Mercedes Roncel

A laser flash photolysis study of the reduction of methyl viologen by conduction band electrons of TiO2 and FeTi oxide photocatalysts

The photochemical activity of undoped and iron-doped TiO2 particles was studied by laser flash photolysis. A short duration laser pulse was used to produce electron-hole pairs; methyl viologen was employed as electron scavenger. The presence of small amounts of iron ions (less than 0.5%) in TiO2 matrices is beneficial to the photoreduction of methyl viologen (MV2+); by contrast, an increase in the amount of iron doping in TiO2 samples (from 0.5% to 5%) sharply reduces the MV+ yield to a value similar to that when pure TiO2 is used. The implications of these results to charge transfer photoreactions, such as dinitrogen reduction to ammonia, are discussed.

Cytochrome c550 in the Cyanobacterium Thermosynechococcus elongatus STUDY OF REDOX MUTANTS

Cytochrome c550 is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c550 we constructed two mutants of Thermosynechococcus elongatus in which the residue His-92, the sixth ligand of the heme, was replaced by a Met or a Cys in order to modify the redox properties of the heme. The H92M and H92C mutations changed the midpoint redox potential of the heme in the isolated cytochrome by +125 mV and –30 mV, respectively, compared with the wild type. The binding-induced increase of the redox potential observed in the wild type and the H92C mutant was absent in the H92M mutant. Both modified cytochromes were more easily detachable from the Photosystem II compared with the wild type. The Photosystem II activity in cells was not modified by the mutations suggesting that the redox potential of the cytochrome c550 is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c550 was also constructed. It showed a lowered affinity for Cl– and Ca2+ as reported earlier for the cytochrome c550-less Synechocystis 6803 mutant, but it showed a shorter lived \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{S}_{2}Q_{B}^{-}\) \end{document} state, rather than a stabilized S2 state and rapid deactivation of the enzyme in the dark, which were characteristic of the Synechocystis mutant. It is suggested that the latter effects may be caused by loss (or weaker binding) of the other extrinsic proteins rather than a direct effect of the absence of the cytochrome c550.

Characterization of a photosynthetic Euglena strain isolated from an acidic hot mud pool of a volcanic area of Costa Rica.

Abstract Conspicuous green patches on the surface of an acidic hot mud pool located near the Rincón de la Vieja volcano (northwestern Costa Rica) consisted of apparently unialgal populations of a chloroplast-bearing euglenoid. Morphological and physiological studies showed that it is a non-flagellated photosynthetic Euglena strain able to grow in defined mineral media at temperatures up to 40 degrees C and exhibiting higher thermotolerance than Euglena gracilis SAG 5/15 in photosynthetic activity analyses. Molecular phylogeny studies using 18S rDNA and GapC genes indicated that this strain is closely related to Euglena mutabilis, another acid-tolerant photosynthetic euglenoid, forming a clade deeply rooted in the Euglenales lineage. To our knowledge this is the most thermotolerant euglenoid described so far and the first Euglenozoan strain reported to inhabit acidic hot aquatic habitats.

Light-induced degradation of cytochrome b559 during photoinhibition of the photosystem II reaction center

The behaviour of cytochrome (cyt) b559 during acceptor‐ and donor‐side photoinhibition has been investigated in oxygen‐evolving and non‐evolving photosystem II (PSII) membranes. Strong illumination at 20°C under aerobiosis induced a strong decrease in the absorbance of the cyt b559 α‐band in the two preparations. This absorbance decline was observed only in non‐oxygen‐evolving PSII samples when illumination was performed under aerobiosis but at 4°C, or under anaerobiosis at 20°C. These results suggest that acceptor‐side photoinhibition induces the degradation of cyt b559 by a mechanism related to an enzymatic reaction mediated by singlet oxygen. Donor‐side photoinhibition may induce, however, a non‐enzymatic photocleavage of the protein.

The role of the high potential form of the cytochrome b559: Study of Thermosynechococcus elongatus mutants.

