Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey 627, mt −

The function of the eyespot in phototaxis of the flagellate green alga Chlamydomonas reinhardtii Dangeard was studied using quantitative reflection confocal laser scanning microscopy and photoelectric measurements. The reflective properties of the eyespot and the photoreceptor current of the C. reinhardtii eyespot mutant ey 627, mt ^– were compared with those of Chlamydomonas strains possessing a well-developed eyespot. Under growth conditions in which strongly disorganized eyespots were observed in the mutant by electron microscopy, there was a significant reduction in the reflection intensity of the eyespot and in the amplitude ratio (500440 nm) of photoreceptor currents induced by flashes of 500- and 440-nm light in non-oriented cells. Photoelectrical responses of pre-oriented cells revealed that the latter effect is caused by an altered directional sensitivity of the antenna complex, whereas the functional state of the photoreceptor pigment is not strongly affected in mutant cells. Both the reflection intensity and the amplitude ratio of photoreceptor currents increased to the level of reference strains under conditions supporting the development of a well-organized eyespot in the mutant. Furthermore, incubation of the mutant with high concentrations of all-trans-retinal (10 M), independent of whether carotenoid biosynthesis was inhibited or not, was found to increase the reflection intensity of the eyespot. An increase in the rate of photoorientation of the mutant occurred concomitant with the increase in the reflective properties of the mutant eyespot. These observations demonstrate the importance of an intact eyespot for interference reflection and absorption of phototactically active light, and thus for the directional sensitivity of the eyespot apparatus.Abbreviations HSM high-salt medium This study was supported by the Deutsche Forschungsgemeinschaft. O. A. Sineshchekov was supported by a Research Fellowship from the Alexander von Humboldt Foundation. The authors wish to thank U. Powalowski (Botanisches Institut, Universität zu Köln) for help with electron microscopy.     

The IleL229 → Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 M atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF=20) in UQ_o reconstituted isolated reaction centers and terbutryne in reaction centers (RF=55) and in chromatophores (RF=85). The amino acid sequence of the Q_B binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile^L229 Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone Q_B. The binding and redox properties of Q_B in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of Q_AQ_B ^– with respect to Q_A ^–Q_B was G_AB=–60 meV and 0 meV in reaction centers and G_AB=–85 meV and –46 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQ_o and semiquinone UQ_o ^– in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of Q_B.Abbreviations BChl bacteriochlorophyll - Ile isoleucine - Met methionin - P primary donor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ_o 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50 This work is dedicated to the memory of Randall Ross Stein (1954–1994) and is, in a small way, a testament to the impact which Randy's ideas have had on the development of the field of competitive herbicide binding.     

Mössbauer studies of the non-heme iron and cytochrome b559 in a Chlamydomonas reinhardtii PSI- mutant and their interactions with alpha-tocopherol quinone

Spin and valence states of the non-heme iron and the heme iron of cytochrome b559, as well as their interactions with alpha-tocopherol quinone (alpha-TQ) in photosystem II (PSII) thylakoid membranes prepared from the Chlamydomonas reinhardtii PSI- mutant have been studied using M?ssbauer spectroscopy. Both of the iron atoms are in low spin ferrous states. The Debye temperature of the non-heme is 194 K and of the heme iron is 182 K. The treatment of alpha-TQ does not change the spin and the valence states of the non-heme iron but enhances the covalence of its bonds. alpha-TQ oxidizes the heme iron into the high spin Fe3+ state. A possible role of the non-heme iron and alpha-TQ in electron flow through the PSII is discussed.     

