Photosynthetic cytochrome c550

01/11/2011 1 Review Photosynthetic cytochrome c550 Mercedes Roncela*, Diana Kirilovskyb, Fernando Guerreroa,c, Aurelio Serranoa and José M. Ortegaa aInstituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Américo Vespucio 49, 41092-Sevilla, Spain. bCommissariat à l’Énergie Atomique, Institut de Biologie et Téchnologies de Saclay (iBiTec-S), 91191 Gif-sur-Yvette, France, and Centre National de la Récherche Scientifique, URA 2096, 91191 Gif-sur-Yvette, France. cPresent adress: Bioenergy Group, Molecular Plant Biology Unit, Department of Biochemistry and Food Chemistry, University of Turku, Tykistökatu 6B, Biocity 4th Floor, 20520, Turku, Finland *Author for correspondence: Mercedes Roncel Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Américo Vespucio 49, 41092-Sevilla, Spain Tel: 34 954 489525 Fax: 34 954 460065 E-mail: [email protected] Keywords: Cytochrome c550; Cyanobacteria; Diatoms; Photosystem II; psbV gene product; Red algae *Manuscript Click here to view linked References Biochim Biophys Acta-Bioenergetics. 2012 Aug;1817(8):1152-63. doi: 10.1016/ j.bbabio.2012.01.008 01/11/2011 2 Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2. Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3. Physical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.1. UV-visible spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.2. Electron paramagnetic resonance spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.3. Resonance Raman spectra . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 11 3.4. Redox properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 4. Protein structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 4.1. Amino Acids Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 4.2. Structural properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 5. Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Acknowledgements . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Abbreviations: cyt, cytochrome; CP43, 43 kDa chlorophyll-binding protein in PSII; DAD, 2,3,5,6-tetramethyl-1,4-phenylenediamine or diaminodurol; D1/D2, homologous PSII reaction center core proteins; Eh, equilibrium redox potential; Em, midpoint redox potential; EPR, electron paramagnetic resonance spectroscopy; OEC, oxygen evolving complex; P680, primary electron donor in PSII; PSI, photosystem I; PSII, photosystem II; QA/QB, redox-active quinones bound to PSII; RR, resonance raman; TMH, transmembrane alpha helix 01/11/2011 3 1. Introduction Cytochromes are hemoproteins with redox activity that function in a variety of electron transport processes ranging from aerobic respiratory chains to photosynthesis and reductive inorganic nutrient assimilation. Depending on the nature of their heme group, several classes of cytochromes can be distinguished [1]. C-type cytochromes, a widespread class of cytochromes, can be defined as having one or several hemes c, bound to the protein moiety by two (or rarely one) thioether bonds generated by reaction of thiol groups of cysteine residues with vinyl groups of the heme [2]. These cysteine residues almost always are found in the consensus heme c binding sequence CXXCH where X denotes any residue and H is one of the His axial ligand to heme iron. Almost any residues (except cysteine) may be found in the XX positions; very rarely there are three or four residues between the two cysteine residues [3]. In c-type cytochromes, the fifth ligand of the heme iron atom is always a histidine. A nitrogen atom of a histidine residue or a sulfur atom of a methionine residue fills the sixth ligand position of the heme iron atom [2]. Cytochrome c550 (cyt c550), encoded by the psbV gene, is a cytochrome with a molecular mass of ~ 15 kDa bearing a covalently bound heme-group protein [4, 5, 6]. Cyt c550 presents a bis-histidine heme coordination which is very unusual for monoheme c-type cytochromes. The only other known structure of bis-histidinyl coordination in a monoheme cyt c fold is found in a domain of the nitrite reductase complex cyt cd1, from Thiosphaera panthotropha [7]. Therefore, cyt c550 is the first structural example of a monodomain, monoheme soluble cyt c with bis-histidinyl axial coordination [8]. Although it was initially isolated as a soluble protein, currently it is clearly established that it is an extra-membrane component of the PSII complex in cyanobacteria and some eukaryotic algae, as red and brown algae [9, 10, 11]. In PSII, the cyt c550 with the other extrinsic proteins stabilizes the binding of Cl- and Ca+ to the oxygen evolving complex and 01/11/2011 4 protects the Mn4Ca cluster from attack by bulk reductants [5, 6, 11, 12]. The role (if there is one) of the heme of the cyt c550 is unknown. The low midpoint redox potential (Em) of the purified soluble form (from -250 to -314 mV) [13, 14, 15, 16] 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 [16, 17]. A recent work has showed an Em value of +200 mV for cyt c550 [18]. These data raised the possibility of a redox function for this protein in electron transfer in PSII [18]. Despite the long distance (22 Å) between cyt c550 and the nearest redox cofactor (Mn4Ca cluster) [19], an electron transfer reaction between these components is possible [18]. This reaction, even with a very slow rate (ms-s time scale) relative to charge separation events, could be potentially significant relative to the lifetime of reversible charge accumulation states in the enzyme (tens of seconds to minutes) [20]. Some kind of protective cycle involving a soluble redox component in the lumen has also been proposed [18]. 