristic for a ferric low-spin bis-histidine coordinated heme. The resonance Raman spectrum of the isolated protein exhibits features characteristic of bis-histidine axial ligation of the iron and a slight ruffling of the heme macrocycle. Together, these results indicate that the heme structure is not very different from most c-type cytochromes, and thus the structure of the heme does not account for its unusually low reduction potential. A direct electrochemical measurement of the reduction potential was performed using square wave voltammetry on a pyrolytic graphite edge electrode, yielding E1.2=-108 mV (vs. NHE) with a peak separation of 5 mV. This value is 150 mV more positive than that previously measured by redox titrations. Because the behavior of the protein in the electrochemistry experiments is indicative of adsorption to the electrode surface, we surmise that binding of the protein to the electrode excludes solvent water from the heme-binding site. We conclude that the degree of solvent exposure makes a significant contribution to the heme reduction potential. Similarly, the binding of cyt c550 to PSII may also reduce the solvent exposure of the heme, and so the direct electrochemical value of the reduction potential may be relevant to the protein in its native state.

Characterization of the O(2)-evolving reaction catalyzed by [(terpy)(H2O)Mn(III)(O)2Mn(IV)(OH2)(terpy)](NO3)3 (terpy = 2,2':6,2"-terpyridine).

The complex [(terpy)(H(2)O)Mn(III)(O)(2)Mn(IV)(OH(2))(terpy)](NO(3))(3) (terpy = 2,2':6,2' '-terpyridine) (1)catalyzes O(2) evolution from either KHSO(5) (potassium oxone) or NaOCl. The reactions follow Michaelis-Menten kinetics where V(max) = 2420 +/- 490 mol O(2) (mol 1)(-1) hr(-1) and K(M) = 53 +/- 5 mM for oxone ([1] = 7.5 microM), and V(max) = 6.5 +/- 0.3 mol O(2) (mol 1)(-1) hr(-1) and K(M) = 39 +/- 4 mM for hypochlorite ([1] = 70 microM), with first-order kinetics observed in 1 for both oxidants. A mechanism is proposed having a preequilibrium between 1 and HSO(5-) or OCl(-), supported by the isolation and structural characterization of [(terpy)(SO(4))Mn(IV)(O)(2)Mn(IV)(O(4)S)(terpy)] (2). Isotope-labeling studies using H(2)(18)O and KHS(16)O(5) show that O(2) evolution proceeds via an intermediate that can exchange with water, where Raman spectroscopy has been used to confirm that the active oxygen of HSO(5-) is nonexchanging (t(1/2) >> 1 h). The amount of label incorporated into O(2) is dependent on the relative concentrations of oxone and 1. (32)O(2):(34)O(2):(36)O(2) is 91.9 +/- 0.3:7.6 +/- 0.3:0.51 +/- 0.48, when [HSO(5-)] = 50 mM (0.5 mM 1), and 49 +/- 21:39 +/- 15:12 +/- 6 when [HSO(5-)] = 15 mM (0.75 mM 1). The rate-limiting step of O(2) evolution is proposed to be formation of a formally Mn(V)=O moiety which could then competitively react with either oxone or water/hydroxide to produce O(2). These results show that 1 serves as a functional model for photosynthetic water oxidation.

High-frequency EPR study of a new mononuclear manganese(III) complex:. [(terpy)Mn(N3)3] (terpy = 2,2':6',2''-terpyridine).

