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In this work we report on the optical properties of specific synthetic carbon nano-dots (CDs) and their suitability for the development of optical biosensors. We examine the photoluminescence behavior of these CDs under different conditions, in their native form, as well as when conjugated to the catalytic protein glucose oxidase (GOx) for the construction of optical glucose biosensors. The effect of pH and hydrogen peroxide on the observed spectra is examined as the basis for the biosensor development. The CDs examined here have inherent surface amino functional groups which allow for easy conjugation to biomolecules via EDC-NHS, providing a well defined platform for biosensing applications. We conclude that the well controlled, stable, and highly efficient fluorescence behavior of the CDs in solution or in conjugate, provides the grounds for this class of materials to be used in a variety of arrangements for the development of optical and optoelectrochemical detection systems.

The UV-A (320-400 nm) component of sunlight is a significant damaging factor of plant photosynthesis, which targets the photosystem II complex. Here we performed a detailed characterization of UV-A-induced damage in photosystem II membrane particles using EPR spectroscopy and chlorophyll fluorescence measurements. UV-A irradiation results in the rapid inhibition of oxygen evolution accompanied by the loss of the multiline EPR signal from the S(2) state of the water-oxidizing complex. Gradual decrease of EPR signals arising from the Q(A)(-)Fe(2+) acceptor complex, Tyr-D degrees, and the ferricyanide-induced oxidation of the non-heme Fe(2+) to Fe(3+) is also observed, but at a significantly slower rate than the inhibition of oxygen evolution and of the multiline signal. The amplitude of Signal II(fast), arising from Tyr-Z degrees in the absence of fast electron donation from the Mn cluster, was gradually increased during the course of UV-A treatment. However, the amount of functional Tyr-Z decreased to a similar extent as Tyr-D as shown by the loss of amplitude of Signal II(fast) that could be measured in the UV-A-treated particles after Tris washing. UV-A irradiation also affects the relaxation of flash-induced variable chlorophyll fluorescence. The amplitudes of the fast (600 micros) and slow (2 s) decaying components, assigned to reoxidation of Q(A)(-) by Q(B) and by recombination of (Q(A)Q(B))(-) with donor side components, respectively, decrease in favor of the 15-20 ms component, which reflects PQ binding to the Q(B) site. In the presence of DCMU, the fluorescence relaxation is dominated by a 1 s component due to recombination of Q(A)(-) with the S(2) state. After UV-A radiation, this is partially replaced by a much faster component (30-70 ms) arising from recombination of Q(A)(-) with a stabilized intermediate PSII donor, most likely Tyr-Z degrees. It is concluded that the primary damage site of UV-A irradiation is the catalytic manganese cluster of the water-oxidizing complex, where electron transfer to Tyr-Z degrees and P(680)(+) becomes inhibited. Modification and/or inactivation of the redox-active tyrosines and the Q(A)Fe(2+) acceptor complex are subsequent events. This damaging mechanism is very similar to that induced by the shorter wavelength UV-B (280-320) radiation, but different from that induced by the longer wavelength photosynthetically active light (400-700 nm).

Fatty acid hydroperoxide (HPO) lyase is a component of the oxylipin pathway and holds a central role in elicited plant defense. HPO lyase from bell pepper has been identified as a heme protein which shares 40% homology with allene oxide synthase, a cytochrome P450 (CYP74A). HPO lyase of immature bell pepper fruits was expressed in Escherichia coli and the enzyme was purified and characterized by spectroscopic techniques. The electronic structure and ligand coordination properties of the heme were investigated by using a series of exogenous ligands. The various complexes were characterized by using UV-visible absorption and electron paramagnetic resonance spectroscopy. The spectroscopic data demonstrated that the isolated recombinant HPO lyase has a pentacoordinate, high-spin heme with thiolate ligation. Addition of the neutral ligand imidazole or the anionic ligand cyanide results in the formation of hexacoordinate adducts that retain thiolate ligation. The striking similarities between both the ferric and ferrous HPO lyase-NO complexes with the analogous P450 complexes, suggest that the active sites of HPO lyase and P450 share common structural features.

