Berta De la Cerda

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.

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.

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.

A proteomic approach to iron and copper homeostasis in cyanobacteria

Cyanobacteria, which are considered to be the chloroplast precursors, are significant contributors to global photosynthetic productivity. The ample variety of membrane and soluble proteins containing different metals (mainly, iron and copper) has made these organisms develop a complex homeostasis with different mechanisms and tight regulation processes to fulfil their metal requirements in a changing environment. Cell metabolism is so adapted as to synthesize alternative proteins depending on the relative metal availabilities. In particular, plastocyanin, a copper protein, and cytochrome c(6), a haem protein, can replace each other to play the same physiological role as electron carriers in photosynthesis and respiration, with the synthesis of one protein or another being regulated by copper concentration in the medium. The unicellular cyanobacterium Synechocystis sp. PCC 6803 has been widely used as a model system because of completion of its genome sequence and the ease of its genetic manipulation, with a lot of proteomic work being done. In this review article, we focus on the functional characterization of knockout Synechocystis mutants for plastocyanin and cytochrome c(6), and discuss the ongoing proteomic analyses performed at varying copper concentrations to investigate the cyanobacterial metal homeostasis and cell response to changing environmental conditions.

Negatively charged residues in the H loop of PsaB subunit in Photosystem I from Synechocystis sp. PCC 6803 appear to be responsible for electrostatic repulsions with plastocyanin

Wild-type plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 does not form any kinetically detectable transient complex with Photosystem I (PS I) during electron transfer, but the D44R/D47R double mutant of copper protein does [De la Cerda et al. (1997) Biochemistry 36: 10125–10130]. To identify the PS I component that is involved in the complex formation with the D44R/D47R plastocyanin, the kinetic efficiency of several PS I mutants, including a PsaF–PsaJ-less PS I and deletion mutants in the lumenal H and J loops of PsaB, were analyzed by laser flash absorption spectroscopy. The experimental data herein suggest that some of the negative charges at the H loop of PsaB are involved in electrostatic repulsions with mutant plastocyanin. Mutations in the J loop demonstrate that this region of PsaB is also critical. The interaction site of PS I is thus not as defined as first expected but much broader, thereby revealing how complex the evolution of intermolecular electron transfer mechanisms in photosynthesis has been.

Reduction of photosystem I by cytochrome c6 and plastocyanin: molecular recognition and reaction mechanism☆

Abstract Molecular recognition and protein-protein electron transfer reactions were studied in a model system in which two structurally different proteins (cytochrome c6 and plastocyanin) are used alternatively to accomplish the same redox event, i.e. reduction of the photo-oxidized chlorophyll molecule P700− in photosystem I (PSI). Laser flash photolysis kinetic analyses were carried out to obtain an understanding, from a structural and functional point of view, of how this interchange ability is accomplished, as well as to obtain increased insight into the electron transfer mechanisms. Our experimental data indicate that the mechanism of reaction of both the copper-and heme-proteins with PSI is similar within the same organism, but different from one organism to another, thereby suggesting convergent evolution of the two donor proteins.

Mutations in both leucine 12 and lysine 33 in plastocyanin from Synechocystis sp. PCC 6803 induce drastic changes in the hydrophobic interactions with Photosystem I

The role played by the residues Leu12 and Lys33 – which are both located at the north hydrophobic patch of plastocyanin – in the interaction of the copper protein with Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 has been investigated by site-directed mutagenesis. A thermodynamic analysis of PS I reduction by wild-type and mutant plastocyanins has been performed by laser-flash absorption spectroscopy. In all cases, the electron transfer is impaired by mutations, which induce drastic changes in the apparent activation entropy of the overall reaction. Substitution of Leu12 by alanine specifically affects the hydrophobic interactions with PS I, whereas replacement of Lys33 by glutamate not only induces local electrostatic changes, but also alters the hydrophobic interactions with the photosystem. The thermodynamic analysis of the reactivity of K33E mutant towards PS I reveals that the effect of the mutation can be reversed by addition of magnesium cations, which probably bind at a place close to Glu33. The electrostatic surface potential does thus modulate the hydrophobic interactions with PS I by altering the solvent accessibility of some surface residues.

From Cytochrome C 6 to Plastocyanin. An Evolutionary Approach

The transfer of electrons from the cytochrome b6 - f complex to photosystem I (PSI) is carried out by any of the two soluble metalloproteins cytochrome c6 and plastocyanin. From an evolutionary point of view, it is noteworthy that the heme protein has been replaced by plastocyanin: Whereas cytochrome c6 seems to be the only electron carrier in primitive oxygen-evolving photosynthetic organisms, there is a number of cyanobacteria and green algae that are able to synthesize both cytochrome c6 and plastocyanin; in higher plants, by contrast, the redox connection between the two membrane complexes is just mediated by the copper protein. An interesting question thus arises: What is the reason why plastocyanin has been chosen over cytochrome c6 along evolution?