The isolation and structural characterization of [(terpy)Mn(III)(N3)3], complex 1, is reported (terpy = 2,2':6',2' '-terpyridine). Complex 1, a product of the reaction between the mixed-valence dimer [(terpy)(H2O)Mn(III)(O)2Mn(IV)(OH2)(terpy)](NO3)3 and NaN3, crystallizes in a triclinic system, space group P1, a = 8.480(1) A, b = 8.9007(2) A, c = 12.109(2) A, alpha = 93.79(1) degrees, beta = 103.17(1) degrees, gamma = 103.11(1) degrees, and Z = 2. Complex 1 exhibits a Jahn-Teller distortion of the octahedron characteristic of a six-coordinated high-spin Mn(III). A vibrational spectroscopic study was performed. The nu(asym)(N3) mode of complex 1 appears in the IR as a strong band at 2035 cm(-1) with a less intense feature at 2072 cm(-1), and in the FT-Raman as a strong band at 2071 cm(-1) with a weaker broad band at 2046 cm(-1). The electronic properties of complex 1 were investigated using a high-field and high-frequency EPR study (190-475 GHz). The different spin Hamiltonian parameters have been determined (D = -3.29 (+/-0.01) cm(-1), E = 0.48 (+/-0.01) cm(-1), E '= 0.53 (+/-0.01) cm(-1), g(x) = 2.00 (+/-0.005), g(y) = 1.98 (+/-0.005), g(z) = 2.01 (+/-0.005)). These parameters are in agreement with the geometry of complex 1 observed in the crystal structure, a D < 0 related to the elongated distortion, and a value of E/D close to 0.2 as expected from the highly distorted octahedron. The two values of the E-parameter are explained by the presence of two slightly different structural forms of complex 1 in the crystal lattice. A second hypothesis was explored to explain the experimental data. The calculation for the simulation was done taking into account that the g and D tensors are not collinear due to the low symmetry of complex 1. In that case, the spin Hamiltonian parameters found are D = -3.29 (+/-0.01) cm(-1), E = 0.51 (+/-0.01) cm(-1), g(x) = 2.00 (+/-0.005), g(y) = 1.98 (+/-0.005), and g(z) = 2.01 (+/-0.005).

Depolarized resonance light scattering by porphyrin and chlorophyll a aggregates.

A quantum mechanical model is developed for the observed resonance enhancement of light scattering by aggregates of electronically interacting chromophores. Aggregate size, monomer oscillator strength, extent of electronic coupling, and aggregate geometry are all important determinants of intensity in resonance light scattering (RLS) spectra. The theory also predicts the value of the depolarization ratio (rho(v)(90)) of RLS for a given aggregate geometry. These results are used to interpret the RLS depolarization ratios of four aggregates: tetrakis(4-sulfonatophenyl)porphine aggregated at low pH (rho(v)(90) = 0.17 at 488 nm), trans-bis(N-methylpyridinium-4-yl)-diphenylporphinato copper(II) aggregated in 0.2 M NaCl solution (rho(v)(90) = 0.13 at 450 nm) and on calf thymus DNA (rho(v)(90) = 0.20 at 454 nm), and chlorophyll a aggregates in formamide/water (rho(v)(90) = 0.23 and 0.32 at 469 and 699 nm, respectively). The analysis is consistent with a J-aggregate geometry for all four systems. Furthermore, the specific values of rho(v)(90) allow us to estimate the orientation of the monomer transition dipoles with respect to the long axis of the aggregate. We conclude that depolarized resonance light scattering spectroscopy is a powerful probe of the geometric and electronic structures of extended aggregates of strong chromophores.

Aggregation of chlorophyll a probed by resonance light scattering spectroscopy.

We report the resonance light scattering (RLS) spectra of chlorophyll a aggregated in a 9:1 solution of formamide and pH 6.8 phosphate buffer. The aggregate formed after 2 h of mixing, referred to as Chl469, shows a strong scattering feature at 469 nm (Soret band) and a much weaker feature at 699 nm (Qy band). A kinetic investigation confirmed that the aggregation process is cooperative, and also detected one intermediate (Chl458) with a strong RLS spectrum but only a weak CD spectrum. We propose that aggregation proceeds via at least three steps: 1) formation of a nucleating species, probably a dimer of chlorophylls; 2) formation of large aggregates with little or no secondary structure (e.g., Chl458); and 3) conformational change to form helical aggregate (Chl469).

Structure-function relationships in the 47-kDa antenna protein and its complex with the photosystem II reaction center core: insights from picosecond fluorescence decay kinetics and resonance Raman spectroscopy.

