The two forms of the S(2) state multiline signal in Photosystem II: effect of methanol and ethanol.

The characteristic Mn hyperfine 'multiline' signal exhibited in the S2 state of the oxygen-evolving complex (OEC) complex of Photosystem II (PSII) has been shown to be heterogeneous in character. In this study, we have explored the effects that influence the proportions of the two forms of the S2 state multiline signal present in any sample. The narrow form of the signal is lost upon storage (weeks) at 77 K, whereas the broad form remains. In particular, we explore the roles of ethanol and methanol as well as effects of the second turnover of the enzyme on storage of the sample at 77 K. We find that in samples containing methanol, the narrow form may predominate upon the first flash, but the broad form predominates on the fifth flash and also in samples containing ethanol.

Evidence for a direct manganese-oxygen ligand in water binding to the S2 state of the photosynthetic water oxidation complex.

The interaction of water with the water oxidizing Mn complex of photosystem II has been investigated using electron spin-echo envelope modulation spectroscopy in the presence of H(2)(17)O. The spectra show interaction of the (17)O with the preparation in the S(2) state induced by 200 K illumination. The modulation is observed only in the center of the multiline spectrum. The inferred hyperfine coupling terms are compatible with water (not hydroxyl) oxygen bound to a particular quasi-axial Mn(III) center in a coupled Mn cluster.

Probing the Mn oxidation states in the OEC. Insights from spectroscopic, computational and kinetic data.

Results from a variety of experimental techniques which have been used to define the oxidation levels of Mn and other components in the S states of the water oxidising complex in Photosystem II are reviewed. A self-consistent interpretation of Mn X-ray absorption near edge spectroscopy, UV-visible and near infrared spectroscopic data suggests that Mn oxidation occurs only on the S0-->S1 transition, and that all four Mn centres have formal oxidation state III thereafter. Ligand oxidation occurs on the transitions to S2 and S3. This is supported by high level quantum chemical calculations and an analysis of the kinetics of substrate water exchange, as recently determined by Wydrzynski et al. (this journal). One type of model for the catalytic site structure and water oxidation mechanism, consistent with these conclusions, is discussed. This model invokes magnetically separate oxo bridged dimers with water oxidation occurring by a concerted 2H+/2e- transfer mechanism, with one H transfer to a bridge oxygen on each dimer.

The gramicidin-based biosensor: a functioning nano-machine.

Biosensors combine a biological recognition mechanism with a physical transduction technique. In nature, the transduction mechanism for high sensitivity molecular detection is the modulation of the cell membrane ionic conductivity through specific ligand-receptor binding-induced switching of ion channels. This effects an inherent signal amplification of six to eight orders of magnitude, corresponding to the total ion flow arising from the single channel gating event. Here we describe the first reduction of this principle to a practical sensing device, which is a planar impedance element composed of a macroscopically supported synthetic bilayer membrane incorporating gramicidin ion channels. The membrane and an ionic reservoir are covalently attached to an evaporated gold surface. The channels have specific receptor groups attached (usually antibodies) that permit switching of gramicidin channels by analyte binding to the receptors. The device may then be made specific for the detection of a wide range of analytes, including proteins, drugs, hormones, antibodies, DNA, etc., currently in the 10(-7)-10(-13) M range. It also lends itself readily to microelectronic fabrication and signal transduction. By adjusting the surface density of the receptors/channel components during fabrication, the optimum sensitivity range of the device may be tuned over several orders of magnitude.

EPR kinetic studies of oxygen release in thylakoids and PSII membranes: a kinetic intermediate in the S3 to S0 transition.

Time-resolved EPR oximetry has been used to determine the oxygen release kinetics in spinach thylakoids and PSII membranes. We observe release kinetics with half-times of approximately 0.85 and approximately 1.45 ms for thylakoids and PSII membranes, respectively, which are in close agreement with the EPR determined Yz decay kinetics for the S3 --> --> S0 transition in these systems. The results show conclusively that water-oxygen chemistry is not a rate-limiting step in the donor side of PSII under normal turnover conditions. By analyzing the oxygen release kinetics in thylakoids under nonphysiological, but still functionally competent conditions (low pH or high salt), we observed an initial delay in the O2 release of up to 200 microseconds following flash turnover from the S3 state. This is the first direct indication of a probable quasi-stable intermediate in the S3 --> --> S0 turnover of PSII, possibly representing the putative S4 state. Under conditions more closely approaching physiological, no such delay was resolved, indicating that the S4 --> O2 transition occurs within 50 microseconds under such circumstances. Two possible reaction sequences for O2 formation consistent with these and other data are discussed. It is suggested that the more probable form of "S4" is in fact the S3 + Yz* combination, which must undergo some molecular rearrangement on the tens to hundreds of microseconds time scale before O2 formation chemistry occurs.

A comparison of low vision clinic data with low vision survey and blindness registration information.