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. For earlier reviews on cyt c550 see articles by Krogmann [21, 22]. Several reviews have been published on photosynthetic soluble cytochromes including cyt c550 [5, 23]. Recent articles on the three-dimensional (3-D) structure of PSII contain also information on the structure of PSII-bound cyt c550 [19, 24, 25, 26, 27]. A number of reviews have been written about extrinsic proteins of PSII and contain information on cyt c550 [28, 29, 30]. 2. Occurrence Cyt c550 was initially found as a soluble protein in the cyanobacterium Anacystis (A.) nidulans [13] and later in Microcystis (M.) aeruginosa, Aphanizomenon (A.) flos-aquae [14] 01/11/2011 5 and Synechocystis sp. PCC 6803 [15]. In the eighties, a membrane bound cytochrome with similar size, spectrum and redox properties was detected in detergent treated photosynthetic membranes from the cyanobacteria Anabaena variabilis and Phormidium laminosum [9, 31]. Later, a similar membrane-associated cytochrome in addition to the soluble form was obtained washing the thylakoids of A. nidulans with low ionic strength or with 1 M NaCl. However, these forms were distinct in their binding in ion-exchange chromatography and in EPR spectra [10, 32]. In the nineties, a PSII core complex purified from the thermophilic cyanobacterium Synechococcus vulcanus was described. This complex contained a stoichiometric amount of cyt c550 as one of the extrinsic components of cyanobacterial PSII [4, 33]. Based on its close interaction with the extrinsic 33- and 12-kDa proteins (PsbO and PsbU), it was concluded that cyt c550 is associated with PSII at the lumenal surface of the thylakoid membrane [11]. Nowadays, it is clearly established that cyt c550 is a component of the PSII complexes in cyanobacteria, which can be extracted by detergent or high salt treatments [9, 10, 33]. The three-dimensional structure of the PSII isolated from two different thermophilic cyanobacteria strains has confirmed that cyt c550 is stoichiometrically bound to the luminal PSII surface in the vicinity of the D1 and CP43 proteins and close to the oxygenevolving complex [19, 24, 25, 26, 27, 34]. Cyt c550 has also been identified in the red algae Cyanidium caldarium, Porphyra purpurea, Porphyridium cruentum and Cyanidioschyzon merolae [14, 28, 35, 36, 37] and in the diatoms Navícula pelliculosa [38], Phaeodactylum tricornutum [39] and Odontella sinensis [40]. The presence of this protein in several eukaryotic algae has also been confirmed by detection of the psbV1 gene in these organisms [35, 37, 40, 41, 42, 43, 44]. There is only one article in which cyt c550 has been detected in a green alga (Bryopsis maxima) by a spectrum assigned to this cytochrome [45]. No further confirmation has been 01/11/2011 6 made by other authors and no cyt c550 protein sequence or psbV gene has been found for any green algae in any of the databases searched. Sequence alignments and psbV gene distribution of cyt c550 among 39 different cyanobacterial genomes has been recenltly reported [23]. In the present review, we did a more extensive study on the distribution of this protein in different photosynthetic organisms using public databases and the Genbank database [htpp://www.ncbi.nlm.nih.gov]. About 90 organisms contain the psbV gene. Most of these organisms are cyanobacteria strains (57), but psbV is also present in different phylogenetic groups of photosynthetic eukaryotes (32) as Rhodophyta (or red algae) (10), Stramenopiles (or heterokonts) (17) and photosynthetic alveolates (9). It also appears in Glaucocystophyta (1), Haptophyta (2), Cryptophyta (2) and Rhizaria (1). A group of selected representatives are shown in Table 1. Evolutionary relationships between the different photosynthetic organisms containing this protein were studied by using the alignment of all sequences available in database. A phylogenetic tree [46] that envisages these evolutionary relationships is presented in Figure 1. Cyanobacterial cyt c550 sequences constitute a well-defined cluster, arranged separately from the assembly of eukaryotic clusters which includes sequences of Glaucophyta, Rhodophyta and diverse photosynthetic protists bearing complex plastids of red algal ancestry (Dinophyta, Cryptophyta, Haptophyta, and Stramenopiles). These data are in agreement with the evolutionary history of these groups of photosynthetic eukaryotes, since Cryptophyta, Haptophyta, Stramenopiles and most photosynthetic alveolates (Chromerida and Dinophyta) are considered as evolutionary descendants of red algae. It is interesting to note that cell ultra-structural and molecular phylogenetic data strongly suggest that they originated by secondary endosymbiosis from rhodophycean ascestors engulfed by different fagotrophic eukaryotes [47]. In accordance with this, cyt c550 is also present in Glaucocystophyta, an