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

Plastocyanin and cytochrome c ₆ are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes b ₆ f and Photosystem I. Despite plastocyanin and cytochrome c ₆ 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 c ₆ 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 c ₆ 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. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of crowding on the electron transfer process from plastocyanin and cytochrome c<Subscript>6</Subscript> to photosystem I: a comparative study from cyanobacteria to green algae

Plastocyanin and cytochrome c ₆, the alternate donor proteins to photosystem I, can be acidic, neutral or basic; the role of electrostatics in their interaction with photosystem I vary accordingly for cyanobacteria, algae and plants. The effect of different crowding agents on the kinetics of the reaction between plastocyanin or cytochrome c ₆ and photosystem I from three different cyanobacteria, Synechocystis PCC 6803, Nostoc PCC 7119 and Arthrospira maxima, and a green alga, Monoraphidium braunii, has been investigated by laser flash photolysis, in order to elucidate how molecular crowding affects the interaction between the two donor proteins and photosystem I. The negative effect of viscosity on the interaction of the two donors with photosystem I for the three cyanobacterial systems is very similar, as studied by increasing sucrose concentration. Bovine serum albumin seems to alter the different systems in a specific way, probably by means of electrostatic interactions with the donor proteins. Ficoll and dextran behave in a parallel manner, favouring the interaction by an average factor of 2, although this effect is somewhat less pronounced in Nostoc. With regards to the eukaryotic system, a strong negative effect of viscosity is able to overcome the favourable effect of any crowding agent, maybe due to stronger donor/photosystem I electrostatic interactions or the structural nature of the eukaryotic photosystem I-enriched membrane particles.     

Resonance Raman fingerprinting of multiheme cytochromes from the cytochrome c 3 family

Resonance Raman (RR) spectroscopy was used to investigate conformational characteristics of the hemes of several ferricytochromes of the cytochrome c ₃ family, electron transfer proteins isolated from the periplasm and membranes of sulfate-reducing bacteria. Our analysis concentrated on the low-frequency region of the RR spectra, a fingerprint region that includes vibrations for heme-protein C–S bonds [ν(C_aS)]. It has been proposed that these bonds are directly involved in the electron transfer process. The three groups of tetraheme cytochrome c ₃ analyzed, namely Type I cytochrome c ₃ (TpIc ₃s), Type II cytochrome c ₃ (TpIIc ₃s) and Desulfomicrobium cytochromes c ₃, display different frequency separations for the two ν(C_aS) lines that are similar among members of each group. These spectral differences correlate with differences in protein structure observed among the three groups of cytochromes c ₃. Two larger cytochromes of the cytochrome c ₃ family display RR spectral characteristics for the ν(C_aS) lines that are closer to TpIIc ₃ than to TpIc ₃. Two other multiheme cytochromes from Desulfovibrio that do not belong to the cytochrome c ₃ family display ν(C_aS) lines with reverse relative areas in comparison with the latter family. This RR study shows that the small differences in protein structure observed among these cytochrome c ₃ correlate to differences on the heme–protein bonds, which are likely to have an impact upon the protein function, making RR spectroscopy a sensitive and useful tool for characterizing these cytochromes.     

Cytochrome c6-like protein as a putative donor of electrons to photosystem I in the cyanobacterium Nostoc sp. PCC 7119

Most organisms performing oxygenic photosynthesis contain either cytochrome c ₆ or plastocyanin, or both, to transfer electrons from cytochrome b ₆-f to photosystem I. Even though plastocyanin has superseded cytochrome c ₆ along evolution, plants contain a modified cytochrome c ₆, the so called cytochrome c _6A, whose function still remains unknown. In this article, we describe a second cytochrome c ₆ (the so called cytochrome c ₆-like protein), which is found in some cyanobacteria but is phylogenetically more related to plant cytochrome c _6A than to cyanobacterial cytochrome c ₆. In this article, we conclude that the cytochrome c ₆-like protein is a putative electron donor to photosystem I, but does play a role different to that of cytochrome c ₆ and plastocyanin as it cannot accept electrons from cytochrome f. The existence of this third electron donor to PSI could explain why some cyanobacteria are able to grow photoautotrophically in the absence of both cytochrome c ₆ and plastocyanin. In any way, the Cyt c ₆-like protein from Nostoc sp. PCC 7119 would be potentially utilized for the biohydrogen production, using cell-free photosystem I catalytic nanoparticles.     

