Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

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 Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, 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 Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. 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 Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Selective extraction of 22 kDa and 10 kDa polypeptides from Photosystem II without removal of 23 kDa and 17 kDa extrinsic proteins

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.Abbreviations Chl- chlorophyll - DCBQ- 2,5-dichloro-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ESR- electron spin resonance - MES- 2-(N-morpholino)ethanesulfonic acid - OTG- octyl--d-thioglucopyranoside - PS II- Photosystem II - PEG- polyethylene glycol, M_r=6000 - Tris- 2-amino-2-hydroxyethylpropane-1,3-diol     

Isolation, characterization, sequencing and crystal structure of charybdin, a type 1 ribosome-inactivating protein from Charybdis maritima agg

A novel, type 1 ribosome-inactivating protein designated charybdin was isolated from bulbs of Charybdis maritima agg. The protein, consisting of a single polypeptide chain with a molecular mass of 29 kDa, inhibited translation in rabbit reticulocytes with an IC50 of 27.2 nm. Plant genomic DNA extracted from the bulb was amplified by PCR between primers based on the N-terminal and C-terminal sequence of the protein from dissolved crystals. The complete mature protein sequence was derived by partial DNA sequencing and terminal protein sequencing, and was confirmed by high-resolution crystal structure analysis. The protein contains Val at position 79 instead of the conserved Tyr residue of the ribosome-inactivating proteins known to date. To our knowledge, this is the first observation of a natural substitution of a catalytic residue at the active site of a natural ribosome-inactivating protein. This substitution in the active site may be responsible for the relatively low in vitro translation inhibitory effect compared with other ribosome-inactivating proteins. Single crystals were grown in the cold room from PEG6000 solutions. Diffraction data collected to 1.6 A resolution were used to determine the protein structure by the molecular replacement method. The fold of the protein comprises two structural domains: an alpha + beta N-terminal domain (residues 4-190) and a mainly alpha-helical C-terminal domain (residues 191-257). The active site is located in the interface between the two domains and comprises residues Val79, Tyr117, Glu167 and Arg170.     

A preparative method for the isolation of the 43 kDa, 47 kDa and the D1-D2-Cyt b 559 species directly from thylakoid membranes

Using the non-ionic detergent dodecyl--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 Q_A.Abbreviations PS II Photosystem II - CF Coupling Factor - LHC Light Harvesting Complex - MES 4-morpholine-ethanesulfonic acid - BIS-TRIS 2-[Bis (2-hydroxyethyl) amino]-2-hydroxymethyl-propane-1,3-diol - SMN 0.4 M sucrose/50 mM MES (pH6)/10 mM NaCl     

Isolation and crystallization of CP47, a Photosystem II chlorophyll binding protein. Degradation of CP47 upon dissociation from the core complex

The CP47 protein was isolated from Photosystem II membranes by using a combination of the detergents n-dodecyl-β-D-maltoside and octyl-β-D-thioglucoside. The purified CP47 was used in a series of crystallization experiments, which yielded highly reproducible hexagonal crystals. Immunoblot analysis revealed that the isolated CP47 undergoes degradation even under dim light conditions. This degradation takes place after the protein has been dissociated from the core complex. Proteolysis experiments with trypsin demonstrated that the dissociation of the CP47 from the PS II core complex results in changes that render the protein sensitive to proteolysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Polyamines in the photosynthetic apparatus

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.Abbreviations Chl chlorophyll - HPLC high performance liquid chromatography - LHC light-harvesting complex - PS II Photosystem II - PS II-RC Photosystem II reaction center - Put putrescine - Spd spermidine - Spm spermine - 10%S-core D₁-D₂-Cyt b559-47 kD-43 kD complex     

EPR characterization of the CP47-D1-D2-cytochrome b-559 complex of photosystem II

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 role of reagents accelerating the deactivation reactions of water-splitting enzyme system Y (ADRY reagents) in destabilizing high-potential oxidizing equivalents generated in chloroplast Photosystem II

A class of compounds, usually referred to as ADRY reagents, destabilize intermediates in the photosynthetic water-oxidizing process. The effects of these species on the reduction kinetics of Z^?, the oxidized donor to P-680, have been monitored in Tris-washed chloroplasts by following the decay of EPR Signal IIf. In the presence of ADRY reagents (e.g., sodium picrate, carbonyl cyanide m-chlorophenylhydrazone) this process follows an exponential time course, the decay half-time of which decreases as the ADRY reagent concentration increases. From this pseudo-first-order behavior, the second-order rate constants for four commonly used ADRY reagents have been extracted. The ADRY-induced acceleration in Z^? reduction proceeds independently of conditions imposed on the acceptor side of Photosystem II and shows no synergism with exogenous electron donors. These observations are most easily rationalized in terms of a model which proposes direct reduction of Z^? by the ADRY reagent followed by regeneration of the reduced ADRY reagent in a nonspecific reaction with membrane components such as carotenoids, chlorophyll or protein. A comparison of the second-order rate constants we obtain for ADRY reagents in their reaction with Z^? in Tris-washed chloroplasts with those obtained from the literature for the ADRY- reagent induced deactivation of states S₂ and S₃ in oxygen-evolving chloroplasts reveals a close similarity between the two processes. From this observation, a general model for the action of ADRY reagents in destabilizing the high-potential oxidizing equivalents generated in Photosystem II is proposed.     

Accessibility of tyrosine Y(.)(Z) to exogenous reductants and Mn(2+) in various Photosystem II preparations

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).     

Polypeptides of photosystem II and their role in oxygen evolution

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.Abbreviations A 23187 ionophore that exchanges divalent cations with H⁺ - Chl chlorophyll - cyt cytochrome - DCPIP dichlorophenolindophenol - DPC diphenylcarbazide - EGTA ethyleneglycoltetraacetic acid - P680 the chlorophyll a reaction center of photosystem II - pheo pheophytin - PQ plastoquinone - PS photosystem - Q_A and Q_B primary and secondary plastoquinone electron acceptors of photosystem II - Sn (n=0, 1, 2, 3, 4) charge accumulating state of the oxygen evolving system - Signals II_vf, II_f and II_s epr-detectable free radicals associated with the oxidizing side of photosystem II - Z primary electron donor to the photosystem II reaction center The survey of literature for this review ended in September, 1984.