early branching protist group of the Viridiplantae assembly, thus 01/11/2011 7 suggesting an ancestral character of this redox protein. It was then probably lost during the subsequent evolution of the green photosynthetic lineage, after divergence of the green and red algae. In accordance with this, cyt c550 is also present in the Rhizarian euglyphid (tecated amoeba) Paulinella chromatophora, a phototrophic protist bearing a cyanobacterium-like organelle named chromatophore. This protist was recently reported as the only representative known so far of an incipient lineage of photosynthetic eukaryotes that underwent a primary endosymbiosis different from that of the Viridiplantae lineage (Chromatophore genome sequence of Paulinella sheds light on acquisition of photosynthesis by eukaryotes) [48]. Some cyanobacteria contain another gene called psbV2 that gives a protein product with 44% identity to cyt c550. This homolog was found in both unicellular and filamentous cyanobacteria [23]. In Thermosynechococcus (T.) elongatus this gene is located between psbV and the gene petJ encoding cyt c6. PsbV2 shows homology with both proteins, being similar to PsbV in the heme binding region and to petJ in the C-terminal region. These data together with the position of the psbV2 gene on the chromosome suggest that this protein was generated during evolution by gene duplication and posterior intergenic recombination [49]. It seems that psbV2 is expressed in T. elongatus but in an amount corresponding to 1% as compared to cyt c550 [50]. PsbV2 lacks a second histidine residue for axial ligation to the heme; however, its EPR spectrum is typical for a FeIII in a low-spin state. EPR and Raman spectra suggested that the sixth ligand could be a tyrosine [50]. Based on a structural model of PsbV2, Tyr 86 appeared to be the most likely sixth axial ligand to the heme [50]. The Em of this cytochrome-like protein must be very low since dithionite was not able to reduce it. Its function is unknown, but it could participate in the stabilization of the OEC. In fact, PSII isolated from a T. elongatus mutant lacking this protein presented a slightig lower oxygen evolving activity than that isolated from the WT [D Kirilovsky, unpublished data]. 01/11/2011 8 3. Physical properties 3.1. UV-visible spectra The UV-visible electronic absorption spectrum of the oxidized soluble cyt c550 presents a broad absorption band with maximum at 524.5 nm, a Soret band peaking at 405.7 nm and other maxima at 350 and 275 nm. Upon reduction with dithionite, typical absorption peaks appear at 550 (-band), 522 (-band) and 417 nm ( or Soret band) [15, 17, 50, 51]. Most optical measurements of cyt c550 focus on the  component of the Q band at 550 nm; this transition has a moderate extinction coeficient, and the interference from the absorbance of other chromophores is relatively small in this region compared to the Soret region [36, 52, 53, 54]. Usually, the changes in the redox state of the soluble cyt c550 are measured from the reduced-minus-oxidized spectrum as the difference between the maximum absorbance at 550 nm and the absorbance at one of the isosbestic points at 530 or 560 nm. The PSII-bound cyt c550 spectrum has also been measured as a reduced-minus-oxidized difference spectrum to remove the overwhelming contribution from the excess of strongly absorbing chlorophylls [16, 18]. Unfortunately, in PSII preparations there is another cytochrome associated with PSII, the cyt b559 (max = 559 nm) which has an absorbance band that overlaps with the 550 nm cyt c550 signal. To avoid this interference, spectra in the /- bands region of both cytochromes have been recorded at different solution redox potentials. Taking into account that the main redox potential forms of cyt b559 in T. elongatus have Em values significantly higher than cyt c550, the spectrum of cyt c550 can be easily distinguished by selective chemical oxidation/reduction and substrating of spectra of cyt b559 [16, 18]. Figure 2 shows a difference absorption spectrum of cyt c550 obtained by subtracting absolute spectra at two different solution redox potentials. Spectrum 1 (–300 mV minus +458 mV) clearly shows that PSII core complexes contain two different cytochrome components with absorption maxima in the -band at 559 and 550 nm. The component with an absorption 01/11/2011 9 maximum in the -band at 559 nm was assigned to cyt b559 [16]. The component with an absorption maximum in the -band at 550 nm that appeared between +55 mV and –300 mV (spectrum 2) can be assigned to cyt c550. Thus, the spectrum of the PSII-bound cyt c550 can be well recorded if cyt b559 is selectively reduced, which makes its absorption contribution in the -band at 550 of the cyt c550 insignificant (Figure 2). 