José A. Navarro

Laser flash kinetic analysis of Synechocystis PCC 6803 cytochrome c6 and plastocyanin oxidation by Photosystem I

Abstract Laser flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from reduced cytochrome c 6 and plastocyanin to photooxidized P700 in Photosystem I (PS I) particles from the cyanobacterium Synechocystis PCC 6803. Data analysis yields second-order rate constants of 1.3 · 10 7 M −1 s −1 and 1.0 · 10 7 M −1 s −1 for the heme- and copper-proteins, respectively. With the two donor proteins, the observed rate constants ( k obs ) present a linear protein-concentration dependence, thus suggesting an apparent one-step bimolecular collisional mechanism. At neutral pH, the k obs values monotonically increase with increasing NaCl or MgCl 2 concentration, which is ascribed to the involvement of repulsive electrostatic interactions between the donor proteins and PS I. The difference in the effective concentration at which MgCl 2 has its maximum effect as compared with that of NaCl is attributed to the specific role played by Mg 2+ ions, which could act as electrostatic bridges between negatively charged groups. At physiological mild acid pH, cytochrome c 6 is a more efficient electron donor than plastocyanin. The inversion of the NaCl and MgCl 2 effect at pH below 5 — that is, decreasing of k obs with increasing ionic strength — is interpreted as arising from the involvement of attractive ionic interactions at pH lower than the isoelectric point of the donor proteins. Some evolutive aspects on the mechanism of electron donation to PS I are discussed.

Photosynthesis: A new function for an old cytochrome?

In many cyanobacteria and algae, cytochrome c6 transports electrons between the cytochrome bf complex and photosystem I, replacing plastocyanin when copper is deficient. Higher plants, however, were thought to lack cytochrome c6 (refs 1,2) until the existence of a modified form in several species was inferred from genomic evidence. By measuring oxygen evolution with inside-out thylakoids, Gupta et al. inferred that heterologously expressed Arabidopsis cytochrome c6 can replace plastocyanin from Synechocystis or Arabidopsis in reconstitution experiments in vitro. From structural and kinetic evidence, however, we find that Arabidopsis cytochrome c6 cannot carry out the same function as Arabidopsis plastocyanin or as cytochrome c6 from the alga Monoraphidium braunii. This suggests that cytochrome c6 in higher plants may have lost its original primitive function in photosynthesis.

Co-evolution of cytochrome c6 and plastocyanin, mobile proteins transferring electrons from cytochrome b6f to photosystem I

Abstract Cytochrome c6 and plastocyanin are soluble metalloproteins that act as mobile carriers transferring electrons between the two membrane-embedded photosynthetic complexes cytochrome b6 f and photosystem I (PSI). First, an account of recent data on structural and functional features of these two membrane complexes is presented. Afterwards, attention is focused on the mobile heme and copper proteins – and, in particular, on the structural factors that allow recognition and confer molecular specificity and control the rates of electron transfer from and to the membrane complexes. The interesting question of why plastocyanin has been chosen over the ancient heme protein is discussed to place emphasis on the evolutionary aspects. In fact, cytochrome c6 and plastocyanin are presented herein as an excellent case study of biological evolution, which is not only convergent (two different structures but the same physiological function), but also parallel (two proteins adapting themselves to vary accordingly to each other within the same organism).

Proteomic analyses of the response of cyanobacteria to different stress conditions

Cyanobacteria are significant contributors to global photosynthetic productivity, thus making it relevant to study how the different environmental stresses can alter their physiological activities. Here, we review the current research work on the response of cyanobacteria to different kinds of stress, mainly focusing on their response to metal stress as studied by using the modern proteomic tools. We also report a proteomic analysis of plastocyanin and cytochrome c 6 deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803 grown under copper or iron deprivation, as compared to wild‐type cells, so as to get a further understanding of the metal homeostasis in cyanobacteria and their response to changing environmental conditions.

Laser flash photolysis studies of the kinetics of reduction of ferredoxins and ferredoxin-NADP+ reductases from Anabaena PCC 7119 and spinach: electrostatic effects on intracomplex electron transfer.

The influence of electrostatic forces on the formation of, and electron transfer within, transient complexes between redox proteins was examined by comparing ionic strength effects on the kinetics of the electron transfer reaction between reduced ferredoxins (Fd) and oxidized ferredoxin-NADP+ reductases (FNR) from Anabaena and from spinach, using laser flash photolysis techniques. With the Anabaena proteins, direct reduction by laser-generated flavin semiquinone of the FNR component was inhibited by complex formation at low ionic strength, whereas Fd reduction was not. The opposite results were obtained with the spinach system. These observations clearly indicate structural differences between the cyanobacterial and higher plant complexes. For the complex formed by the Anabaena proteins, the results indicate that electrostatic forces are not a major contributor to complex stability. However, the rate constant for intracomplex electron transfer had a biphasic dependence on ionic strength, suggesting that structural rearrangements within the transient complex facilitate electron transfer. In contrast to the Anabaena complex, electrostatic forces are important for the stabilization of the spinach Fd:FNR complex, and changes in ionic strength had little effect on the limiting rate constant for intracomplex electron transfer. This suggests that in this case the geometry of the initial collisional complex is optimal for reaction. These results provide a clear illustration of the differing roles that electrostatic interactions may play in controlling electron transfer between two redox proteins.

