RESUMEN
A chlorophyll-protein complex has been isolated from the cyanobacterium Synechocystis sp. PCC 6803 that closely resembles higher plant photosystem II reaction centers in spectral properties. The Synechocystis complex has a pigment content of 5-7 chlorophyll a molecules:1 Cyt b559:2 pheophytins; an optical absorption redmost transition at approximately 675 nm; and a nonconservative circular dichroism red signal, with extrema at 682 (+) and 652 (-) nm. Upon illumination, the Synechocystis D1/D2/Cyt b559 complex accumulates reduced pheophytin. LDS-PAGE and/or immunoblotting showed the D1, D2, and Cyt b559 proteins, aggregated and degraded forms of D1 and possibly D2, and traces of ATP synthase and the CP47 photosystem II chlorophyll protein. The availability of such a Synechocystis preparation opens the way for employing site-directed mutagenesis in studying primary reactions of oxygenic photosynthesis.
Asunto(s)
Cianobacterias/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/aislamiento & purificación , Clorofila/aislamiento & purificación , Clorofila/metabolismo , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Dicroismo Circular , Ditionita , Electroforesis en Gel de Poliacrilamida , Indicadores y Reactivos , Complejos de Proteína Captadores de Luz , Peso Molecular , Oxidación-Reducción , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Complejo de Proteína del Fotosistema II , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
The region between helices D and E (D-E region) of the D1 protein of photosystem II (PSII) is exposed at the stromal side of the photosynthetic membrane, contains the secondary plastoquinone (QB) binding niche, and is involved in processes at the reducing side of PSII. The role of the D-E region was studied in 27 site-directed mutants generated in the psbAII gene of the cyanobacterium Synechocystis sp. PCC 6803. The photochemical performance of the modified PSII reaction centers was assessed with respect to photoautotrophic growth, oxygen evolution, fluorescence induction, and herbicide inhibition. A few mutations, located at positions presumably involved in essential interactions in the QB binding niche, greatly interfered with PSII performance. On the other hand, mutations in the presumptive loop region between helices D and de resulted in relatively minor effects, indicating a flexible region not critical for photochemical function. Indeed, although more than 80% of the D-E region is phylogenetically invariant, the bulk of the mutations affected the measured parameters only moderately. The significance of the conserved residues appears to be in subtle interactions that optimize the thermodynamic balance between some of the redox components of PSII, as indicated by mild changes in the steady state fluorescence. Many mutations modified tolerances to PSII herbicides. The dispersion of these mutations throughout the D-E region indicates the complex nature of the interactions, direct and indirect, affecting herbicide binding in the QB niche. Mutation of codons Ser221 and Ser222 to Leu221 and Ala222 revealed a new location coordinating the herbicide diuron in the D1 protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Herbicidas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Quinonas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cianobacterias/genética , Cianobacterias/metabolismo , ADN Bacteriano/genética , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Oxígeno/metabolismo , Fotoquímica , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Homología de Secuencia de AminoácidoRESUMEN
The D2 protein contains an extended loop (the D-de loop) between helices D and de at the reducing side of photosystem II (PS II). Characterization of D2 mutants of the cyanobacterium Synechocystis sp. PCC 6803 has indicated that the length and amino acid composition of the D-de loop are not critical for basic PS II functions, although most of the residues in that region are conserved phylogenetically. Here we show using herbicide binding and electron-flow inhibition measurements that drastic modifications in the D-de loop of the D2 protein modify the interaction of some PS II-directed herbicides with their binding niche. The stability of (semi)-reduced QB in its binding pocket is altered in at least two of the mutants, as indicated by a shifted peak temperature of the thermoluminescence signal originating from charge recombination involving QB. These results suggest a close functional association between the D-de loop of the D2 protein and the QB/herbicide-binding environment, which is viewed as being coordinated mostly by residues of the D1 protein. This represents one of the first examples of modification of the QB/herbicide-binding domain by mutations in the D2 protein.