A comparative study of the thermal stability of plastocyanin, cytochrome c6 and Photosystem I in thermophilic and mesophilic cyanobacteria

Cytochrome c₆ (Cyt) from the thermophilic cyanobacterium Phormidium laminosum has been purified and characterized. It is a mildly acidic protein, with physicochemical properties very similar to those of plastocyanin (Pc). This is in agreement with the functional interchangeability of the two metalloproteins as electron donors to Photosystem I (PS I). The kinetic analyses of the interaction of Pc and Cyt with Photosystem I show that both metalloproteins reduce PS I with similar efficiencies, according to an oriented collisional kinetic model involving repulsive electrostatic interactions. The thermostability study of the Phormidium Pc/PS I system compared with those from mesophilic cyanobacteria (Synechocystis, Anabaena and Pseudanabaena) reveals that Pc is the partner limiting the thermostability of the Phormidium couple. The cross-reactions between Pc and PS I from different organisms demonstrate not only that Phormidium Pc enhances the stability of the Pc/PS I system using PS I from mesophilic cyanobacteria, but also that Phormidium PS I possesses a higher thermostability than the other photosystems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Lumenal proteins involved in respiratory electron transport in the cyanobacterium Synechocystis sp. PCC6803

Cyanobacterial thylakoids catalyze both photosynthetic and respiratory activities. In a photosystem I-less Synechocystis sp. PCC 6803 strain, electrons generated by photosystem II appear to be utilized by cytochrome oxidase. To identify the lumenal electron carriers (plastocyanin and/or cytochromes c ₅₅₃, c ₅₅₀, and possibly c _M) that are involved in transfer of photosystem II-generated electrons to the terminal oxidase, deletion constructs for genes coding for these components were introduced into a photosystem I-less Synechocystis sp. PCC 6803 strain, and electron flow out of photosystem II was monitored in resulting strains through chlorophyll fluorescence yields. Loss of cytochrome c ₅₅₃ or plastocyanin, but not of cytochrome c ₅₅₀, decreased the rate of electron flow out of photosystem II. Surprisingly, cytochrome c _M could not be deleted in a photosystem I-less background strain, and also a double-deletion mutant lacking both plastocyanin and cytochromec ₅₅₃ could not be obtained. Cytochrome c _M has some homology with the cytochrome c-binding regions of the cytochromecaa₃ -type cytochrome oxidase from Bacillus spp. and Thermus thermophilus. We suggest that cytochrome c _M is a component of cytochrome oxidase in cyanobacteria that serves as redox intermediate between soluble electron carriers and the cytochromeaa₃ complex, and that either plastocyanin or cytochrome c ₅₅₃ can shuttle electrons from the cytochrome b_6f complex to cytochrome c _M.     

Cytochrome c6B of Synechococcus sp. WH 8102 – Crystal structure and basic properties of novel c6-like family representative

Cytochromes c are soluble electron carriers of relatively low molecular weight, containing single heme moiety. In cyanobacteria cytochrome c₆ participates in electron transfer from cytochrome b₆f complex to photosystem I. Recent phylogenetic analysis revealed the existence of a few families of proteins homologous to the previously mentioned. Cytochrome c_6A from Arabidopsis thaliana was identified as a protein responsible for disulfide bond formation in response to intracellular redox state changes and c₅₅₀ is well known element of photosystem II. However, function of cytochromes marked as c_6B, c_6C and c_M as well as the physiological process in which they take a part still remain unidentified. Here we present the first structural and biophysical analysis of cytochrome from the c_6B family from mesophilic cyanobacteria Synechococcus sp. WH 8102. Purified protein was crystallized and its structure was refined at 1.4 Å resolution. Overall architecture of this polypeptide resembles typical I-class cytochromes c. The main features, that distinguish described protein from cytochrome c₆, are slightly red-shifted α band of UV–Vis spectrum as well as relatively low midpoint potential (113.2 ± 2.2 mV). Although, physiological function of cytochrome c_6B has yet to be determined its properties probably exclude the participation of this protein in electron trafficking between b₆f complex and photosystem I.     