3.2. Electron paramagnetic resonance spectra EPR spectroscopy has been used to observe the oxidized Fe (III) form of soluble and PSII-bound cyt c550, since it is a technique sensitive to changes in the coordination environment of metal ions with unpaired electrons. Typical EPR spectra for both soluble and PSII-bound forms from different cyanobacteria have been published by different authors [10, 15, 16, 17, 50, 52] and in both forms, the g values obtained are typical for a c-type low spin heme [17]. The g-values found (gz 2.94, gy 2.24 and gx 1.49) in T. elongatus for the soluble cyt c550 [17, 16, 50] were quite similar to those found in A. nidulans (gz 2.98, gy 2.24 and gx 1.46) [10, 50] and Arthrospira (A.) maxima (gz 2.90, gy 2.27 and gx 1.54) [55, 50], but slightly differ from those measured in the isolated cyt c550 from Synechocystis sp. PCC 6803 (gz 2.87, gy 2.28 and gx 1.57) [8, 15, 16, 17]. The differences in the g values are related to subtle differences in the geometry and the environment of the heme in the different cytochromes [50]. EPR spectral properties of PSII-bound cyt c550 have also been also studied. Vrettos et al. [17] used isolated PSII from Synechocystis oxidized with ferrycianide. In this type of preparations, EPR spectra of PSII display overlapping EPR signals from cyt b559 and cyt c550 since the g tensors of these cytochromes are very similar. To avoid this overlap in the EPR signals, the difference in reduction potentials between these cytochromes was exploited. 01/11/2011 16 41 and 92 and two cysteines, Cys37 and Cys40 that appear to be conserved in all sequences. These residues correspond to the heme attachment site CXXCH and the sixth ligand of the iron. Of the 137 positions describing the total sequence, twenty (Thr9, Phe33, Gly44, Thr46, Lys47, Leu54, Leu59, Ala62, Pro64, Leu72, Pro79, Tyr82, Asp83, Gly84, Pro93, Arg105, Asp111, Trp130, Gly131, Gly132) are completely conserved residues giving an overall identity conservation of 15%. The most highly conserved amino acids are predominantly found in the region close to the heme pocket. 4.2. Structural properties Three crystallographic structures of the soluble form of the cyt c550 are available: isolated from Synechocystis (PDB: 1E29) [8], A. maxima (PDB: 1F1C) [55] and T. elongatus (PDB: 1MZ4) [50]. The three proteins show a similar overall folding, being predominantly alphahelical with a short two-stranded -sheet near the N-terminus, similar to other monoheme ctype cytochromes. A bis-histidine heme coordination previously proposed from EPR studies [10, 15], which is unusual for monoheme c-type cytochromes, has been confirmed. The 3-D structure of soluble cyt c550 from T. elongatus (Fig. 4A) shows the typical hydrophobic inner core of monoheme cyt c, with three helices (residues 22-42, 68-78, 108-126) forming a nest for the prosthetic group [8]. It also presents a fourth helical segment (55-61) in the N-terminal domain, similarly to what is found in cyt c5 and c6, further protecting the heme from solvent [8]. An anti-parallel -sheet (residues 8-12 and 17-21) connected by a -hairpin turn (residues 13-16) near the N-terminus was also observed. This is an unusual feature in a monoheme ctype cytochrome that is almost exclusively composed of -structures [2]. All sequences analyzed present three conserved residues of cis-proline (Pro64, Pro79 and Pro93) which correspond to turns on the structure of the molecule at the end of one alpha helix [55]. Proline residue at position 93 orients its carbonyl oxygen to form a hydrogen bond with 01/11/2011 17 the Natom of the axial His92 heme ligand [51]. All soluble cyt c550 structures have similar comparable Fe-axial ligand distances. The polypeptide chain largely envelops the heme prosthetic group with the exception of the propionate D oxygen atoms, showing comparable amounts of the buried heme [55]. Comparison of the three structures also shows that the porphyrin rings and the propionate A presents a similar position in the three species while the propionate D is found in different orientations, the difference being up to approximately 45°. Structural alignment is usually applied for the structural comparison of proteins with low sequence similarity, where evolutionary relationships between them cannot be easily detected by standard sequence alignment techniques. The minimal root mean square deviation (rmsd) of two aligned structures is the measure of the average distance between the atoms (usually the backbone atoms) of superimposed proteins, indicating their divergence from one another. Comparison of the three available structures of soluble cyt c550 shows that T. elongatus cyt c550 superimposes on Synechocystis cyt c550 with an rmsd of 1.0 Å (126 C atoms) and on A. maxima cyt c550 with an rmsd of 0.79 Å (128 C atoms) [50]. Cyt c550 from the thermophilic cyanobacteria T. elongatus contains more intramolecular hydrogen bonds than its mesophilic counterparts: 102 compared to 81 and 82 in A. maxima and Synechocystis cyt c550, respectively [50]. This observation agrees with the proposal that increased numbers of hydrogen bonds and ion pairs, additional helices, and shorter loops may play a role in the structural basis of thermostability [50, 73]. No other thermostability-enhancing structural differences could be detected by comparing the three structures. To date, two crystallographic structures of PSII-bound cyt c550 are available. These have been obtained from T. elongatus (PDB: 1W5C; 2AXT; 3BZ2) [19, 25, 74] and T. vulcanus (PDB: 3ARC) [27]. The resolution of the 3-D structure of these two PSII-bound forms of cyt c550 has shown that the N-terminal helix seems to be important for the interactions with other PSII subunits [26, 34]. Figure 4B shows the structure of PSII-bound cyt c550 from T. 01/11/2011 18 elongatus. It is noteworthy that two conserved residues, Thr46 and Lys47, are shielding the pyrrole A, D, and C rings of the heme [55] and seems to participate in the electrostatic interaction of the cytochrome with other components of the PSII. Lys47 is in the proximity of PsbC-Glu413 (Fig. 4B). The side chains of the two highly conserved hydrophobic amino acids, Leu54 and Leu59, which are located in a short alpha-helical region in the proximity of the propionate and facing the heme, probably contribute to keep the hydrophobicity in the heme pocket [55]. Another hydrophobic conserved amino acid is Ala62, which is found in the periphery of the cytochrome, next to Tyr82 and psbU-Arg39, hence suggesting an inter- and intra-molecular structural role [8, 55]. Gly44 and Gly84 are located in different turns of loops in the cyt c550 tertiary structure. Gly44 is at the end of the first alpha-helix, which is part of the nest for the localization of the heme group, in close proximity to the conserved Leu59. On the other hand, Gly84 is found at the turn of the loop formed by the polar residues Tyr82 and Asp83, which can be crucial in the contact with PsbU [8, 55]. Gly131 and Gly132 are highly conserved probably due to the importance of the flexibility of the C-terminus for the recognition and binding to PSII. Likewise, Trp130 may be important in keeping the structure of this C-terminus anchoring region. Between conserved residues, the positively charged Arg105 is in the “upper” part, probably contributing to the cyt c550 binding to the PSII by interacting with PsbC-Glu104 [55]. The N-terminus of PsbV points towards the loop region between TMH-e and the membrane-attached α-helix eC of D1 as well as to the α-chain of cyt b559 [75]. A twostranded β-sheet (β1 and β2) is found near the N-terminus followed by α-helix (α1) in the neighbourhood of CP43 [75]. Close to the Fe2+ coordinating residue PsbV-His41, located on a short loop segment between α1 and α2 (Fig. 4B), an H-bond is formed from PsbV-Asn49 to CP43-Arg320 [75]. The α-helix (α3) is located in the N-terminal of the second antiparallel βsheet (β3 and β4). The β3 and the short loop linking β3 and β4 is in contact with the N- 01/11/2011 19 terminal part of PsbU [75]. The loop between β4 and α4 is close to ef (3) in the extended loop of CP43. PsbV-His92, coordinating the Fe2+ is located downstream of two short α-helices (α4 and α5). The C terminus of PsbV is in close vicinity (~8 Å) to the currently modelled C- terminus of PsbU [75]. The heme group of PsbV is well defined in the electron density, tilted by 62° to the membrane plane (angle between the heterocycle plane normal and the pseudo-C2 (Fe2+) axis) and shielded from solvent by hydrophobic residues of PsbV and the extended lumenal domain of CP43 (Fig. 4B). Fe2+ is coordinated by PsbV-His41Nε and PsbV-His92Nε that is H-bonded with NδH to PsbV-Pro93 (2.7 Å) [75]. The hydrophobic environment of the heme and the proximity of charged residues from other PSII components (PsbC-Lys323) could modulate the heme redox properties [75]. The redox potential difference between bound and isolated PsbV (-240 mV and +200 mV, respectively at pH 6.0) [18] could be caused by the additional shielding of the heme group by CP43 in the bound state. In the structure of soluble cyt c550 the C-terminal region is highly disordered, whereas in the PSII-bound cyt c550 structure, it is stabilised upon binding to the complex and points towards the Mn4Ca cluster. While the edge- to-edge distance from the heme to the manganese cluster (22 Å) may be too high to consider a direct electron transfer reaction, the residue PsbV-Lys134 is at a distance of 10.2 Å from the Mn1 of the Mn4Ca cluster. This lysine 134, together with the two C-terminal tyrosine residues (PsbV-Tyr136 and PsbV-Tyr137) could have functional importance in possible electron transfer reactions involving the cyt c550 and the S states of the OEC [18]. It is not clear how cyt c550 binds to PSII, or if significant structural changes occur during the binding process. By analogy with other cytochromes that bind photosynthetic membrane complexes, like cyt c6 (in association with the PSI) and cyt f (in association with the cyt b6f) [76, 77] it is possible to speculate that the binding of the cyt c550 to PSII could be due to electrostatic and hydrophobic interactions. The distribution of charges on the surface of the 01/11/2011 20 cyt c550 supports this hypothesis [78]. Furthermore, the last residues in the carboxy-terminus of the cyt c550 (-GGKVYY) are not resolved in the soluble structure, while they are visible in the crystals when cyt c550 is bound to PSII. This indicates that this region is much more flexible when the cytochrome is in its soluble form [79]. Furthermore, the B-factors of the atoms in the C-terminus together with the section from His41 to Ser51 (-HVGGITKTNPS-) are the lowest of the cyt c550 structure. These regions are located between the heme and the contact with the PSII (PsbU, CP43 and D1). The degree of conservation of several of these residues and the low flexibility shown in the crystal structures (indicated by low B-factors and the lack of