A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c 6 as alternative electron donors to Photosystem I

Plastocyanin and cytochrome c6 are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes b6f and Photosystem I. Despite plastocyanin and cytochrome c6 differing in the nature of their redox center (one is a copper protein, the other is a heme protein) and folding pattern (one is a β-barrel, the other consists of α-helices), they are exchangeable in green algae and cyanobacteria. In fact, the two proteins share a number of structural similarities that allow them to interact with the same membrane complexes in a similar way. The kinetic and thermodynamic analysis of Photosystem I reduction by plastocyanin and cytochrome c6 reveals that the same factors govern the reaction mechanism within the same organism, but differ from one another. In cyanobacteria, in particular, the electrostatic and hydrophobic interactions between Photosystem I and its electron donors have been analyzed using the wild-type protein species and site-directed mutants. A number of residues similarly conserved in the two proteins have been shown to be critical for the electron transfer reaction. Cytochrome c6 does contain two functional areas that are equivalent to those previously described in plastocyanin: one is a hydrophobic patch for electron transfer (site 1), and the other is an electrically charged area for complex formation (site 2). Each cyanobacterial protein contains just one arginyl residue, similarly located between sites 1 and 2, that is essential for the redox interaction with Photosystem I.

A single arginyl residue in plastocyanin and in cytochrome c(6) from the cyanobacterium Anabaena sp. PCC 7119 is required for efficient reduction of photosystem I.

Positively charged plastocyanin fromAnabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c 6 from Anabaena(Molina-Heredia, F. P., Dı́az-Quintana, A., Hervás, M., Navarro, J. A., and De la Rosa, M. A. (1999)J. Biol. Chem. 274, 33565–33570) andSynechocystis (De la Cerda, B., Dı́az-Quintana, A., Navarro, J. A., Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292–13297). Other mutations concern specific residues of plastocyanin either at its positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E). Mutations altering the surface electrostatic potential distribution allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no effect on the kinetics of photosystem I reduction, Leu-12 and Lys-33 are essential for the reactivity of plastocyanin.

Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures

Acetylsalicylic acid (ASA), a derivative from the plant hormone salicylic acid (SA), is a commonly used drug that has a dual role in animal organisms as an anti-inflammatory and anticancer agent. It acts as an inhibitor of cyclooxygenases (COXs), which catalyze prostaglandins production. It is known that ASA serves as an apoptotic agent on cancer cells through the inhibition of the COX-2 enzyme. Here, we provide evidences that ASA also behaves as an agent inducing programmed cell death (PCD) in cell cultures of the model plant Arabidopsis thaliana, in a similar way than the well-established PCD-inducing agent H2O2, although the induction of PCD by ASA requires much lower inducer concentrations. Moreover, ASA is herein shown to be a more efficient PCD-inducing agent than salicylic acid. ASA treatment of Arabidopsis cells induces typical PCD-linked morphological and biochemical changes, namely cell shrinkage, nuclear DNA degradation, loss of mitochondrial membrane potential, cytochrome c release from mitochondria and induction of caspase-like activity. However, the ASA effect can be partially reverted by jasmonic acid. Taking together, these results reveal the existence of common features in ASA-induced animal apoptosis and plant PCD, and also suggest that there are similarities between the pathways of synthesis and function of prostanoid-like lipid mediators in animal and plant organisms.

Site-directed Mutagenesis of Cytochromec 6 from Synechocystis sp. PCC 6803 THE HEME PROTEIN POSSESSES A NEGATIVELY CHARGED AREA THAT MAY BE ISOFUNCTIONAL WITH THE ACIDIC PATCH OF PLASTOCYANIN

This paper reports the first site-directed mutagenesis analysis of any cytochrome c 6, a heme protein that performs the same function as the copper-protein plastocyanin in the electron transport chain of photosynthetic organisms. Photosystem I reduction by the mutants of cytochromec 6 from the cyanobacteriumSynechocystis sp. PCC 6803 has been studied by laser flash absorption spectroscopy. Their kinetic efficiency and thermodynamic properties have been compared with those of plastocyanin mutants from the same organism. Such a comparative study reveals that aspartates at positions 70 and 72 in cytochrome c 6 are located in an acidic patch that may be isofunctional with the well known “south-east” patch of plastocyanin. Calculations of surface electrostatic potential distribution in the mutants of cytochromec 6 and plastocyanin indicate that the changes in protein reactivity depend on the surface electrostatic potential pattern rather than on the net charge modification induced by mutagenesis. Phe-64, which is close to the heme group and may be the counterpart of Tyr-83 in plastocyanin, does not appear to be involved in the electron transfer to photosystem I. In contrast, Arg-67, which is at the edge of the cytochrome c 6 acidic area, seems to be crucial for the interaction with the reaction center.

Comparative infection progress analysis of Lettuce big-vein virus and Mirafiori Lettuce virus in Lettuce crops by developed molecular diagnosis techniques

ABSTRACT Nonisotopic molecular dot blot hybridization technique and multiplex reverse transcription-polymerase chain reaction assay for the specific detection of Lettuce big-vein virus (LBVV) and Mirafiori lettuce virus (MiLV) in lettuce tissue were developed. Both procedures were suitable for the specific detection of both viruses in a range of naturally infected lettuce plants from various Spanish production areas and seven different cultivars. The study of the distribution of both viruses in the plant revealed that the highest concentration of LBVV and MiLV occurred in roots and old leaves, respectively. LBVV infection progress in a lettuce production area was faster than that observed for MiLV. In spite of different rates of virus infection progress, most lettuce plants became infected with both viruses about 100 days posttransplant. The appearance of both viruses in lettuce crops was preceded by a peak in the concentration of resting spores and zoosporangia of the fungus vector Olpidium brassicae in lettuce roots.