A comparative analysis of the spin state distribution of in vitro and in vivo mutants of PsaC. A biochemical argument for the sequence of electron transfer in Photosystem I as FX → FA → FB → ferredoxin/flavodoxin

The EPR properties of in vivo and in vitro C14X–PS I and C51X–PS I (X = D, S, A or G) mutants of PsaC are compared in an attempt to extract information about electron transfer not contained in any one of these studies in isolation. This analysis indicates that 1) sulfur from an external 'rescue thiolate' is preferred over oxygen from an aspartate or serine replacement amino acid as a ligand to the F_A and F_B iron-sulfur clusters; 2) the inherent spectroscopic symmetry in the F_A and F_B clusters of unbound PsaC is lost when PsaC is docked to its site on the PsaA/PsaB heterodimer; 3) the bound 'rescue thiolate' ligand in the modified site of the F_A cluster, but not the F_B, cluster is displaced when PsaC is docked to its site on the PsaA/PsaB heterodimer; 4) the free energy of binding PsaC to the PsaA/PsaB heterodimer drives the otherwise-unfavorable ligand replacement in the F_A site. These and other findings argue that the substitute ligands support a [4Fe–4S] cluster at the modified site, but the cluster is in either a ground spin state of S 3/2 or S = 1/2 depending on the chemical identity of the ligand, on whether PsaC is unbound or bound, and on the reduction state of the cluster in the unmodified site. By a comparative analysis of the spin state distribution of the in vivo and in vitro C14X–PS I and C51X–PS I (X = D, S, A or G) mutants, and with knowledge from the X-ray crystal structure that PsaC is bound asymmetrically to the PS I reaction center, an independent case is made that PsaC is oriented so that the F_A cluster is proximal to F_X and the F_B cluster is distal to F_X. These results are compared and contrasted with the results of in vivo mutagenesis studies of PsaC in Anabaena variabilis ATCC 29413 and in Chlamydomonas reinhardtii. In all cases, the primary data can be interpreted to support the sequence of electron transfer as F_X F_A F_B ferredoxin.     

Development of Photosystem II in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PS IIβ, QB-nonreducing centers to the PS IIα, QB-reducing form

The green alga Chlamydomonas reinhardtii is a facultative heterotroph and, when cultured in the presence of acetate, will synthesize chlorophyll (Chl) and photosystem (PS) components in the dark. Analysis of the thylakoid membrane composition and function in dark grown C. reinhardtii revealed that photochemically competent PS II complexes were synthesized and assembled in the thylakoid membrane. These PS II centers were impaired in the electron-transport reaction from the primary-quinone electron acceptor, Q_A, to the secondary-quinone electron acceptor, Q_B (Q_B-nonreducing centers). Both complements of the PS II Chl a–b light harvesting antenna (LHC II-inner and LHC II-peripheral) were synthesized and assembled in the thylakoid membrane of dark grown C. reinhardtii cells. However, the LHC II-peripheral was energetically uncoupled from the PS II reaction center. Thus, PS II units in dark grown cells had a -type Chl antenna size with only 130 Chl (a and b) molecules (by definition, PS II units lack LHC II-peripheral). Illumination of dark grown C. reinhardtii caused pronounced changes in the organization and function of PS II. With a half-time of about 30 min, PS II centers were converted froma Q_B-nonreducing form in the dark, to a Q_B-reducing form in the light. Concomitant with this change, PS II units were energetically coupled with the LHC II-peripheral complement in the thylakoid membrane and were converted to a PS II form. The functional antenna of the latter contained more than 250 Chl(a+b) molecules. The results are discussed in terms of a light-dependent activation of the Q_A-Q_B electron-transfer reaction which is followed by association of the PS II unit with a LHC II-peripheral antenna and by inclusion of the mature form of PS II (PS II) in the membrane of the grana partition region.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F₀ non-variable fluorescence yield - F_plf intermediate fluorescence yield plateau leyel - F_max maximum fluorescence yield - F_i initial fluorescence yield increase from F₀ to F_pl (F_pl–F₀) - F_v total variable fluorescence yield (F_m–F0) - DCMU dichlorophenyl-dimethylurea     

Oligomeric form of the light-harvesting chlorophyll a + b-protein complex CP II,phosphatidyldiacylglycerol, Δ3-trans-hexadecenoic acid and energy transfer in Chlamydomonas reinhardtii,wild type and mutants