resolution in the soluble form) may indicate that the regions mentioned are important in the recognition and attachment between cyt c550 and the PSII [78]. The resolution of the 3-D structure of soluble and PSII-bound cyt c550 forms has confirmed that these are similar with different conformations found for only few amino acid side chains located at the interface formed by cyt c550 with D1, CP43, and PsbU and the six C-terminal amino acids. The soluble cyt c550 (PDB: 1MZ4) superimposes on PSII-bound form (PDB: 3BZ2) with an rmsd of 0.3 Å for 131 Cα atom pairs [75]. Comparison with other bacterial c-type cytochromes has been made using structural alignment between soluble cyt c550 and the structures of other class I cytochromes. All of these cytochromes are very distantly related: cyt c550 superimposes on yeast cyt c [80] with an rmsd of 3.4 Å, on Rhodobacter sphaeroides cyt c2 [81] with an rmsd of 4.2 Å (over 281 backbone atoms), on Rhodopseudomonas viridis cyt c2 [82] with an rmsd of 3.5 Å (over 281 backbone atoms), and on Pseudomonas cyt c551 [83] with an rmsd of 3.5 Å (over 273 backbone atoms) [55]. Comparison with cyt c6 has also been reported [55]. Cyt c6 is a soluble c-type cytochrome which donates electrons to PSI in cyanobacteria and algae but not in higher plants. The examination of the sequences of these cytochromes recognized two regions of sequence 01/11/2011 21 similarity in the two proteins. The cyt c550 sequence from residue 28 to residue 45 is similar to the sequence of residue 1 to residue 18 in cyt c6. This includes an alignment of the hemebinding sites of cyt c550 of residues 37 and 40 with cyt c6 at residues 10 and 13. Another region of similarity is at the carboxy terminus of the two proteins. It has been suggested that the gene for one of the proteins provided segments for the assembly of a gene for the second cytochrome [22]. The superimposed structures of cyt c550 and cyt c6 from A. maxima (over 263 backbone atoms) showed an rmsd of 0.7 Å, despite only 32% identity between their primary structures [55]. This structural comparison between the helical cores of both A. maxima cyt c550 and cyt c6 shows great similarity. The longest helical segments closely superimpose on the four helices of cyt c6. The fold of the two proteins in the vicinity of the first axial ligand (His40 in cyt c550, His18 in cyt c6) is strikingly similar [55]. Cyt c550 has 22 additional residues in the N-terminal. In part they form a short, two-stranded  -sheet that precedes the first helix and the CXXCH heme coordination sequence (residues 37-41; Table 3). A second major difference between the two structures is the insert in the primary structure of cyt c550 that is not found in cyt c6 (between residues 89 and 103). It contains the sixth axial ligand, His92. The insert lacks secondary structure with the exception of the turn formed by residues 102-104. The protein backbone conformation in the region adjacent to the sixth axial ligand (His92 in cyt c550, Met61 in cyt c6) in the two structures is quite dissimilar. These results thereby reinforce the idea that cyt c550 and cyt c6 are actually close relatives [55]. 5. Function Cyt c550 was initially identified as a soluble protein with an Em of about -240 mV [13, 14] suggesting functions not related to PSII for this protein. Several non-photosynthetic roles have been proposed for cyt c550, mostly in anaerobic carbon and hydrogen metabolism [22, 84, 85] (summarized in Table 4). It was suggested that the cyt c550 was an endogenous cofactor of 01/11/2011 22 cyclic photophosphorylation [86]. Since cyt c550 was abundant in cells grown on high levels of nitrate and was absent from cells grown on ammonia it was proposed that it could participate in the reduction of nitrate to ammonia [14]. Krogmann and Smith [21] suggested that the function of cyt c550 could be related to anaerobic disposal of electrons from carbohydrate reserves or fermentation to sustain an organism during prolonged dark and anaerobic conditions. A very low rate of cyt c550 enzymatic reduction using NADPH and the spinach ferredoxin: NADP oxidoreductase (FNR) was observed under anaerobic conditions. A modest increase in the rate of this cytochrome was observed when ferredoxin II from cyanobacteria was added to the reaction mixture. The specificity for ferredoxin II in the reduction of cyt c550 and the often coincident appearance of ferredoxin II and cyt c550 in cells experiencing dark, anaerobic conditions suggested that these proteins may link carbohydrate breakdown to disposal of electrons in a fermentative pathway [21]. The low Em of this cytochrome was similar to that of cyt c3 of Desulfovibrio desulfuricans [2] and so it could have similar functions. A few unicellular cyanobacteria and diatoms experience dark, anaerobic conditions as a result of bloom formation. Under these conditions, they acquire an hibernation ability and the appearance of the low-potential cyt c550 facilitating perhaps one fermentation pathway would make sense [22]. In the nineties, it was clearly demonstrated that the cyt c550 is one of the extrinsic proteins of the cyanobacterial PSII [10, 33]. It was shown that cyt c550 was stoichiometrically bound to the PS II, activated oxygen evolving