Serum concentration-guided intravenous magnesium sulfate administration for neuroprotection in patients with aneurysmal subarachnoid hemorrhage: a retrospective evaluation of a 12-year single-center experience.

Delayed cerebral infarction (DCI) is a major cause of morbidity and mortality in patients with aneurysmal subarachnoid hemorrhage (aSAH). The benefits of magnesium sulfate as an alternative treatment are controversial, and most previous studies examined its benefits only as adjunctive treatment to traditional nimodipine. We retrospectively analyzed aSAH patients records with magnesium sulfate between 2010 and 2021. We aimed for a serum magnesium concentration of 2-2.5 mmol/l between post-hemorrhage days 3 and 12. The patients were separated in three groups based on average serum magnesium concentration (magnesium >2 mmol/l, reduced magnesium 1.1-1.9 mmol/l, and no magnesium). Additionally, we assessed delayed cerebral infarction (DCI) and clinical outcome at follow-up, using the modified Rankin Scale (mRS), categorized in favorable (0-3) and unfavorable outcome (4-5). In this analysis, 548 patients were included. Hereof, radiological evidence of DCI could be found in 23.0% (n = 126) of patients. DCI rates were lower if patients' average serum magnesium was higher than 2 mmol/l (magnesium 18.8%, n = 85; reduced magnesium 38.3%, n = 23; no magnesium 51.4%, n = 18; p < 0.001). Also, at the last follow-up, patients in the group with a higher serum magnesium concentration had better outcome (favorable outcome: magnesium 64.7%, n = 293; reduced magnesium 50.0%, n = 30; no magnesium 34.3%, n = 12; p < 0.001). This 12-year study reveals the value of serum concentration-guided magnesium administration in aSAH patients. Our findings demonstrate the safety and efficacy when titrated to a serum concentration of 2-2.5 mmol/l. We observed higher rates of delayed cerebral infarction and unfavorable outcomes in patients with serum concentrations below 2 mmol/l.

A new instrument for kinetics and branching ratio studies of gas phase collisional processes at very low temperatures.

A new instrument dedicated to the kinetic study of low-temperature gas phase neutral-neutral reactions, including clustering processes, is presented. It combines a supersonic flow reactor with vacuum ultra-violet synchrotron photoionization time-of-flight mass spectrometry. A photoion-photoelectron coincidence detection scheme has been adopted to optimize the particle counting efficiency. The characteristics of the instrument are detailed along with its capabilities illustrated through a few results obtained at low temperatures (<100 K) including a photoionization spectrum of n-butane, the detection of formic acid dimer formation, and the observation of diacetylene molecules formed by the reaction between the C2H radical and C2H2.

Preliminary Findings Associate Hippocampal 1H-MR Spectroscopic Metabolite Concentrations with Psychotic and Manic Symptoms in Patients with Schizophrenia.

BACKGROUND AND PURPOSE: Previous hippocampal proton MR spectroscopic imaging distinguished patients with schizophrenia from controls by elevated Cr levels and significantly more variable NAA and Cho concentrations. This goal of this study was to ascertain whether this metabolic variability is associated with clinical features of the syndrome, possibly reflecting heterogeneous hippocampal pathologies and perhaps variability in its "positive" (psychotic) and "negative" (social and emotional deficits) symptoms. MATERIALS AND METHODS: In a sample of 15 patients with schizophrenia according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, we examined the association of NAA and Cho levels with research diagnostic interviews and clinical symptom ratings of the patients. Metabolite concentrations were previously obtained with 3D proton MR spectroscopic imaging at 3T, a technique that facilitates complete coverage of this small, irregularly shaped, bilateral, temporal lobe structure. RESULTS: The patient cohort comprised 8 men and 7 women (mean age, 39.1 [SD, 10.8] years, with a mean disease duration of 17.2 [SD, 10.8] years. Despite the relatively modest cohort size, we found the following: 1) Elevated Cho levels predict the positive (psychotic, r = 0.590, P = .021) and manic (r = 0.686, P = .005) symptom severity; and 2) lower NAA levels trend toward negative symptoms (r = 0.484, P = .08). No clinical symptoms were associated with Cr level or hippocampal volume (all, P ≥ .055). CONCLUSIONS: These preliminary findings suggest that NAA and Cho variations reflect different pathophysiologic processes, consistent with microgliosis/astrogliosis and/or lower vitality (reduced NAA) and demyelination (elevated Cho). In particular, the active state-related symptoms, including psychosis and mania, were associated with demyelination. Consequently, their deviations from the means of healthy controls may be a marker that may benefit precision medicine in selection and monitoring of schizophrenia treatment.