Analyses of chlorophyll-protein complexes and of lipids were performed with the wild type of Chlamydomonas reinhardtii and three non-photosynthetic mutants: Fl 39, which was a ‘classical’ high-fluorescent Photosystem II (PS II)-lacking mutant, and mf 1 and mf 2, which lacked also functional PS II but were low-fluorescent and showed an abnormally predominant energy transfer from the main light-harvesting antenna towards Photosystem I. An oligomeric form of the chlorophyll a + b-protein complex CP II was clearly isolated from the wild type and the mutant Fl 39 but it was not detected in the mutants mf 1 and mf 2. The three mutants showed total lipid contents close to or greater than that of the wild type. Their phosphatidyldiacylglycerol (PG) contents, on a chlorophyll basis, were higher (Fl 39) or 1.4- (mf 1) and 2.0- (mf 2) times lower than that of the wild type. The fatty acid compositions of the wild type and of the mutant Fl 39 were comparable, showing about equal amounts of a C18 series and a C16 series which included the Δ3-trans-hexadecenoic acid (C16:1-trans). This C16:1-trans was not detected in the mutants mf 1 and mf 2 which contained the other fatty acids. These results indicate correlations between lack of C16:1-trans-containing PG, lack of an oligomeric form of CP II and an impaired mechanism of the regulation of excitation energy transfer from the main chlorophyll a + b antenna.     

Characterization of second site mutations show that fast proton transfer to QB − is restored in bacterial reaction centers of Rhodobacter sphaeroides containing the Asp-L213 → Asn lesion

The structural basis for proton coupled electron transfer to Q_B in bacterial reaction centers (RCs) was studied by investigating RCs containing second site suppressor mutations (Asn M44 Asp, Arg M233 Cys, Arg H177 His) that complement the effects of the deleterious Asp L213 Asn mutation [DN(L213)]. The suppressor RCs all showed an increased proton coupled electron transfer rate k _AB ⁽²⁾(Q_A ^–Q_B ^– + H⁺ Q_AQ_BH^–) by at least 10³ (pH 7.5) and a recombination rate k _BD (D⁺Q_AQ_B ^– DQ_AQ_B) 15–40 times larger than the value found in DN(L213) RCs. Proton transfer was studied by measuring the dependence of k _AB ⁽²⁾ on the free energy for electron transfer (G_et). k _AB ⁽²⁾ was independent of G_et in DN(L213) RCs, but dependent on G_et in native and all suppressor RCs. This shows that proton transfer limits the k _AB ⁽²⁾ reaction with a rate of 0.1s^–1 in DN(L213) RCs but is not rate limiting and at least 10⁸-fold faster in native and 10⁵-fold faster in the suppressor RCs. The increased rate of proton transfer by the suppressor mutations are proposed to be due to: (i) a reduction in the barrier to proton transfer by providing a more negative electrostatic potential near Q_B ^–; and/or (ii) structural changes that permit fast proton transfer through the network of protonatable residues and water molecules near Q_B.     

The hypo-osmolarity-sensitive phenotype of the Saccharomyces cerevisiae hpo2 mutant is due to a mutation in PKC1, which regulates expression of β-glucanase

To obtain more information about the cell wall organization of Saccharomyces cerevisiae, we have developed a novel screening system to obtain cell wall-defective mutants, using a density gradient centrifugation method. Nine hypo-osmolarity-sensitive mutants were classified into two complementation groups, hpo1 and hpo2. Phase contrast microscopic observation showed that mutant cells bearing lesions at either locus became abnormally large. A gene that complemented the mutant phenotype of hpo2 was cloned and sequenced. This gene turned out to be identical to PKC1, which encodes the yeast homologue of mammalian protein kinase C. Complementation tests with pkc1Δ showed that hpo2 is allelic to pkc1. To study the reason for the fragility of hpo2 cells, cell wall was isolated and the glucan was analyzed. The amount of alkali, acid-insoluble glucan, which is responsible for the rigidity of the cell wall, was reduced to about 30% that of the wild-type cell and this may be the major cause of the fragility of the hpo2 mutant cell. Analysis of total wall proteins in hpo2 mutant cells on SDS-polyacrylamide gels revealed that a 33 kDa protein was overproduced two- to threefold relative to the wild-type level. This 33 kDa protein was identified as a β-glucanase, encoded by BGL2. Disruption of BGL2 in the hpo2 mutant partially rescued the growth rate defect. This suggests that the PKC1 kinase cascade regulates BGL2 expression negatively and overproduction of the β-glucanase is partially responsible for the growth defect. Since the bgl2 disruption did not rescue the hypo-osmolarty-sensitive phenotype of the hpo2 mutant, PKC1 must negatively regulate other enzymes involved in the biosynthesis and metabolism of the cell wall.     