Evidence for a dual role of cytochrome c-553 and plastocyanin in photosynthesis and respiration of the cyanobacterium,Anabaena variabilis

The cytochrome oxidase activity (oxygen uptake in the dark) of a membrane preparation from Anabaena variabilis was found to be stimulated by cytochrome c-553 and plastocyanin obtained from this alga. Cytochrome c from horse heart was as active as cytochrome c-553, whereas little or no stimulation of oxygen uptake was obtained with cytochromes c ₂ from two Rhodospirillaceae, the plastidic cytochrome c-552 from Euglena, and plastocyanin from spinach. Cytochrome c-553 (A. variabilis) stimulated photosystem 1 activity in the same preparation much more than cytochrome c (horse heart). The results indicate that cytochrome c-553 and plastocyanin, besides their established function as electron donors of photosystem 1, participate in respiratory electron transport as reductants of a terminal oxidase. Photooxidation and dark oxidation show a different donor specificity.Abbreviations Chl chlorophyll a - TMPD N,N,N,N-tetramethyl-p-phenylenediamine     

Early research on the role of plastocyanin in photosynthesis

A review is presented of the early history of investigations into the function of the blue copper-protein plastocyanin in photosynthesis. The controversy or confusion that arose as to the function of plastoycanin in conjunction with cytochrome f and cytochrome c ₆ is discussed and investigations contributing to the establishment of the role of plastocyanin as the mobile electron carrier between the Photosystem I reaction center complex and the cytochrome b ₆/f complex are described. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Targeted deletion of psaJ from the cyanobacterium Synechocystis sp. PCC 6803 indicates structural interactions between the PsaJ and PsaF subunits of photosystem I

Photosystem I catalyzes the light-driven oxidation of plastocyanin or cytochrome c ₆ and the reduction of ferredoxin or flavodoxin. PsaJ is a 4.4 kDa hydrophobic subunit of photosystem I from cyanobacteria and chloroplasts. To investigate the function of PsaJ, we generated a mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 in which the psaJ gene is replaced by a gene for chloramphenicol resistance. Deletion of psaJ led to a reduction in the steady state RNA level from psaF which is located upstream from psaJ. Immunoquantification using an anti-PsaF antibody revealed a significant decrease in the amount of PsaF in membranes of the mutant strain. Trimeric photosystem I complexes isolated from the mutant strain using n-dodecyl -D-maltoside lacked PsaJ, contained ca. 80% less PsaF, but maintained wild-type levels of other photosystem I subunits. In contrast, the photosystem I purified using Triton X-100 contained less than 2% PsaF when compared to the wild type, showing the more extractable nature of PsaF in PsaJ-less photosystem I in the presence of Triton X-100. PsaE was more accessible to removal by NaI in a mutant strain lacking PsaF and PsaJ than in the wild type. The presence of PsaF in photosystem I from the PsaJ-less strain did not alter the increased susceptibility of PsaE to removal by NaI. These results indicate an interaction between PsaJ and PsaF in the organization of the complex.     

Photoreactions of cytochrome b6 in systems using resolved chloroplast electron-transfer complexes

Photoreactions of cytochrome b₆ have been studied using resolved chloroplast electron-transfer complexes. In the presence of Photosystem (PS) II and the cytochrome b₆-f complex, photoreduction of the cytochrome can be observed. No soluble components are required for this reaction. Cytochrome b₆ photoreduction was found to be inhibited by quinone analogs, which inhibit at the Rieske iron-sulfur center of the cytochrome complex, by the addition of ascorbate and by depletion of the Rieske center and bound plastoquinone from the cytochrome complex. Photoreduction of cytochrome b₆ can also be demonstrated in the presence of the cytochrome complex and PS I. This photoreduction requires plastocyanin and a low-potential electron donor, such as durohydroquinone. Cytochrome b₆ photoreduction in the presence of PS I is inhibited by quinone analogs which interact with the Rieske iron-sulfur center. These results are discussed in terms of a Q-cycle mechanism in which plastosemiquinone serves as the reductant for cytochrome b₆ via an oxidant-induced reductive pathway.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Isolation of cytochrome bc 1 complexes from the photosynthetic bacteria Rhodopseudomonas viridis and Rhodospirillum rubrum