PURPOSE: To determine whether low vision demographic data provided by low vision clinic data are comparable to that provided by blindness registration and disability questionnaire information. METHODS: Low vision demographic data for Canada and Ontario within the postcensus Health and Activity Limitation Survey (HALS 1991) were obtained from Statistics Canada. These data were compared with 4744 reports of low vision examinations obtained in a multi-center low vision clinic study in Ontario, Canada (1991-1994) and appropriate annual figures from the Canadian National Institute for the Blind (CNIB). RESULTS: Data from the low vision clinic study and the CNIB were similar. The low vision clinic study (and CNIB) reported far fewer adults (15 to 64 years) and far more seniors (65+ years) obtaining low vision examinations than suggested by HALS. CONCLUSIONS: HALS does not report on patients with low vision, as defined in low vision clinics. The differences between survey, low vision clinic, and blindness registration data are presented.

Yz. reduction kinetics in the absence of the manganese-stabilizing protein of photosystem II.

Decay of Signal IIvf of photosystem II (PSII), under repetitive flash conditions, was examined in whole cells of wild-type Synechocystis sp. PCC6803 and in cells of an engineered strain, delta psbO, which lacks the extrinsic 33 kDa manganese-stabilizing protein (MSP). Previous polarographic analysis had shown that O2 release during the S3-->[S4]-->S0 transition of the catalytic cycle is significantly retarded in the delta psbO strain relative to the wild-type [Burnap et al. (1992) Biochemistry 31, 7404-7410]. The present experiments provide evidence that a parallel retardation in the rate of reduction of photooxidized Yz by the H2O oxidation complex is due to the absence of MSP. The half-time of the Signal IIvf component, corresponding to Yz. reduction during the S3-->[S4]-->S0 transition, was estimated to be 1.2 and 6.0 ms in the wild-type and delta psbO cells, respectively.

A biosensor that uses ion-channel switches.

Biosensors are molecular sensors that combine a biological recognition mechanism with a physical transduction technique. They provide a new class of inexpensive, portable instrument that permit sophisticated analytical measurements to be undertaken rapidly at decentralized locations. However, the adoption of biosensors for practical applications other than the measurement of blood glucose is currently limited by the expense, insensitivity and inflexibility of the available transduction methods. Here we describe the development of a biosensing technique in which the conductance of a population of molecular ion channels is switched by the recognition event. The approach mimics biological sensory functions and can be used with most types of receptor, including antibodies and nucleotides. The technique is very flexible and even in its simplest form it is sensitive to picomolar concentrations of proteins. The sensor is essentially an impedance element whose dimensions can readily be reduced to become an integral component of a microelectronic circuit. It may be used in a wide range of applications and in complex media, including blood. These uses might include cell typing, the detection of large proteins, viruses, antibodies, DNA, electrolytes, drugs, pesticides and other low-molecular-weight compounds.

Electron paramagnetic resonance kinetic studies of the S states in spinach thylakoids.

The Tyrz+ decay kinetics have been analyzed by using time-resolved EPR to determine the half-time of each Si-->S(i + 1) transition in the O2-evolving complex of spinach thylakoids under physiological conditions. Using dark-adapted thylakoids and appropriate single-turnover flash sequences, we were able to detect the signal IIvf kinetics of the Tyrz+ S0-->Tyrz S1, Tyrz+ S1-->Tyrz S2, Tyrz+ S2-->Tyrz S3, and Tyrz+ S3-->(S4)-->Tyrz S0 transitions. To correct for damping of the S state synchronization during the flash sequence, the Kok parameters were estimated by measuring the oxygen flash pattern in situ using nitroxide-based EPR oximetry. Following deconvolution of the individual S state contributions, the signal IIvf decay kinetics yield the following half-times for the S state transitions: S0-->S1 in 40-60 microseconds, S1-->S2 in 85 microseconds, S2-->S3 in 140 microseconds, and S3-->(S4)-->S0 in 750 microseconds. Preliminary results with detergent-solubilized PSII membranes suggest that the S3-->S0 transition at least is slowed by a factor of approximately 2 in this system. Ramifications of these half-times in terms of electron transfer events on the donor site of PSII are discussed.

Simulation of the S2 state multiline electron paramagnetic resonance signal of photosystem II: a multifrequency approach.