Limited nucleotide pools restrict Epstein-Barr virus-mediated B-cell immortalization.

Activation of cellular oncogenes as well as infection with tumor viruses can promote aberrant proliferation and activation of the host DNA damage response. Epstein-Barr virus (EBV) infection of primary human B cells induces a transient period of hyper-proliferation, but many of these infected cells succumb to an ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/Chk2)-mediated senescence-like growth arrest. In this study, we assessed the role of DNA replicative stress and nucleotide pool levels in limiting EBV-infected B-cell outgrowth. We found that EBV triggered activation of the ataxia telangiectasia and Rad3-related (ATR) signaling pathway in the early rapidly proliferating cells, which were also significantly more sensitive to inhibition of the ATR pathway than late attenuated proliferating cells. Through nuclear halo assays, we determined that early EBV-infected cells displayed increased replicative stress and DNA damage relative to late proliferating cells. Finally, we found that early after infection, hyper-proliferating B cells exhibited limited deoxyribonucleotide triphosphate (dNTP) pools compared with late proliferating and EBV-immortalized lymphoblastoid cell lines with a specific loss of purine dNTPs. Importantly, supplementation with exogenous nucleosides before the period of hyper-proliferation markedly enhanced B-cell immortalization by EBV and rescued replicative stress. Together our results suggest that purine dNTP biosynthesis has a critical role in the early stages of EBV-mediated B-cell immortalization.

Towards characterization of photo-excited electron transfer and catalysis in natural and artificial systems using XFELs.

The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting X-ray diffraction data and X-ray emission spectra, using an energy dispersive spectrometer, at ambient conditions, and used this approach to study the room temperature structure and intermediate states of the photosynthetic water oxidizing metallo-protein, photosystem II. Moreover, we have also used this setup to simultaneously collect the X-ray emission spectra from multiple metals to follow the ultrafast dynamics of light-induced charge transfer between multiple metal sites. A Mn-Ti containing system was studied at an XFEL to demonstrate the efficacy and potential of this method.

Ionized hypercalcemia in dogs: a retrospective study of 109 cases (1998-2003).

BACKGROUND: Serum hypercalcemia in dogs has been reported in association with a variety of diseases. Serum-ionized calcium (iCa) concentration is a more accurate measure of hypercalcemia than total serum calcium or corrected serum calcium concentrations. The severity of hypercalcemia has been utilized to suggest the most likely differential diagnosis for the hypercalcemia. HYPOTHESIS: Diseases causing ionized hypercalcemia may be different than those that cause increases in total or corrected serum calcium concentrations. The severity of ionized hypercalcemia in specific diseases cannot be used to determine the most likely differential diagnosis for ionized hypercalcemia. ANIMALS: One-hundred and nine client-owned dogs with a definitive cause for their ionized hypercalcemia evaluated between 1998 and 2003 were included in this study. METHODS: Retrospective, medical records review. RESULTS: Neoplasia, specifically lymphosarcoma, followed by renal failure, hyperparathyroidism, and hypoadrenocorticism were the most common causes of ionized hypercalcemia. Dogs with lymphoma and anal sac adenocarcinoma have higher serum iCa concentrations than those with renal failure, hypoadrenocorticism, and other types of neoplasia. The magnitude of serum-ionized hypercalcemia did not predict specific disease states. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum-ionized hypercalcemia was most commonly associated with neoplasia, specifically lymphosarcoma. Although dogs with lymphosarcoma and anal sac adenocarcinoma had higher serum iCa concentrations than dogs with other diseases, the magnitude of the serum iCa concentration could not be used to predict the cause of hypercalcemia. Total serum calcium and corrected calcium concentrations did not accurately reflect the calcium status of the dogs in this study.

Evaluation of inhibitors for 17beta-hydroxysteroid dehydrogenase type 1 in vivo in immunodeficient mice inoculated with MCF-7 cells stably expressing the recombinant human enzyme.