Cytochrome bc ₁ complexes have been isolated from wild type Rhodopseudomonas viridis and Rhodospirillum rubrum and purified by affinity chromatography on cytochrome c-Sepharose 4B. Both complexes are largely free of bacteriochlorophyll and carotenoids and contain cytochromes b and c ₁ in a 2:1 molar ratio. For the Rps. viridis complex, evidence has been obtained for two spectrally distinct b-cytochromes. The R. rubrum complex contains a Rieske iron-sulfur protein (present in approximately 1:1 molar ratio to cytochrome c ₁) and catalyzes an antimycin A- and myxothiazol-sensitive electron transfer from duroquinol to equine cytochrome c or R. rubrum cytochrome c ₂. Although an attempt to prepare a cytochrome bc ₁ complex from the gliding green bacterium Chloroflexus aurantiacus was not successful, membranes isolated from phototrophically grown Cfl. aurantiacus were shown to contain a Rieske iron-sulfur protein and protoheme (the prosthetic group of b-type cytochromes).Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

How rapid are the internal reactions of the ubiquinol:cytochrome c 2 oxidoreductase?

The temperature dependence of the partial reactions leading to turn-over of the UQH₂:cyt c ₂ oxidoreductase of Rhodobacter sphaeroides have been studied. The redox properties of the cytochrome components show a weak temperature dependence over the range 280–330 K, with coefficients of about 1 m V per degree; our results suggest that the other components show similar dependencies, so that no significant change in the gradient of standard free-energy between components occurs over this temperature range. The rates of the reactions of the high potential chain (the Rieske iron sulfur center, cytochromes c ₁ and c ₂, reaction center primary donor) show a weak temperature dependence, indicating an activation energy < 8 kJ per mole for electron transfer in this chain. The oxidation of ubiquinol at the Q_z-site of the complex showed a strong temperature dependence, with an activation energy of about 32 kJ mole^–1. The electron transfer from cytochrome b-566 to cytochrome b-561 was not rate determining at any temperature, and did not contribute to the energy barrier. The activation energy of 32 kJ mole^–1 for quinol oxidation was the same for all states of the quinone pool (fully oxidized, partially reduced, or fully reduced before the flash). We suggest that the activation barrier is in the reaction by which ubiquinol at the catalytic site is oxidized to semiquinone. The most economical scheme for this reaction would have the semiquinone intermediate at the energy level indicated by the activation barrier. We discuss the plausibility of this simple model, and the values for rate constants, stability constant, the redox potentials of the intermediate couples, and the binding constant for the semiquinone, which are pertinent to the mechanism of the ubiquinol oxidizing site.Abbreviations (BChl)₂ P870, primary donor of the photochemical reaction center - b/c ₁ complex ubiquinol: cytochrome c ₂ oxidoreductase - cyt b _H cytochrome b-561 or higher potential cytochrome b - cyt b _L cytochrome b-566, or low potential cytochrome b - cyt c ₁, cyt c ₂, cyt c _t cytochromes c ₁ and c ₂, and total cytochrome c (cyt c ₁ and cyt c ₂) - Fe.S Rieske-type iron sulfur center, Q - QH₂ ubiquinone, ubiquinol - Q_z, Q_zH₂, Q_z ^– ubiquinone, ubiquinol, and semiquinone anion of ubiquinone, bound at quinol oxidizing site - Q_z-site ubiquinol oxidizing site (also called Q_o-(outside) - Q_o (Oxidizing) - Q_P (Positive proton potential) site) - Q_c-site uubiquinone reductase site (also called the Q_i-(inside) - Q_R (Reducing), or - Q_N (Negative proton potential) site) - UHDBT 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazol     

In vivo photosystem I reduction in thermophilic and mesophilic cyanobacteria: the thermal resistance of the process is limited by factors other than the unfolding of the partners

Photosystem I reduction by plastocyanin and cytochrome c(6) in cyanobacteria has been extensively studied in vitro, but much less information is provided on this process inside the cell. Here, we report an analysis of the electron transfer from both plastocyanin and cytochrome c(6) to photosystem I in intact cells of several cyanobacterial species, including a comparative study of the temperature effect in mesophilic and thermophilic organisms. Our data show that cytochrome c(6) reduces photosystem I by following a reaction mechanism involving complex formation, whereas the copper-protein follows a simpler collisional mechanism. These results contrast with previous kinetic studies in vitro. The effect of temperature on photosystem I reduction leads us to conclude that the thermal resistance of this process is determined by factors other than the proper stability of the protein partners.     