The S2 state electron paramagnetic resonance (EPR) multiline signal of Photosystem II has been simulated at Q-band (35 Ghz), X-band (9 GHz) and S-band (4 GHz) frequencies. The model used for the simulation assumes that the signal arises from an essentially magnetically isolated MnIII-MnIV dimer, with a ground state electronic spin ST = 1/2. The spectra are generated from exact numerical solution of a general spin Hamiltonian containing anisotropic hyperfine and quadrupolar interactions at both Mn nuclei. The features that distinguish the multiline from the EPR spectra of model manganese dimer complexes (additional width of the spectrum (195 mT), additional peaks (22), internal "superhyperfine" structure) are plausibly explained assuming an unusual ligand geometry at both Mn nuclei, giving rise to normally forbidden transitions from quadrupole interactions as well as hyperfine anisotropy. The fitted parameters indicate that the hyperfine and quadrupole interactions arise from Mn ions in low symmetry environments, corresponding approximately to the removal of one ligand from an octahedral geometry in both cases. For a quadrupole interaction of the magnitude indicated here to be present, the MnIII ion must be 5-coordinate and the MnIV 5-coordinate or possibly have a sixth, weakly bound ligand. The hyperfine parameters indicate a quasi-axial anisotropy at MnIII, which while consistent with Jahn-Teller distortion as expected for a d4 ion, corresponds here to the unpaired spin being in the ligand deficient, z direction of the molecular reference axis. The fitted parameters for MnIV are very unusual, showing a high degree of anisotropy not expected in a d3 ion. This degree of anisotropy could be qualitatively accounted for by a histidine ligand providing pi backbonding into the metal dxy orbital, together with a weakly bound or absent ligand in the x direction.

Chloride binding to the anion transport binding sites of band 3. A 35Cl NMR study.

Band 3 is an integral membrane protein that exchanges anions across the red cell membrane. Due to the abundance and the high turnover rate of the band 3 transport unit, the band 3 system is the most heavily used ion-transport system in a typical vertebrate organism. Here we show that 35Cl NMR enables direct and specific observation of substrate Cl- binding to band 3 transport sites, which are identified by a variety of criteria: (a) the sites are inhibited by 4,4'- dinitrostilbene -2,2'-disulfonate, which is known to inhibit competitively Cl- binding to band 3 transport sites; (b) the sites have affinities for 4,4'- dinitrostilbene -2,2'-disulfonate and Cl- that are quantitatively similar to the known affinities of band 3 transport sites for these anions; and (c) the sites have relative affinities for Cl-, HCO-3, F-, and I- that are quantitatively similar to the known relative affinities of band 3 transport sites for these anions. The 35Cl NMR assay also reveals a class of low affinity Cl- binding sites (KD much greater than 0.5 M) that are not affected by 4,4'- dinitrostilbene -2,2'-disulfonate. These low affinity sites may be responsible for the inhibition of band 3 catalyzed anion exchange that has been previously observed at high [Cl-]. In the following paper the 35Cl NMR assay is used to resolve the band 3 transport sites on opposite sides of the membrane, thereby enabling direct observation of the transmembrane recruitment of transport sites.

Direct observation of the transmembrane recruitment of band 3 transport sites by competitive inhibitors. A 35Cl NMR study.

Numerous models describing anion exchange across the red cell membrane by band 3 have been discussed in literature. These models are readily distinguished from one another by an experiment which tests the ability of band 3 transport sites to be recruited to one side of the membrane. In order to observe directly the transmembrane recruitment of transport sites, we have developed 35Cl NMR techniques that resolve the two transport site populations on opposite sides of the membrane. Using these techniques, we show that the inhibitors 4,4'- dinitrostilbene -2,2'-disulfonate and p- nitrobenzensulfonate each recruit all of the transport sites on both sides of the membrane to the extracellular facing conformation. This result indicates that band 3 has an alternating site transport mechanism: each band 3 transport unit possesses a single functional transport site which is alternately exposed first to one side of the membrane then to the other.

Cholesterol-phospholipid interaction in membranes. 2. Stoichiometry and molecular packing of cholesterol-rich domains.

A model for the molecular interaction between cholesterol and phospholipid in bilayer membranes is presented. We propose that cholesterol forms associations with phospholipids with stoichiometries of both 1:1 and 1:2. A hydrogen bond between the beta-OH of cholesterol and the glycerol ester oxygen of a phospholipid is suggested as a likely mechanism for tight binding in a 1:1 complex. A second phospholipid molecule is loosely associated with the complex to form domains of 1:2 stoichiometry, which may coexist with pure phospholipid domains. Interfacial boundary phospholipid separates these two domains. Under conditions in which interfacial phospholipid is maximal, the perturbed phospholipid assumes a composition of 20 mol % cholesterol. To account for the phase behavior and surface properties of cholesterol-lipid membranes, we propose a molecular packing model for linear arrays within the cholesterol-rich domains. In this arrangement, two rows of 1:1 complex run antiparallel with loosely associated phospholipid intercalated between them. The loosely associated phospholipid can pack in the nearly hexagonal manner in which pure crystalline phospholipid is known to pack. The model provides maximal van der Waals contact in the hydrocarbon region of the bilayer and can maintain phospholipids as cholesterol's nearest neighbors at all concentrations up to 50 mol % cholesterol. The model is compatible with the diverse experimental observations compiled by many investigators over the past decade.