The reduction of tyrosine Y(.)(Z) by benzidine and exogenous Mn(2+) was studied by kinetic EPR experiments in various Photosystem II (PSII) preparations. Using lanthanide treated PSII membranes it was demonstrated that neither the extrinsic polypeptides (17, 23 and 33 kDa) nor the Mn complex block the accessibility of Y(.)(Z) to exogenous reductants, such as benzidine. In addition, it was shown that in the presence of the native Mn complex exogenous Mn(2+) does not reduce Y(.)(Z).

The photosystem II (PSII) reaction center (RC) complex was isolated from spinach and characterized by gel electrophoresis, gel filtration and analytical ultracentrifugation. The purified complex contained the PsbA, PsbD, PsbE, PsbF and PsbI subunits. Gel filtration and analytical ultracentrifugation indicated the presence of a homogeneous complex. The mass of the RC complexes was found to be 107 kDa by analytical ultracentrifugation and 132 kDa by scanning transmission electron microscopy (STEM). The mass obtained showed the isolated complex to exist as a monomer and only one cytochrome b559 (cyt b559) to be associated with the RC complex. Digital images of negatively stained RC complexes were recorded by STEM and analyzed by single-particle averaging. The complex was 9 nm long and 5 nm wide, and exhibited a pronounced quasi-twofold symmetry. This supports the symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex.

Depletion of Ca2+ and/or Cl- ions from PSII membranes blocks the electron-transfer reactions that precede O2 evolution on the oxidizing side of the enzyme. Illumination of these inhibited preparations at 273 K generates a paramagnetic species that is detectable by low-temperature (T < 20 K) EPR as a signal in the g = 2 region, 90-230 G wide, depending on the treatment that PSII has undergone. This signal has recently been assigned to YZ* in magnetic interaction with the manganese cluster in its S2 state [Gilchrist et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9545-9549]. This view, however, is not universal, owing, in part, to the fact that its spectroscopic properties depend on the preparation and the experimental conditions used for its study and, in part, to uncertainties as to the room temperature behavior of YZ* in inhibited preparations. Here, we report time-resolved and conventional EPR data showing that, at room temperature and at 273 K, YZ* can be accumulated in its 20 G form in high yields in both Ca2+-depleted and acetate-inhibited preparations, and that the kinetics of its decay match the decay kinetics of the low-temperature signal generated in corresponding samples. The properties of the YZ* signal, however, are shown to depend on the polypeptide content, the temperature, and the electron donors and acceptors present in the sample under examination. Our results support assignment of the EPR signal in inhibited preparations to S2 YZ* and demonstrate a protective role of the 17 and 23 kDa extrinsic polypeptides for the manganese cluster against externally added reductants.

Oxygen evolving photosystem II (PSII-OEC) complexes and PSII core complexes were isolated from spinach and the thermophilic cyanobacterium Synechococcus sp. OD24 and characterized by gel electrophoresis, immunoblotting, and absorbance spectroscopy. The mass of the core complexes was determined by scanning transmission electron microscopy (STEM) and found to be 281 &plusmn; 65 kDa for spinach and 313 &plusmn; 52 kDa for Synechococcus sp. OD24. The mass of the spinach PSII-OEC complex was 327 &plusmn; 64 kDa. Digital images of negatively stained PSII-OEC and PSII core complexes were recorded by STEM and analyzed by single particle averaging. All monomeric complexes showed similar morphologies and were of comparable length (14 nm) and width (10 nm). The averages revealed a pseudo-twofold symmetry axis, which is a prominent structural element of the monomeric form. Difference maps between the averaged projections of the oxygen evolving complexes and the core complexes from both species indicated where the 33-kDa extrinsic manganese stabilizing protein is bound. A symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex, is proposed.