Structural and functional studies on the tetraheme cytochrome subunit and its electron donor proteins: the possible docking mechanisms during the electron transfer reaction

The photosynthetic reaction centers (RCs) classified as the group II possess a peripheral cytochrome (Cyt) subunit, which serves as the electron mediator to the special-pair. In the cycle of the photosynthetic electron transfer reactions, the Cyt subunit accepts electrons from soluble electron carrier proteins, and re-reduces the photo-oxidized special-pair of the bacteriochlorophyll. Physiologically, high-potential cytochromes such as the cytochrome c₂ and the high-potential iron–sulfur protein (HiPIP) function as the electron donors to the Cyt subunit. Most of the Cyt subunits possess four heme c groups, and it was unclear which heme group first accepts the electron from the electron donor. The most distal heme to the special-pair, the heme-1, has a lower redox potential than the electron donors, which makes it difficult to understand the electron transfer mechanism mediated by the Cyt subunit. Extensive mutagenesis combined with kinetic studies has made a great contribution to our understanding of the molecular interaction mechanisms, and has demonstrated the importance of the region close to the heme-1 in the electron transfer. Moreover, crystallographic studies have elucidated two high-resolution three-dimensional structures for the RCs containing the Cyt subunit, the Blastochloris viridis and Thermochromatium tepidum RCs, as well as the structures of their electron donors. An examination of the structural data also suggested that the binding sites for both the cytochrome c₂ and the HiPIP are located adjacent to the solvent-accessible edge of the heme-1. In addition, it is also indicated by the structural and biochemical data that the cytochrome c₂ and the HiPIP dock with the Cyt subunit by different mechanisms although the two electron donors utilize the same region for the interactions; cytochrome c₂ is recognized through electrostatic interactions while hydrophobic interactions are important in the HiPIP docking.     

Antigenic diversity of cytochromes c3 from the anaerobic,sulfate-reducing bacteria,Desulfovibrio

An indirect enzyme-linked immunoadsorption assay (ELISA) was developed for cytochrome c₃ using antisera to the cytochromes fromDesulfovibrio africanus Benghazi, Desulfovibrio vulgaris Hildenborough andDesulfovibrio salexigens British Guiana. The ELISA system was used to test for cross-reactions between these antisera and the heterologous antigens. In contrast to previous experiments using the Ouchterlony technique, all of the cytochromes c₃ tested exhibited some degree of cross-reaction. Considerable variation was seen in cross-reactions for cytochromes c₃ from differing strains ofD. desulfuricans. This observation raises questions about the taxonomic relatedness of these strains. No cross-reaction was seen with eukaryotic cytochrome c or withD. vulgaris cytochrome c₅₅₃. The data demonstrate that cytochrome c₃ is capable of undergoing nonprecipitating cross-reactions, and thus may not be as immunologically unique as was once thought.Abbreviations ELISA Enzyme-linked immunoadsorption assay     

Strong homology between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase of two species of Acetabularia and the occurrence of unusual codon usage

Summary The complete nucleotide sequence of the genes encoding the Rieske FeS, the cytochrome b and the cytochrome c ₁ subunits of the ubiquinol-cytochrome c ₂ oxidoreductase from the photosynthetic purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequences are presented. These three genes, fbcF, fbcB and fbcC, are located at contiguous sites of the genome. The DNA-deduced amino acid sequences are compared with known primary structures of corresponding proteins from other purple photosynthetic bacteria, as well as mitochondria, cyanobacteria and chloroplasts.Abbreviations BSA bovine serum albumin - Rb Rhodobacter - Rps Rhodopseudomonas