17Beta-hydroxysteroid dehydrogenase (17HSD1) is an enzyme activating estrone (E1) to estradiol (E2). In the present study, a mechanistic animal model was set up for evaluating putative inhibitors for the human enzyme in vivo. Estrogen-dependent MCF-7 human breast carcinoma cells were stably transfected with a plasmid expressing human 17HSD1. These cells formed estrogen-dependent tumors in immunodeficient mice. In the optimized model, tumor sizes were decreased in both ovariectomized and intact vehicle-treated mice, whereas they were maintained or slightly increased in mice supplemented 2 weeks with an appropriate dose of the 17HSD1-substrate E1. Tumor sizes in mice treated with 0.1 micromol/kg/d of E1 were reduced by administering 5 micromol/kg/d of different 17HSD1-inhibitors and a 86% reduction in size was detected with the most potent inhibitor. A dose-response relationship in the inhibitory effect of this compound further confirmed the validity of the model for testing the drug candidates in vivo.

Electron spin-lattice relaxation of the S0 state of the oxygen-evolving complex in photosystem II and of dinuclear manganese model complexes.

The temperature dependence of the electron spin-lattice relaxation time T1 was measured for the S0 state of the oxygen-evolving complex (OEC) in photosystem II and for two dinuclear manganese model complexes by pulse EPR using the inversion-recovery method. For [Mn(III)Mn(IV)(mu-O)2 bipy4]ClO4, the Raman relaxation process dominates at temperatures below 50 K. In contrast, Orbach type relaxation was found for [Mn(II)Mn(III)(mu-OH)(mu-piv)2(Me3 tacn)2](ClO4)2 between 4.3 and 9 K. For the latter complex, an energy separation of 24.7-28.0 cm(-1) between the ground and the first excited electronic state was determined. In the S0 state of photosystem II, the T1 relaxation times were measured in the range of 4.3-6.5 K. A comparison with the relaxation data (rate and pre-exponential factor) of the two model complexes and of the S2 state of photosystem II indicates that the Orbach relaxation process is dominant for the S0 state and that its first excited state lies 21.7 +/- 0.4 cm(-1) above its ground state. The results are discussed with respect to the structure of the OEC in photosystem II.

High-resolution X-ray spectroscopy of rare events: a different look at local structure and chemistry.

The combination of large-acceptance high-resolution X-ray optics with bright synchrotron sources permits quantitative analysis of rare events such as X-ray fluorescence from very dilute systems, weak fluorescence transitions or X-ray Raman scattering. Transition-metal Kbeta fluorescence contains information about spin and oxidation state; examples of the characterization of the Mn oxidation states in the oxygen-evolving complex of photosystem II and Mn-consuming spores from the marine bacillus SG- are presented. Weaker features of the Kbeta spectrum resulting from valence-level and 'interatomic' ligand to metal transitions contain detailed information on the ligand- atom type, distance and orientation. Applications of this spectral region to characterize the local structure of model compounds are presented. X-ray Raman scattering (XRS) is an extremely rare event, but also represents a unique technique to obtain bulk-sensitive low-energy (<600 eV) X-ray absorption fine structure (XAFS) spectra using hard (approximately 10 keV) X-rays. A photon is inelastically scattered, losing part of its energy to promote an electron into an unoccupied level. In many cases, the cross section is proportional to that of the corresponding absorption process yielding the same X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) features. XRS finds application for systems that defy XAFS analysis at low energies, e.g. liquids or highly concentrated complex systems, reactive compounds and samples under extreme conditions (pressure, temperature). Recent results are discussed.

Absence of Mn-centered oxidation in the S(2) --> S(3) transition: implications for the mechanism of photosynthetic water oxidation.

A key question for the understanding of photosynthetic water oxidation is whether the four oxidizing equivalents necessary to oxidize water to dioxygen are accumulated on the four Mn ions of the oxygen-evolving complex (OEC), or whether some ligand-centered oxidations take place before the formation and release of dioxygen during the S(3) --> [S(4)] --> S(0) transition. Progress in instrumentation and flash sample preparation allowed us to apply Mn Kbeta X-ray emission spectroscopy (Kbeta XES) to this problem for the first time. The Kbeta XES results, in combination with Mn X-ray absorption near-edge structure (XANES) and electron paramagnetic resonance (EPR) data obtained from the same set of samples, show that the S(2) --> S(3) transition, in contrast to the S(0) --> S(1) and S(1) --> S(2) transitions, does not involve a Mn-centered oxidation. On the basis of new structural data from the S(3)-state, manganese mu-oxo bridge radical formation is proposed for the S(2) --> S(3) transition, and three possible mechanisms for the O-O bond formation are presented.