Asunto(s)
Cianobacterias/metabolismo , Herbicidas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Clorofila/metabolismo , Cianobacterias/genética , Diurona/metabolismo , Diurona/farmacología , Transporte de Electrón/efectos de los fármacos , Herbicidas/farmacología , Cinética , Complejos de Proteína Captadores de Luz , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fotosíntesis/efectos de los fármacos , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema II , Estructura Secundaria de Proteína , Transformación GenéticaRESUMEN
Plant geneticists have determined that the color of ripe fruits of sweet peppers (Capsicum annuum L.) is determined by four genes: y, c1, c2 and cl. We have compared the electrophoretic behavior of chromoplast membrane proteins of seven varieties of C. annuum which differ in these genes. ChrA was detected only in the varieties that had a y+ genotype, and was not affected by variations in the other three genes. The identity of ChrA was verified by probing blots of SDS gels with antiserum to ChrA. The second known chromoplast-specific protein, ChrB, was found to be independent of all four genes. No proteins correlating with c1, c2 or cl were detected in either one- or two-dimensional gels.
Asunto(s)
Capsicum/genética , Dimetilaliltranstransferasa/análisis , Orgánulos/enzimología , Plantas Medicinales , Capsicum/enzimología , Color , GenotipoRESUMEN
A fluorescent dye sensitive to membrane potential was used to follow the plasma-membrane potential in the unicellular halo-tolerant alga Dunaliella salina. The signal observed during dissipation of the plasma membrane potential by the addition of excess K(+) and valinomycin, or a protonophore, was taken as a measure of the preexisting potential. A resting potential of -85 to -100 millivolts (negative inside) was calculated. Following a hypertonic shock, the plasma membrane was rapidly hyperpolarized. This hyperpolarization was transient, and the algae resumed their resting potential about 30 minutes after the shock. The resting plasma membrane potential was decreased by vanadate and is concluded to be generated mostly by the plasma membrane ATPase of Dunaliella. The transient hyperpolarization following a hypertonic shock indicates, therefore, a transient activation of the ATPase. This is further corroborated by a rapid transient decrease in the intracellular ATP following a hypertonic shock and its inhibition by vanadate. It is suggested that activation of the plasma membrane ATPase may be the trigger for osmoregulation in Dunaliella.
RESUMEN
The unicellular halotolerant alga Dunaliella salina recovers normally from a hypertonic shock even when suspended in NaCl and buffer only. Furthermore, addition of Cu(2+), valinomycin and KCl, or permeable ions such as methyltriphenylphosphonium or thiocyanate, do not affect the recovery. However, treatment with two specific inhibitors of the plasma membrane adenosine triphosphatase (ATPase), diethylstilbestrol, or vanadate, fully inhibit the recovery. The inhibition is manifested by the inability of the cells to both synthesize glycerol and return to their original volume. The inhibitions are nonlethal, reversible and equally effective in the dark or the light. Since the plasma membrane ATPase is the only enzyme known to be inhibited by both diethylstilbestrol and vanadate, it is concluded that its activity is essential for the recovery of Dunaliella from a hypertonic shock. Mechanisms by which the plasma membrane ATPase may participate in the activation of glycerol production in the algae are discussed.
RESUMEN
The intracellular phosphorus and carbon metabolites in the halotolerant alga Dunaliella salina adapted to different salinities were monitored in living cells by (31)P- and (13)C-nuclear magnetic resonance (NMR) spectroscopy. The (13)C-NMR studies showed that the composition of the visible intracellular carbon metabolites other than glycerol is not significantly affected by the salinity of the growth medium. The T(1) relaxation rates of the (13)C-glycerol signals in intact cells were enhanced with increasing salinity of the growth medium, in parallel to the expected increase in the intracellular viscosity due to the increase in intracellular glycerol. The (31)P-NMR studies showed that cells adapted to the various salinities contained inorganic phosphate, phosphomonoesters, high energy phosphate compounds, and long chain polyphosphates. In addition, cells grown in media containing up to 1 molar NaCl contained tripolyphosphates. The tripolyphosphate content was also controlled by the availability of inorganic phosphate during cell growth. Phosphate-depleted D. salina contained no detectable tripolyphosphate signal. Excess phosphate, however, did not result in the appearance of tripolyphosphate in (31)P-NMR spectra of cells adapted to high (>1.5 molar NaCl) salinites.