Dke1—structure, dynamics, and function: a theoretical and experimental study elucidating the role of the binding site shape and the hydrogen-bonding network in catalysis

This study elucidates the role of the protein structure in the catalysis of β-diketone cleavage at the three-histidine metal center of diketone cleaving enzyme (Dke1) by computational methods in correlation with kinetic and mutational analyses. Molecular dynamics simulations, using quantum mechanically deduced parameters for the nonheme Fe(II) cofactor, were performed and showed a distinct organization of the hydrophilic triad in the free and substrate-ligated wild-type enzyme. It is shown that in the free species, the Fe(II) center is coordinated to three histidines and one glutamate, whereas the substrate-ligated, catalytically competent enzyme-substrate complex has an Fe(II) center with three-histidine coordination, with a small fraction of three-histidine, one-glutamate coordination. The substrate binding modes and channels for the traffic of water and ligands (2,4-pentandionyl anion, methylglyoxal, and acetate) were identified. To characterize the impact of the hydrophobic protein environment around the metal center on catalysis, a set of hydrophobic residues close to the active site were targeted. The variations resulted in an up to tenfold decrease of the O(2) reduction rates for the mutants. Molecular dynamics studies revealed an impact of the hydrophobic residues on the substrate stabilization in the active site as well as on the orientations of Glu98 and Arg80, which have previously been shown to be crucial for catalysis. Consequently, the Glu98-His104 interaction in the variants is weaker than in the wild-type complex. The role of protein structure in stabilizing the primary O(2) reduction step in Dke1 is discussed on the basis of our results.     

Influence of the PsbA1/PsbA3, Ca2+/Sr2+ and Cl−/Br− exchanges on the redox potential of the primary quinone QA in Photosystem II from Thermosynechococcus elongatus as revealed by spectroelectrochemistry

Ca²⁺ and Cl^? ions are essential elements for the oxygen evolution activity of photosystem II (PSII). It has been demonstrated that these ions can be exchanged with Sr²⁺ and Br^?, respectively, and that these ion exchanges modify the kinetics of some electron transfer reactions at the Mn₄Ca cluster level (Ishida et al., J. Biol. Chem. 283 (2008) 13330–13340). It has been proposed from thermoluminescence experiments that the kinetic effects arise, at least in part, from a decrease in the free energy level of the Mn₄Ca cluster in the S₃ state though some changes on the acceptor side were also observed. Therefore, in the present work, by using thin-layer cell spectroelectrochemistry, the effects of the Ca²⁺/Sr²⁺ and Cl^?/Br^? exchanges on the redox potential of the primary quinone electron acceptor Q_A, E_m(Q_A/Q_A^?), were investigated. Since the previous studies on the Ca²⁺/Sr²⁺ and Cl^?/Br^? exchanges were performed in PsbA3-containing PSII purified from the thermophilic cyanobacterium Thermosynechococcus elongatus, we first investigated the influences of the PsbA1/PsbA3 exchange on E_m(Q_A/Q_A^?). Here we show that i) the E_m(Q_A/Q_A^?) was up-shifted by ca. + 38 mV in PsbA3-PSII when compared to PsbA1-PSII and ii) the Ca²⁺/Sr²⁺ exchange up-shifted the E_m(Q_A/Q_A^?) by ca. + 27 mV, whereas the Cl^?/Br^? exchange hardly influenced E_m(Q_A/Q_A^?). On the basis of the results of E_m(Q_A/Q_A^?) together with previous thermoluminescence measurements, the ion-exchange effects on the energetics in PSII are discussed.     