Towards understanding the chemistry of photosynthetic oxygen evolution: dynamic structural changes, redox states and substrate water binding of the Mn cluster in photosystem II.

This mini-review summarizes my postdoctoral research in the labs of T. Wydrzynski/C.B. Osmond, J.H.A. Nugent/M.C.W. Evans and V.K. Yachandra/K. Sauer/M.P. Klein. The results are reported in the context of selected data from the literature. Special emphasis is given to the mode of substrate water binding, Mn oxidation states and the structures of the Mn cluster in the four (meta)stable redox states of the oxygen evolving complex. The paper concludes with a working model for the mechanism of photosynthetic water oxidation that combines mu-oxo bridge oxidation in the S(3) state (V.K. Yachandra, K. Sauer, M.P. Klein, Chem. Rev. 96 (1996) 2927-2950) with O-O bond formation between two terminal Mn-hydroxo ligands during the S(3)-->(S(4))-->S(0) transition.

Kinetic determination of the fast exchanging substrate water molecule in the S3 state of photosystem II.

In a previous communication we showed from rapid isotopic exchange measurements that the exchangeability of the substrate water at the water oxidation catalytic site in the S3 state undergoes biphasic kinetics although the fast phase could not be fully resolved at that time [Messinger, J., Badger, M., and Wydrzynski, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3209-3213]. We have since improved the time resolution for these measurements by a further factor of 3 and report here the first detailed kinetics for the fast phase of exchange. First-order exchange kinetics were determined from mass spectrometric measurements of photogenerated O2 as a function of time after injection of H218O into spinach thylakoid samples preset in the S3 state at 10 degreesC. For measurements made at m/e = 34 (i. e., for the mixed labeled 16,18O2 product), the two kinetic components are observed: a slow component with k1 = 2.2 +/- 0.1 s-1 (t1/2 approximately 315 ms) and a fast component with k2 = 38 +/- 4 s-1 (t1/2 approximately 18 ms). When the isotopic exchange is measured at m/e = 36 (i.e., for the double labeled 18,18O2 product), only the slow component (k1) is observed, clearly indicating that the substrate water undergoing slow isotopic exchange provides the rate-limiting step in the formation of the double labeled 18,18O2 product. When the isotopic exchange is measured as a function of temperature, the two kinetic components reveal different temperature dependencies in which k1 increases by a factor of 10 over the range 0-20 degreesC while k2 increases by only a factor of 3. Assuming simple Arrhenius behavior, the activation energies are estimated to be 78 +/- 10 kJ mol-1 for the slow component and 39 +/- 5 kJ mol-1 for the fast component. The different kinetic components in the 18O isotopic exchange provide firm evidence that the two substrate water molecules undergo separate exchange processes at two different chemical sites in the S3 state, prior to the O2 release step (t1/2 approximately 1 ms at 20 degreesC). The results are discussed in terms of how the substrate water may be bound at two separate metal sites.

Synthesis, pharmacological characterization, and quantitative structure-activity relationship analyses of 3,7,9,9-tetraalkylbispidines: derivatives with specific bradycardic activity.

A series of 3,7,9,9-tetraalkyl-3,7-diazabicyclo[3.3.1]nonane derivatives (bispidines) was synthesized and identified as potential antiischemic agents. Pharmacological experiments in vitro as well as in vivo are described, and the results are listed. For selection of those compounds fitting best to the desired profile of a specific bradycardic antianginal agent--decrease in heart rate without affecting contractility and blood pressure--these results were scored and ranked. Quantitative structure--activity relationship (QSAR) analyses were performed and discussed a posteriori by means of Hansch, nonelementary discriminant and factor analysis to get insight into the molecular features determining the biological profile. Highly significant equations were obtained, indicating hydrophobic and steric effects. Both pharmacological ranking and QSAR considerations showed compound 6 as the optimum within the structural class under investigation. Compound 6 (tedisamil, KC8857) has been selected as the most promising compound and was chosen for further pharmacological and clinical investigations as an antiischemic drug.

Detection of an EPR multiline signal for the S0* state in photosystem II.