In recent years Photosystem II, and in particular the oxygen evolving component of the enzyme, have been the subject of intense biochemical and biophysical analysis. To date no high resolution structural model of the complex has been produced. As a consequence unambiguous interpretation of much experimental data has proven difficult, leading to a lack of consensus over many basic questions regarding the mechanisms involved, the oligomerization state of the enzyme in vivo and even the exact biochemical composition.This review is a summary of the progress towards the production of a structural model of PS II-derived from either X-ray crystallography or electron microscopy based techniques-and the current opinions, which have arisen from these structural analyses, on the structural topology and assemblage of the various subunits that constitute the complex.

Inhibition of photosystem II electron transport by UV-B radiation has been studied in isolated spinach photosystem II membrane particles using low-temperature EPR spectroscopy and chlorophyll fluorescence measurements. UV-B irradiation results in the rapid inhibition of oxygen evolution and the decline of variable chlorophyll fluorescence. These effects are accompanied by the loss of the multiline EPR signal arising from the S2 state of the water-oxidizing complex and the induction of Signal IIfast originating from stabilized Try-Z+. The EPR signals from the QA-Fe2+ acceptor complex, Tyr-D+, and the oxidized non-heme iron (Fe3+) are also decreased during the course of UV-B irradiation, but at a significantly slower rate than oxygen evolution and the multiline signal. The decrease of the Fe3+ signal at high g values (g = 8.06, g = 5.6) is accompanied by the induction of another EPR signal at g = 4.26 that arises most likely from the same Fe3+ ion in a modified ligand environment. UV-B irradiation also affects cytochrome b-559. The g = 2.94 EPR signal that arises from the dark- oxidized form is enhanced, whereas the light inducible g = 3.04 signal that arises from the photo-oxidizable population of cytochrome b-559 is diminished. UV-B irradiation also induces the degradation of the D1 reaction center protein. The rate of the D1 protein loss is slower than the inhibition of oxygen evolution and of the multiline signal but follows closely the loss of Signal IIslow, the QA-Fe2+ and the Fe3+ EPR signals, as well as the release of protein-bound manganese. It is concluded from the results that UV-B radiation affects photosystem II redox components at both the donor and acceptor side. The primary damage occurs at the water-oxidizing complex. Modification and/or inactivation of tyrosine-D, cytochrome b-559, and the QAFe2+ acceptor complex are subsequent events that coincide more closely with the UV-B-induced damage to the protein structure of the photosystem II reaction center.

An oxygen evolving photosystem II core complex containing all three extrinsic proteins (33, 23, 17 kDa) was isolated from spinach and reconstituted into tubular two-dimensional crystals of 72.9 nm diameter and 1-2 micrometers length. While the 17 and 23 kDa polypeptides were lost during crystallization, the extrinsic 33 kDa protein was retained. The optical spectrum of the crystallized core was characteristic of an intact PSII core complex. Immunoelectron microscopy revealed that the lumenal surface of the PSII complex was exposed at the outside of the cylindrical tubes. The projection of the complex was determined from flattened tubular crystals by negative stain electron microscopy and image analysis to 2.0 nm resolution. Rhombic unit cells (a = 16.2 nm, b = 13.7 nm; gamma = 142.4 degrees) contained one PSII complex.

A highly purified oxygen evolving Photosystem II core complex was isolated from PS II membranes solubilized with the non-ionic detergent n-octyl-β-D-thioglucoside. The three extrinsic proteins (33, 23 and 17 kDa) were functionally bound to the PS II core complex. Selective extraction of the 22, 10 kDa, CP 26 and CP 29 proteins demonstrated that these species are not involved in the binding of the extrinsic proteins (33, 23 and 17 kDa) or the DCMU sensitivity of the Photosystem II complex.

The three main polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) were characterized by HPLC in intact spinach leaf cells, intact chloroplasts, thylakoid membranes, Photosystem II membranes, the light-harvesting complex and the PS II complex. All contain the three polyamines in various ratios; the HPLC polyamine profiles of highly resolved PS II species (a Photosystem II core and the rection center) suggest an enrichment in the polyamine Spm.

A photosystem II core from spinach containing the chlorophyll-binding proteins 47 kDa, 43 kDa, the reaction center proteins D1, D2 and cytochromeb 559 and three low molecular weight polypeptides (MW < 10 kDa) was isolated, its three-dimensional crystals were prepared, and both core and crystals were studied by spectroscopic techniques and electron microscopy. The absorption spectra of the crystallized form of the core indicate a specific orientation of the various pigments within the crystal.