Critical evaluation of electron transfer kinetics in P700–FA/FB, P700–FX, and P700–A1 Photosystem I core complexes in liquid and in trehalose glass

This work aims to fully elucidate the effects of a trehalose glassy matrix on electron transfer reactions in cyanobacterial Photosystem I (PS I). Forward and backward electron transfer rates from A_1A^? and A_1B^? to F_X, and charge recombination rates from A₀^?, A_1B^?, A_1A^?, F_X^?, and [F_A/F_B]^? to P₇₀₀⁺ were measured in P₇₀₀–F_A/F_B complexes, P₇₀₀–F_X cores, and P₇₀₀–A₁ cores, both in liquid and in a trehalose glassy matrix at 11% humidity. By comparing CONTIN-resolved kinetic events over 6 orders of time in increasingly simplified versions of PS I at 480?nm, a wavelength that reports primarily A_1A^?/A_1B^? oxidation, and over 9 orders of time at 830?nm, a wavelength that reports P₇₀₀⁺ reduction and A₀^? oxidation, assignments could be made for nearly all of the resolved kinetic phases. Trehalose-embedded PS I samples demonstrated partially arrested forward electron transfer. The fractions of complexes in which electron transfer did not proceed beyond A₀, A₁ and F_X were 53%, 16% and 22%, respectively, with only 10% of electrons reaching the terminal F_A/F_B clusters. The ~10?μs and ~150?μs components in both liquid and trehalose-embedded PS I were assigned to recombination between A_1B^? and P₇₀₀⁺ and between A_1A^? and P₇₀₀⁺, respectively. The kinetics and amplitudes of these resolved kinetic phases in liquid and trehalose-embedded PS I samples could be well-fitted by a kinetic model that allowed us to calculate the asymmetrical contribution of the A_1A^? and A_1B^? quinones to the electrochromic signal at 480?nm. Possible reasons for these effects are discussed.     

Variation of the electron population by four units in the cluster series [(η-Cp′)3Mo3S4Co(L)] (L = I, CO, PPh3, NO; n = 0, 1)

The versatility of cuboidal Mo₃S₄Co clusters for the preparation of complexes with different numbers of valence shell electrons (VSE) in the cluster is described. The reaction of the geometrically incomplete cuboidal cluster salt [(η⁵-Cp′)₃Mo₃S₄][pts] (pts = p-toluenesulfonate) with one molar equivalent of [Co₂(CO)₈] afforded almost quantitatively the electroneutral 60 VSE cluster [(η⁵-Cp′)₃Mo₃S₄Co(CO)] (1), which previously has been prepared in low yield by Curtis et al. in autoclave syntheses [M.D. Curtis, U. Riaz, O.J. Curnow, J.W. Kampf, Organometallics 14 (1995) 5337]. Cluster 1 was also obtained in high yield by reaction of [(η⁵-Cp′)₃Mo₃S₄][pts] with [(η⁵-Cp^∗)Co(CO)₂]. Reaction of [(η⁵-Cp′)₃Mo₃S₄][pts] with two molar equivalents of [Co(I)(CO)₃(PPh₃)] led to a complex mixture of products, of which the electron deficient 58 VSE cluster salt [(η⁵-Cp′)₃Mo₃S₄Co(I)][Co(I)₃(thf)] ([2][Co(I)₃(thf)]) was isolated as single crystals. In the crystal structures of 1 and [2][Co(I)₃(thf)], the Co-Mo bond lengths are almost identical, indicating a delocalization of the electron deficiency in [2]⁺. The reduced form of [2]⁺, [(η⁵-Cp′)₃Mo₃S₄Co(I)] (2), was prepared by oxidative substitution of the carbonyl ligand in 1 by I₂. Further reactions of 1 with PPh₃ and NO leading to the 60 and 61 VSE cluster complexes [(η⁵-Cp′)₃Mo₃S₄Co(PPh₃)] (3) and [(η⁵-Cp′)₃Mo₃S₄Co(NO)] (4), respectively, enabled the preparation of Mo₃S₄Co clusters in altogether four different oxidation states.