The S0* state was generated by incubation of dark-adapted (S1 state) photosystem II membranes either with the exogenous two electron reductant hydrazine and subsequent 273 K illumination in the presence of DCMU or by dark incubation with low amounts of the one electron reductant hydroxylamine. In agreement with earlier reports, the S1 and S-1 states were found to be electron paramagnetic resonance (EPR) silent. However, in the presence of 0.5-1.5% methanol, a weak EPR multiline signal centered around g = 2.0 was observed at 7 K for the S0* states generated by both procedures. This signal has a similar average line splitting to the well-characterized S2 state multiline EPR signal, but can be clearly distinguished from that and other modified S2 multiline signals by differences in line position and intensities. In addition, at 4 K it can be seen that the S0* multiline has a greater spectral breadth than the S2 multilines and is composed of up to 26 peaks. The S0* signal is not seen in the absence of methanol and is not affected by 1 mM EDTA in the buffer medium. We assign the S0* multiline signal to the manganese cluster of the oxygen evolving complex in a mixed valence state of the form MnIIMnIIIMnIIIMnIII,MnIIMnIIIMnIVMnIV, or MnIIIMnIIIMnIIIMnIV. Addition of methanol may be helpful in future to find an EPR signal originating form the natural S0 state.

S(-3) state of the water oxidase in photosystem II.

The effect of the reductant hydrazine on the flash-induced oxygen oscillation patterns of spinach thylakoids was used to characterize a new super-reduced redox state of the water oxidase in photosystem II. The formation of a discrete S(-3) state is evident from the shift of the first maximum of oxygen evolution from the 3rd flash through the 5th flash to the 7th flash during a 90 min incubation of dark-adapted thylakoids with 10 mM hydrazine sulfate at pH 6.8 on ice. A distinct period four oscillation with further maxima on the 11th and 15th flashes is still observed at this stage of the incubation. The data analysis within the framework of an extended Kok model reveals that a S(-3) state population of almost 50% can be achieved by this treatment. A prolonged incubation of the S(-3) sample with 10 mM hydrazine (and even 100 mM) does not lead to a further shift of the first maximum toward the 9th flash that could reflect the formation of the S(-5) state. Instead, a slow oxidation of S(-3) to S(-2) takes place by an as yet unidentified electron acceptor. A consistent simulation of all the measured oxygen oscillation patterns of this study could, however, only be achieved by including the formal redox states S(-4) and S(-5) in the fits (S(-4) + S(-5) up to 35%). The implications of these findings for the oxidation states of the manganese in the tetranuclear cluster of the water oxidase are discussed.

Bicarbonate-dependent proton extrusion in Chinese hamster ovary (CHO) cells adapted to growth at pH 6.7.

A CHO cell model is described for in vitro studies of the mechanisms underlying heat resistance in cells adapted to growth in acidic environments. Adaptation is defined as a loss of pH 6.7-induced sensitization to 42.0 degrees C cytotoxicity and it is accompanied with an elevation of steady-state intracellular pH (pHi). CHO cells cultured between 75 and 100 days at pH 6.7 became fully adapted (6.7G cells), and the adapted phenotype was maintained for at least 100 additional days of culture at pH 6.7. The surviving fraction (SF) of 6.7G cells heated (42.0 degrees C) at pH 6.7 was comparable with that of cells cultured at pH 7.3 (7.3G cells) and heated at pH 7.3, while the SF of 7.3G cells acutely acidified to pH 6.7 and heated was an order of magnitude less. Although this resistance of 6.7G cells to killing was observed at 42.0 degrees C, it was not observed at 43.0 and 45.0 degrees C. Both 6.7G and 7.3G cells were able to develop comparable levels of thermotolerance during 42.0 degrees C at their growth pHs. However, in agreement with the literature, development of thermotolerance was reduced in acutely acidified 7.3G cells. An acute acidification of only 0.2 pH unit from pH 6.7 to 6.5 also reduced the ability of 6.7G cells to develop thermotolerance during heating at 42.0 degrees C. The acquired 6.7G phenotype reverted to the 7.3G phenotype following 17 days of culture at pH 7.3. Amiloride (0.5 mM), an inhibitor of the Na+/H+ exchanger (NHE), did not sensitize 7.3G and 6.7G cells to 42.0 degrees at their growth pHs. However, sensitization was observed for acutely acidified 7.3G cells. This is consistent with the hypothesis that extracellular acute acidification causes a decrease in pHi, and that the recovery from that decrease is achieved in part by activation of the NHE. An elevation of steady-state pHi, measured by analysing intracellular BCECF excitation spectra, was documented in a suspension assay for cells grown at pH 6.7 for 180 days. The elevation was bicarbonate-dependent (negligible in the absence of HCO3-, +0.17 pH units in the presence of HCO3-). These results suggest that the altered regulation of pHi in CHO cells adapted to pHe 6.7 is maintained by bicarbonate-dependent processes.