activity and allowed the binding of the 12 kDa protein, another extrinsic component of the cyanobacterial PSII involved in oxygen evolution [33, 4, 11]. Cyt c550 was thus suggested to play the same role as the other extrinsic proteins. By stabilising the neighbouring proteins and protecting the Mn cluster from external reductants, it stabilizes the oxygen-evolving complex [12, 5]. Studies on the phenotype of the cyt c550-less mutant (PsbV) and the cyt c550-less and 12 kDa protein-less double mutant 01/11/2011 23 (PsbV-PsbU) of Synechocystis showed that both the cyt c550 and the 12 kDa protein stabilize the binding of the Ca2+ and Clions which are essential for the oxygen-evolving activity of PSII, in a manner analogous to the extrinsic 17 and 24 kDa polypeptides of higher plants [6, 49, 87]. Mutations of the axial ligand His-92 of cyt c550 to methionine or cysteine changed the Em by +125 mV or -30 mV, respectively. Nevertheless, the activity of this mutated PSII in cells from T. elongatus was not modified, suggesting that under normal growth conditions the Em of cyt c550 is not important for PSII function [52]. Since then, all the studies about the role of the cyt c550 have been related to its function in the PSII. However, it is still possible that two different populations of cyt c550 are present in cyanobacterial cells: one bound to the PSII and the second one soluble in the cytoplasm or in the lumen. EPR spectra of the cyt c550 recorded using whole T. elongatus cells suggested the presence of a significant concentration of soluble cyt c550 in cells: the gx value observed in whole cells corresponded to the isolated cyt c550 with a shoulder that could be from the bound cyt c550 [52]. It was calculated that the soluble fraction could represent between 40 to 60% of the bound population. The role and cellular localization of the soluble cyt c550 is still unknown. However, the fact that the N-terminal amino-acid sequence of both cyt c550 populations was “AELTPE” indicates the loss of the 26 amino acids transit sequence also in the soluble fraction, and suggests a localization of this fraction in the lumen and not in the cytoplasm as suggested by its very low Em [52]. A summary of the different functions proposed for cyt c550 is shown in Table 4. Since the Em of a cytochrome is one of the key parameters for elucidating its function, the discovery that the Em of the PSII-bound cyt c550 is +200-215 mV opens the possibility that it plays a redox function [18]. Although the change of the Em of the cyt c550 by replacing one of the histidines did not change the PSII activity in T. elongatus cells, the sensitivity to high light intensities of the mutant cells increased (Kirilovsky unpublished data). This Em value of about 01/11/2011 24 +200 mV opens the possibility that cyt c550 plays a role as an electron donor to the Mn4Ca cluster in a photoprotective cycle involving a soluble redox component in the lumen [18]. The distance between the heme of the cyt c550 and the Mn4Ca cluster is long (22 Å) meaning that the electron transfer will be in the ms-sec scale. This rate is slow relative to charge separation reactions but it remains potentially significant relative to charge recombination reactions (tens of seconds to min) occurring in PSII. This proposition must be demonstrated experimentally: it must be showed that cyt c550 is indeed capable to give electrons to the S2 and S3 states of the Mn4Ca cluster and that it is reduced by a lumen component. Acknowledgements This work was supported by Grants: BFU2007-68107-C02-01 from the Spanish Ministry of Science and Innovation (MICINN) and PADI CVI-261 from the Andalusia Government. We would like to thank Dr. Anna Lindahl for critically reading the manuscript. References [1] A. Dolla, L. Blanchard, F. Guerlesquin, M. Bruschi, The protein moiety modulates the redox potential in cytochromes c, Biochimie 76 (1994) 471-479. [2] P.D. Barker, S.J. Ferguson, Still a puzzle: why is haem covalently attached in c-type cytochromes?, Struct. Fold Des.7 (1999) 281-290. [3] J.W.A. Allen, O. Daltrop, J.M. 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Guerrero, Citocromos asociados al Fotosistema II en la cianobacteria termófila Thermosynechococcus elongatus. Estudio de mutantes puntuales del citocromo b559 y análisis redox del citocromo c550, Ph.D. Thesis, 2011, Universidad de Sevilla, Sevilla, Spain. [79] L. Wang, A.B. Cowley, S. Terzyan, X. Zhang, D.R. Benson, Comparison of cytochromes b5 from insects and vertebrales, Proteins 67 (2007) 293-304. [80] G.V. Louie, G.D. Brayer, High-resolution refinement of yeast iso-1-cytochrome c and comparisons with other eukaryotic cytochromes c, J. Mol. Biol. 214 (1990) 527-555. [81] H.L. Axelrod, G. Feher, J. P. Allen, A.J. Chirino, M.W. Day, B.T. Hsu, D.C. Rees Crystallization and X-ray structure determination of cytochrome c2 from Rhodobacter sphaeroides in three crystal forms. Acta Crystallogr. D Biol. Crystallogr. 50 (1994) 596- 602. [82] S. Sogabe and K. Miki, Refined crystal structure of ferrocytochrome c2 from Rhodopseudomonas viridis at 1.6 A resolution, J. Mol. 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PCC 7002, Plant Physiol. 105 (1994) 1313-1319. 