We report the fluorescence decay kinetics and the vibrational properties of chlorophyll a bound to the 47-kDa antenna protein (CP47) of spinach photosystem II. The chlorophyll fluorescence of CP47 samples decays with four lifetimes (tau = 75.8 ps, 1.05 ns, 3.22 ns, and 5.41 ns). The 75.8-ps and 3.22-ns components are associated with chlorophyll a bound to relatively intact centers, the 1.05-ns component corresponds to chlorophyll bound to centers that are slightly perturbed, and the the 5.41-ns phase probably originates from centers that are severely denatured. The resonance Raman spectrum of CP47 at 441.6 nm (this work) and at 406.7 nm [de Paula, J. C., Ghanotakis, D. F., Bowlby, N. R., Dekker, J. P., Yocum, C. F., & Babcock, G. T. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.), Vol. I, pp 643-646, Kluwer Academic Publishers, Dordrecht, The Netherlands] shows heterogeneity in the C = O stretching region. This part of the spectrum monitors the environment of the keto group at position 9 of the chlorophyll a molecule. We show that several structurally distinct pools of chlorophyll a are bound to CP47. Four of these may be distinguished by their C9 = O stretching frequencies (nu C = O = 1670, 1688, 1693, and 1701 cm-1). By analyzing the resonance enhancement pattern of these modes, we ascribe the 1693-cm-1 vibration to denatured centers. Of the remaining populations, we propose that the 1670-cm-1 vibration is consistent with a hydrogen bond between the C9 = O group of chlorophyll a and the protein. We elaborate on the role of this chromophore-protein interaction in the mechanism of energy transfer within the 47-kDa antenna protein.

Hemes a and a3 environments of plant cytochrome c oxidase.

The structures of hemes a and a3 of maize and wheat germ cytochrome c oxidase were investigated by resonance Raman spectroscopy. Comparison between the plant and mammalian cytochrome oxidases revealed that (i) the vinyl groups associated with hemes a and a3 vibrate at higher frequencies in the plant enzyme than in the mammalian enzyme, suggesting different degrees of interaction between the heme cores and their periphery; (ii) aside from the geometry of the vinyl group, the structure of heme a3 in plant cytochrome oxidase is essentially unchanged from that of its mammalian counterpart; (iii) the vibrational band associated with the formyl group of reduced heme a shows relatively weak enhancement in the Soret-excited resonance Raman spectra of maize and wheat germ cytochrome oxidase, suggesting that the formyl group of ferrous heme a in the plant enzymes is conjugated only slightly to the porphyrin ring; and (iv) for oxidized heme a, the formyl vibration is strongly enhanced, but its frequency indicates a weaker interaction with the protein milieu relative to the mammalian enzyme. These observations suggest that the local environment around the formyl position of the heme a chromophore differs in the plant and mammalian cytochrome oxidases. The implication of the latter feature in the mechanism of proton pumping by cytochrome oxidase is discussed.

Characterization of the multiple forms of cytochrome b559 in photosystem II.

Cytochrome b559 is an essential component of the photosystem II (PSII) protein complex. Its function, which has long been an unsolved puzzle, is likely to be related to the unique ability of PSII to oxidize water. We have used EPR spectroscopy and spectrophotometric redox titrations to probe the structure of cytochrome b559 in PSII samples that have been treated to remove specific components of the complex. The results of these experiments indicate that the low-temperature photooxidation of cytochrome b559 does not require the presence of the 17-, 23-, or 33-kDa extrinsic polypeptides or the Mn complex (the active site in water oxidation). We observe a shift in the g value of the EPR signal of cytochrome b559 upon warming a low-temperature photooxidized sample, which presumably reflects a change in conformation to accommodate the oxidized state. At least three redox forms of cytochrome b559 are observed. Untreated PSII membranes contain one high-potential (375 mV) and one intermediate-potential (230 mV) cytochrome b559 per PSII. Thylakoid membranes also appear to contain one high-potential and one intermediate-potential cytochrome b559 per PSII, although this measurement is more difficult due to interference from other cytochromes. Removal of the 17- and 23-kDa extrinsic polypeptides from PSII membranes shifts the composition to one intermediate-potential (170 mV) and one low-potential (5 mV) cytochrome b559. This large decrease in potential is accompanied by a very small g-value change (0.04 at gz), indicating that it is the environment and not the ligand field of the heme which changes significantly upon the removal of the 17- and 23-kDa polypeptides.

Isolation and characterization of the 47 kDa protein and the D1-D2-cytochrome b-559 complex.