Selective solubilization of Photosystem II membranes with the non-ionic detergent octyl thioglucopyranoside has allowed the isolation of a PS II system which has been depleted of the 22 and 10 kDa polypeptides but retains all three extrinsic proteins (33, 23 and 17 kDa). The PS II membranes which have been depleted of the 22 and 10 kDa species show high rates of oxygen evolution activity, external calcium is not required for activity and the manganese complex is not destroyed by exogenous reductants. When we compared this system to control PS II membranes, we observed a minor modification of the reducing side, and a conversion of the high-potential to the low-potential form of cytochrome b 559.

Light-induced degradation of the D1 protein in isolated spinach photosystem II core preparations was studied after addition of various protease inhibitors. The degradation was selectively inhibited by several serine protease inhibitors in particular diisopropylfluorophosphate. The results demonstrate that the D1 protein is degraded by a serine-type of proteolytic activity that is an integral part of photosystem II.

Photoinhibitory illumination of isolated oxygen evolving photosystem II core complexes results in a substantial degradation of the D1-protein which is accompanied by the appearance of high amounts of at least 4 different degradation products. It is suggested that the degradation is due to a protease that is an integral part of the photosystem II complex.

Using the non-ionic detergent dodecyl-β-D-maltoside we have developed a preparative method for the isolation of the 43 kDa, 47 kDa and D1-D2-Cyt b 559 species directly from thylakoid membranes. In contrast to previous procedures the photosynthetic membrane was exposed only to one mild detergent and that resulted in more stable preparations. The isolated species were examined spectroscopically and it was found that even under these mild conditions the D1-D2-Cyt b 559 did not retain the primary quinone QA.

A photosystem II complex consisting of a 47-kDa chlorophyll-binding protein (CP47), the reaction center proteins D1 and D2, and cytochrome b-559 was characterized. Trace amounts of plastoquinone were found, indicating that the primary acceptor quinone QA has been extracted during purification. However, in the presence of ferricyanide, an EPR signal with the characteristic line shape and g value of the tyrosine radicals associated with photosystem II could be photoaccumulated in the majority of the reaction centers; in the absence of ferricyanide, or under low-temperature illumination conditions, a 9.5-11-G wide signal with a Gaussian line shape was observed at g = 2.003. Neither signal is observed in D1-D2-b-559 complexes, indicating that retention of CP47 produces a more native, but quinone-depleted photosystem II reaction center. The tyrosine radical photogenerated at room temperature can be trapped at cryogenic temperatures; results are presented showing that this radical can arise from tyrosine YZ, from tyrosine YD, or from both species. Low-temperature EPR spectroscopy also revealed a pronounced split signal with contributions at g = 6.05 and g = 5.75, which is attributed to high-spin, non-heme Fe3+ with axial ligation symmetry which is probably the non-heme iron associated with the acceptor side of photosystem II.

The linear, four-step oxidation of water to molecular oxygen by photosystem II requires cooperation between redox reactions driven by light and a set of redox reactions involving the S-states within the oxygen-evolving complex. The oxygenevolving complex is a highly ordered structure in which a number of polypeptides interact with one another to provide the appropriate environment for productive binding of cofactors such as manganese, chloride and calcium, as well as for productive electron transfer within the photoact. A number of recent advances in the knowledge of the polypeptide structure of photosystem II has revealed a correlation between primary photochemical events and a 'core' complex of five hydrophobic polypeptides which provide binding sites for chlorophyll a, pheophytin a, the reaction center chlorophyll (P680), and its immediate donor, denoted Z. Although the 'core' complex of photosystem II is photochemically active, it does not possess the capacity to evolve oxygen. A second set of polypeptides, which are water-soluble, have been discovered to be associated with photosystem II; these polypeptides are now proposed to be the structural elements of a special domain which promotes the activities of the loosely-bound cofactors (manganese, chloride, calcium) that participate in oxygen evolution activity. Two of these proteins (whose molecular weights are 23 and 17 kDa) can be released from photosystem II without concurrent loss of functional manganese; studies on these proteins and on the membranes from which they have been removed indicate that the 23 and 17 kDa species from part of the structure which promotes retention of chloride and calcium within the oxygen-evolving complex. A third water-soluble polypeptide of molecular weight 33 kDa is held to the photosystem II 'core' complex by a series of forces which in some circumstances may include ligation to manganese. The 33 kDa protein has been studied in some detail and appears to promote the formation of the environment which is required for optimal participation by manganese in the oxygen evolving reaction. This minireview describes the polypeptides of photosystem II, places an emphasis on the current state of knowledge concerning these species, and discusses current areas of uncertainty concerning these important polypeptides.