Cytochrome b559 is an essential component of the photosystem II reaction center in photosynthetic oxygen-evolving organisms, but its function still remains unclear. The use of photosystem II preparations from Thermosynechococcus elongatus of high integrity and activity allowed us to measure for the first time the influence of cytochrome b559 mutations on its midpoint redox potential and on the reduction of the cytochrome b559 by the plastoquinone pool (or QB). In this work, five mutants having a mutation in the α-subunit (I14A, I14S, R18S, I27A and I27T) and one in the β-subunit (F32Y) of cytochrome b559 have been investigated. All the mutations led to a destabilization of the high potential form of the cytochrome b559. The midpoint redox potential of the high potential form was significantly altered in the αR18S and αI27T mutant strains. The αR18S strain also showed a high sensitivity to photoinhibitory illumination and an altered oxidase activity. This was suggested by measurements of light induced oxidation and dark re-reduction of the cytochrome b559 showing that under conditions of a non-functional water oxidation system, once the cytochrome is oxidized by P680(+), the yield of its reduction by QB or the PQ pool was smaller and the kinetic slower in the αR18S mutant than in the wild-type strain. Thus, the extremely positive redox potential of the high potential form of cytochrome b559 could be necessary to ensure efficient oxidation of the PQ pool and to function as an electron reservoir replacing the water oxidation system when it is not operating.

Photosynthetic cytochrome c550

Cytochrome c550 (cyt c550) is a membrane component of the PSII complex in cyanobacteria and some eukaryotic algae, such as red and brown algae. Cyt c550 presents a bis-histidine heme coordination which is very unusual for monoheme c-type cytochromes. In PSII, the cyt c550 with the other extrinsic proteins stabilizes the binding of Cl(-) and Ca(2+) ions to the oxygen evolving complex and protects the Mn(4)Ca cluster from attack by bulk reductants. The role (if there is one) of the heme of the cyt c550 is unknown. The low midpoint redox potential (E(m)) of the purified soluble form (from -250 to -314mV) is incompatible with a redox function in PSII. However, more positive values for the Em have been obtained for the cyt c550 bound to the PSII. A very recent work has shown an E(m) value of +200mV. These data open the possibility of a redox function for this protein in electron transfer in PSII. Despite the long distance (22Å) between cyt c550 and the nearest redox cofactor (Mn(4)Ca cluster), an electron transfer reaction between these components is possible. Some kind of protective cycle involving a soluble redox component in the lumen has also been proposed. The aim of this article is to review previous studies done on cyt c550 and to consider its function in the light of the new results obtained in recent years. The emphasis is on the physical properties of the heme and its redox properties. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Flavin-mediated photoregulation of nitrate reductase: A key point of control in inorganic nitrogen photosynthetic metabolism☆

The first step in the photosynthetic reduction of nitrate to ammonia, as carried out by plants and green algae, is catalyzed by the enzyme nitrate reductase. This enzyme serves an assimilatory function in which it catalyzes the exergonic two-electron reduction of nitrate to nitrite with electrons donated by pyridine nucleotides, which are in turn reduced at the end of the photosynthetic electron transport chain by electrons derived originally from water. Although the assimilation of nitrate by photosynthetic organisms appears to be regulated primarily at the nitrate uptake level, the nitrate reductase activity seems to be another key point of control for this pathway. In fact, nitrate reductase from eukaryotic organisms has been demonstrated to exist in two metabolically interconvertible forms, one oxidized/active and the other reduced/inactive. To account for such a redox interconversion of nitrate reductase, several mechanisms have been proposed. One of them is based on the characteristic photochemical properties of flavins, which can be reduced under illumination by a variety of electron donors, e.g. EDTA (or ethylenediaminetetraacetic acid), semicarbazide and amino acids, which, on the other hand, are unable to reduce flavins in the dark. This redox photoregulation of nitrate reductase activity has been studied in our laboratory using the enzyme from the green alga Monoraphidium braunii. Inactivation has been proposed to involve three steps: (i) reduction of flavins upon irradiation under reducing conditions, (ii) reduction of the enzyme-bound molybdenum centers, and (iii) formation of a stable complex with a nucleophilic agent such as cyanide, superoxide, or other. The reactivation process would also take place in three steps: (i) photoexcitation of flavins under oxidizing conditions, (ii) deoxidation of the regulatory centers by photoexcited flavins, and (iii) release of the nucleophilic agent bound to the enzyme. The redox conditions of the cells when flavins are photoexcited can thereby regulate nitrate reductase activity and hence nitrate assimilation.