The 47 kDa polypeptide and a protein complex consisting of the D1 (32 kDa), D2 (34 kDa) and cytochrome b-559 (9 kDa) species were isolated from a Tris-washed Photosystem II core complex solubilized with dodecylmaltoside in the presence of LiClO4. Although the 43 kDa chlorophyll-binding protein is readily dissociated from the Photosystem II complex under our conditions, two cycles of exposure to high concentrations of detergent and LiClO4 were required for complete removal of the 47 kDa chlorophyll-binding protein from the D1-D2-cytochrome b-559 complex. Spectroscopic characterization of these two species revealed that the 47 kDa protein binds chlorophyll a, whereas the D1-D2-cytochrome b-559 complex shows an enrichment in Pheo a and heme on a chlorophyll basis. A spin-polarized EPR triplet can be observed at liquid helium temperatures in the D1-D2-cytochrome b-559 complex, but no such triplet is observed in the purified 47 kDa species. The zero-field splitting parameters of the P-680+ triplet indicate that the triplet spin is localized onto one chlorophyll molecule. Resonance Raman spectroscopy showed that: (i) beta-carotene is bound to the reaction center in its all-trans conformation; (ii) all chlorophyll a molecules are five-coordinate; and (iii) the C-9 keto group of one of the chlorine pigments is hydrogen-bonded. Our results support the proposal that the D1-D2 complex binds the P-680+ and Pheo a species that are involved in the primary charge separation.

Formation of the S2 state and structure of the Mn complex in photosystem II lacking the extrinsic 33 kilodalton polypeptide.

Electron paramagnetic resonance (EPR) spectroscopy and O2 evolution assays were performed on photosystem II (PSII) membranes which had been treated with 1 M CaCl2 to release the 17, 23 and 33 kilodalton (kDa) extrinsic polypeptides. Manganese was not released from PSII membranes by this treatment as long as a high concentration of chloride was maintained. We have quantitated the EPR signals of the several electron donors and acceptors of PSII that are photooxidized or reduced in a single stable charge separation over the temperature range of 77 to 240 K. The behavior of the samples was qualitatively similar to that observed in samples depleted of only the 17 and 23 kDa polypeptides (de Paula et al. (1986) Biochemistry25, 6487-6494). In both cases, the S2 state multiline EPR signal was observed in high yield and its formation required bound Ca(2+). The lineshape of the S2 state multiline EPR signal and the magnetic properties of the manganese site were virtually identical to those of untreated PSII membranes. These results suggest that the structure of the manganese site is unaffected by removal of the 33 kDa polypeptide. Nevertheless, in samples lacking the 33 kDa polypeptide a stable charge separation could only be produced in about one half of the reaction centers below 160 K, in contrast to the result obtained in untreated or 17 and 23 kDa polypeptide-depleted PSII membranes. This suggests that one function of the 33 kDa polypeptide is to stabilize conformations of PSII that are active in secondary electron transfer events.

Effect of the 17- and 23-kilodalton polypeptides, calcium, and chloride on electron transfer in photosystem II.

Electron paramagnetic resonance (EPR) measurements were performed on photosystem II (PSII) membranes that were treated with 2 M NaCl to release the 17- and 23-kilodalton (kDa) polypeptides. By using 75 microM 3-(3,4-dichlorophenyl)-1,1-dimethylurea to limit the photosystem II samples to one stable charge separation in the temperature range of 77-273 K, we have quantitated the EPR signals of the several electron donors and acceptors of photosystem II. It was found that removal of the 17- and 23-kDa polypeptides caused low potential cytochrome b559 to become fully oxidized during the course of dark adaptation. Following illumination at 77-130 K, one chlorophyll molecule per reaction center was oxidized. Between 130 and 200 K, both a chlorophyll molecule and the S1 state were photooxidized and, together, accounted for one oxidation per reaction center. Above 200 K, the chlorophyll radical was unstable. Oxidation of the S1 state gave rise to the S2-state multiline EPR signal, which arises from the Mn site of the O2-evolving center. The yield of the S2-state multiline EPR signal in NaCl-washed PSII membranes was as high as 93% of the control, untreated PSII membranes, provided that both Ca2+ and Cl- were bound. Furthermore, the 55Mn nuclear hyperfine structure of the S2-state multiline EPR signal was unaltered upon depletion of the 17- and 23-kDa polypeptides. In NaCl-washed PSII samples where Ca2+ and/or Cl- were removed, however, the intensity of the S2-state multiline EPR signal decreased in parallel with the fraction of PSII lacking bound Ca2+ and Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)