A highly resolved PSII reaction center complex has been prepared by exposure of PSII membranes to the detergent octylglucopyranoside at elevated ionic strengths; oxygen evolution activity is about 1,000 μmoles O2/hr/mg Chl in the presence of CaCl2. A Mn quantitation and a kinetic study of Z, the donor to P680, reveals that on a Chl basis this new preparation shows an almost four-fold enrichment in Mn and the electron transport components of PSII.

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.

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn(2+). Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Yz (+), the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Yz (+) reduction by benzidine was a linear function of benzidine concentration. The rate of Yz (+) reduction by Mn(2+) at pH 6 increased linearly at low Mn(2+) concentrations and reached a maximum at the Mn(2+) concentrations equal to several times the reaction center concentration. The rate was inhibited by K(+), Ca(2+) and Mg(2+). These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Yz (+) reduction at pH 7.5 was biphasic with a fast 400 μs phase that suggests binding of Mn(2+) near Yz (+) at a site that may be one of the native manganese binding sites.

Cytochrome b559 in various Photosystem II preparations was studled by using low temperature ESR spectroscopy. This technique was used because it is able to distinguish high from low potential forms of the cytochrome owing to the g-value differences between these species. Moreover, by using low temperature irradiation to oxidize cyt b559 we have avoided the use of redox mediators. Previous work (Ghanotakis DF., Topper J.N. and Yocum, C.F. (1984) Biochim. Biophys. Acta 767, 524-531) demonstrated that reduction and extraction of manganese of the oxygen evolving complex, which might be expected to alter the redox properties of cyt b559, occurs when certain PSII preparations are exposed to reductants. The ESR data presented here show that a mixture of high potential and lower potential cyt b559 species is observed in the oxygen evolving Photosystem II complex. Treatment of PSII membranes with 0.8 M Tris converts the high potential form(s) to those of lower potential. Exposure of the membranes to 2M NaCl shifts a significant amount of high potential cyt b559 to lower potential form(s); addition of CaCl2 reconstituted oxygen evolution activity but did not restore cyt b559 to its high potential form(s).

The conditions for steady-state Signal IIf formation in response to single turnover flashes in Tris-treated, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited chloroplasts have been investigated. DCMU inhibits Signal IIf generation as the photoinactive state, Z P680 Q-A, accumulates. Potassium ferricyanide relieves this inhibition so that Signal IIf can be fully developed on each flash in a flash series. The effectiveness of ferricyanide in stimulating Signal IIf formation is dependent on its concentration, the flash repetition rate, and the salt composition of the chloroplast suspension. There are two models in the literature for Q-A oxidation under these inhibitory conditions: direct oxidation of Q-A by exogenous acceptors like ferricyanide or oxidation of Q-A by an endogenous acceptor, AH, which has a midpoint potential of approximately 400 mV. It is found that the direct exogenous acceptor model accounts well for these data, whereas the AH model does not explain several of these results. The apparent rate constant for the direct oxidation of Q-A by ferricyanide at various concentrations of salt has been calculated from our electron paramagnetic resonance (EPR) data and compared with the corresponding rate constant determined by S. Itoh from fluorescence data (Biochim, Biophys. Acta 504, 324-340, 1978); good agreement is found for the two different experimental approaches.