Structural changes in the water-oxidizing complex monitored via the pH dependence of the reduction rate of redox state S1 by hydrazine and hydroxylamine in isolated spinach thylakoids.

A detailed kinetic analysis is presented for the pH dependence of the reduction of the water-oxidizing complex (WOC) in redox state S1 by hydrophilic amines NH2R (R = NH2, OH) in suspensions of isolated thylakoids. Measurements of patterns of the oxygen yield induced by a train of single-turnover flashes and evaluation of the data within the framework of an extended Kok model [Messinger, J., Wacker, U., & Renger, G. (1991) Biochemistry 30, 7852-7862] led to the following results: (a) the rate constants kS1(NH2R) exhibit strikingly similar pH dependencies for NH2OH and NH2NH2 with "titration waves" at pH 5.3-5.6; 6.2-6.5, and above a critical pH value of about 7.4; (b) the differences in the reaction mechanism between NH2OH (1-electron reduction) and NH2NH2 (2-electron reduction) are almost pH-independent; (c) the ratio of the rate constants, kS1(NH2OH)/kS1(NH2NH2), decreases by a factor of about 9 within the range 5 < pH < 8.5. A detailed analysis reveals that these data cannot be consistently explained by the assumption that the unprotonated forms NH2OH and NH2NH2 are the active species while the protonated cations [NH3OH]+ and [N2H5]+ are nonreactive. A quantitative description is achieved by the additional postulate that pH-dependent structural changes take place in the WOC, thereby modulating the reactivity toward exogenous redox active amines of the type NH2R. On the basis of the results of this study and a recent report [Messinger, J., & Renger, G. (1994) Biochemistry 33, 10896-10905], it is inferred that the WOC undergoes three specific structural changes, with characteristic pH values of 5.3-5.5, 6.2-6.5, and above 7.4.

Detection of one slowly exchanging substrate water molecule in the S3 state of photosystem II.

The exchangeability of the substrate water molecules at the catalytic site of water oxidation in photosystem II has been probed by isotope-exchange measurements using mass spectrometric detection of flash-induced oxygen evolution. A stirred sample chamber was constructed to reduce the lag time between injection of H2(18)O and the detecting flash by a factor of more than 1000 compared to the original experiments by R. Radmer and O. Ollinger [(1986) FEBS Lett. 195, 285-289]. Our data show that there is a slow (t1/2 approximately 500 ms, 10 degrees C) and a fast (t1/2 <25 ms, 10 degrees C) exchanging substrate water molecule in the S3 state of photosystem II. The slow exchange is coupled with an activation energy of about 75 kJ/mol and is discussed in terms of a terminal manganese oxo ligand, while the faster exchanging substrate molecule may represent a water molecule not directly bound to the manganese center.

Analyses of pH-induced modifications of the period four oscillation of flash-induced oxygen evolution reveal distinct structural changes of the photosystem II donor side at characteristic pH values.

This study presents a thorough analysis of the reaction pattern of flash-induced oxygen evolution in spinach thylakoids as a function of pH (4.5 < or = pH < or = 9) and the redox state of tyrosine YD in polypeptide D2. Evaluation of the experimental data within the conventional Kok model [Kok, B., Forbush, B., & McGloin, M. (1970) Photochem. Photobiol. 11, 457-475] led to the following results: (1) the probability of the miss factor is strongly pH dependent (with a pronounced minimum near neutral pH) while the double hit factor is less affected; (2) a marked increase of the apparent S0 population arises at alkaline pH in dark-adapted samples where most of the YD is reduced, but this effect is absent if the percentage of PS II containing the oxidized form YDox is high; and (3) the lifetimes of S2 and S3 exhibit a characteristic pH dependence that is indicative of conformational changes of functional relevance within the water-oxidizing complex and its environment; (4) the kinetic interaction of redox states S2 and S3 with YD is characterized by a change of its behavior at a threshold pH of 6.5-7.0; and (5) at acidic pH values the extent of S2 and S3 reduction by YD decreases concomitant with the occurrence of a very fast decay kinetics. On the basis of a detailed discussion of these results and data from the literature, the water oxidase is inferred to undergo structural changes at pH values of 5-5.5 and 6.5-7.0. These transitions are almost independent of the redox state Si and modify the reaction coordinates of the water oxidase toward endogenous reductants.