A High Redox Potential Form of Cytochrome c550 in Photosystem II from Thermosynechococcus elongatus

Cytochrome c550 (cyt c550) is a component of photosystem II (PSII) from cyanobacteria, red algae, and some other eukaryotic algae. Its physiological role remains unclear. In the present work, measurements of the midpoint redox potential (Em) were performed using intact PSII core complexes preparations from a histidine-tagged PSII mutant strain of the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus. When redox titrations were done in the absence of redox mediators, an Em value of +200 mV was obtained for cyt c550. This value is ∼300 mV more positive than that previously measured in the presence of mediators (Em = −80 mV). The shift from the high potential form (Em = +200 mV) to the low potential form (Em = −80 mV) of cyt c550 is attributed to conformational changes, triggered by the reduction of a component of PSII that is sequestered and out of equilibrium with the medium, most likely the Mn4Ca cluster. This reduction can occur when reduced low potential redox mediators are present or under highly reducing conditions even in the absence of mediators. Based on these observations, it is suggested that the Em of +200 mV obtained without mediators could be the physiological redox potential of the cyt c550 in PSII. This value opens the possibility of a redox function for cyt c550 in PSII.

Spectroscopic and functional characterization of cyanobacterium Synechocystis PCC 6803 mutants on the cytoplasmic-side of cytochrome b559 in photosystem II.

We performed spectroscopic and functional characterization on cyanobacterium Synechocystis PCC6803 with mutations of charged residues of the cytoplasmic side of cytochrome (Cyt) b559 in photosystem II (PSII). All of the mutant cells grew photoautotrophically and assembled stable PSII. However, R7Eα, R17Eα and R17Lβ mutant cells grew significantly slower and were more susceptible to photoinhibition than wild-type cells. The adverse effects of the arginine mutations on the activity and the stability of PSII were in the following order (R17Lβ>R7Eα>R17Eα and R17Aα). All these arginine mutants exhibited normal period-four oscillation in oxygen yield. Thermoluminescence characteristics indicated a slight decrease in the stability of the S3QB(-)/S2QB(-) charge pairs in the R7Eα and R17Lβ mutant cells. R7Eα and R17Lβ PSII core complexes contained predominantly the low potential form of Cyt b559. EPR results indicated the displacement of one of the two axial ligands to the heme of Cyt b559 in R7Eα and R17Lβ mutant reaction centers. Our results demonstrate that the electrostatic interactions between these arginine residues and the heme propionates of Cyt b559 are important to the structure and redox properties of Cyt b559. In addition, the blue light-induced nonphotochemical quenching was significantly attenuated and its recovery was accelerated in the R7Lα and R17Lβ mutant cells. Furthermore, ultra performance liquid chromatography-mass spectrometry results showed that the PQ pool was more reduced in the R7Eα and R17Lβ mutant cells than wild-type cells in the dark. Our data support a functional role of Cyt b559 in protection of PSII under photoinhibition conditions in vivo.

Peculiar properties of chlorophyll thermoluminescence emission of autotrophically or mixotrophically grown Chlamydomonas reinhardtii.

The microalgae Chlamydomonas reinhardtii and Chlorella sp. CCAP 211/84 were grown autotrophically and mixotrophically and their thermoluminescence emissions were recorded above 0 °C after excitation by 1, 2 or 3 xenon flashes or by continuous far-red light. An oscillation of the B band intensity according to the number of flashes was always observed, with a maximum after 2 flashes, accompanied by a downshift of the B band temperature maximum in mixotrophic compared to autotrophic grown cells, indicative of a dark stable pH gradient. Moreover, new flash-induced bands emerged in mixotrophic Chlamydomonas grown cells, at temperatures higher than that of the B band. In contrast to the afterglow band observed in higher plants, in Chlamydomonas these bands were not inducible by far-red light, were fully suppressed by 2 μM antimycin A, and peaked at different temperatures depending on the flash number and growth stage, with higher temperature maxima in cells at a stationary compared to an exponential growth stage. These differences are discussed according to the particular properties of cyclic electron transfer pathways in C. reinhardtii.