01/11/2011 35 Figure legends Figure1. Phylogenetic tree of the evolutionary relationships between cytochromes c550 of cyanobacteria and photosynthetic eukaryotes. Phylogenetic analyses were conducted in MEGA4 program using the Minimum Evolution method [46]. The bootstrap consensus tree was inferred from 1000 replicates. The evolutionary distances are in the units of number of amino acid substitutions per site. Accession numbers of annotated and non-annotated sequences identified in databases are shown after the corresponding species names. Oval: the phototrophic amoeba Paulinella chormatophora. Boxed: two sequences of dinotoms (Dinophyta having endosymbiotic diatoms). Figure 2. Difference absorption spectra of the cytochromes bound to the photosystem II reaction center in T. elongatus. Redox difference absorption spectra of cytochromes b559 and c550 in PSII core complexes from T. elongatus at pH 6.5 were obtained during the course of a redox titration by subtracting absolute spectra recorded at –300 mV minus +458 mV (spectrum 1) and at –300 mV minus +55 mV (spectrum 2). Redox titration procedure and sample composition are described [16, 18]. Solid and dotted vertical lines indicate 559 and 550 nm wavelengths, respectively. Figure 3. Reductive potentiometric titrations of cytochrome c550 in its purified state and bound to PSII Plots of the percentages of reduced cyt c550 obtained from the absorbance differences at 550-538 nm versus ambient redox potentials in its purified state (A) and bond to PSII in the presence (B) and in the absence of redox mediators (C). Redox titrations were carried out at 01/11/2011 36 pH 6.0 as described in [16, 18]. The solid curves represent the best fits of the experimental data to the Nernst equation in accordance with one-electron processes (n=1) for one component. Figure 4. Partial representation of the three dimensional structure of cytochrome c550 in its soluble form (A) and in its native state bound to PSII (B). Fifteen of the completely conserved residues of cyt c550 are shown (Thr9, Lys47, Leu54, Leu59, Ala62, Pro64, Pro79, Tyr82, Asp83, Gly84, Pro93, Arg105, Trp130, Gly131, Gly132). N and C indicate the amino- and carboxy-terminus of the cyt c550. The heme and the coordinating histidines are shown as sticks. All other subunits are shown as “cartoon” in different colours. OEC is shown as spheres. Figures have been made using PyMol and the PDB files 3BZ2 and 1MZ4. Table 1. List of selected organisms containing psbV gen from GenBank Database (http://www.ncbi.nlm.nih.gov) (27th October 2011) CYANOBACTERIA EUKARYOTA Section I Thermosynechococcus elongatus BP-1 Synechococcus elongatus PCC 6301 Microcystis aeruginosa NIES-843 Cyanothece sp. PCC 7425 Synechocystis sp. PCC 6803 Acaryochloris marina MBIC11017 Aphanothece halophytica Prochlorococcus marinus str. MIT 9303 Gloeobacter violaceus PCC 7421 Cyanobium PCC 7001 Aphanizomenon flos-aquae Cylindrospermopsis raciborskii CS-505 Crocosphaera watsonii WH501 Nodularia spumigena CCY9414 Raphidiopsis brookii D9 Mycrocoleus vaginatus FGP-2 Section III Trichodesmium erythraeum IMS101 Arthrospira maximaCS-328 Lyngbya PCC 8106 Section IV Nostoc sp. PCC 7120 Nostoc punctiforme PCC 73102 Anabaena variabilis ATCC 29413 Oscillatoria PCC 6506 Glaucocystophyta Cyanophora paradoxa Rhodophyta or Red algae Gracilaria tenuistipitata var. Liui Cyanidioschyzon merolae 10D Cyanidium caldarium Porphyra yezoensis Porphyra purpurea Stramenopiles Diatoms Fistulifera sp. JPCC DA0580 Thalassiosira pseudonana Thalassiosira oceanica Phaeodactylum tricornutum Odontella sinensis Pelagophyceae Aureococcus anophagefferens Aureoumbra lagunensis Phaeophyceae Ectocarpus siliculosus Fucus vesiculosus Raphidophyceae Heterosigma akashiwo Xanthophyceae Vaucheria litorea Haptophyta Emiliania huxleyi Alveolata Chromerida Chromera velia Chromerida sp. RM11 Dinophyceae Durinskia baltica Kryptoperidinium foliaceum Karlodinium veneficum Cryptophyta Rhodomonas salina Guillardia theta Rhizaria Paulinella chromatophora Table 1 Table 2. Summary of midpoint redox potentials of cytochrome c550 Cyanobacteria Em Ref. Soluble form Spirulina máxima* -260 mV [54] Synechococcus sp. PCC 6301** -280 mV [10] Synechocystis sp. PCC 6803 -250 mV [15] Thermosynechoccocus elongatus -240 mV [16] Synechocystis sp. PCC 6803 (adsorbed to an electrode) -108 mV [17] Bound form Thermosynechoccocus elongatus (with 14 redox mediators) -80 mV [16] Thermosynechoccocus elongatus (with 8 redox mediators) -20 mV [18] Thermosynechoccocus elongatus (with DAD) +215 mV [18] Thermosynechoccocus elongatus (without redox mediators) +200 mV [18] *Arthrospira maxima; ** Anacystis nidulans Table 2 Table 3. Cytochrome c550 Primary Structure Alignment 10 20 30 40 50 60 70 T. elongatus AELTPEVLTV PLNSEGKTIT LTEKQYLEGK RLFQYACASC HVGGITKTNP SLDLRTETLA LATPPRDNIE Synechococcus AELTAETRTV KLNPQGDNVT LSLKQVAEGK QLFAYACGQC HVGGITKTDP NVGLDPEALA LATPPRDSVE Nostoc LELDEATRTV PLNAQGDTVT LSLKQVKEGK RLFQYACAQC HVGGVTKTNQ NVGLEPEALA LATPNRNNIE A. variabilis LELDEATRTV PLNAQGDTVT LSLKQVKEGK RLFQYACAQC HVGGVTKTNQ NVGLEPEALA LATPNRNNIE P. marinus AQWDAETLTV PAGSGGQQVT FSESEIKSAS KLFKSNCATC HNQGVTKTNQ NVGLDLEALS LASPARDNVD A. marina AELDDATRTV ALNEG-STVT LSTQQAKEGQ RLFNFACANC HIGGDTKTNP SINLSSASLA GANPRRDNVE A. maxima LELTEELRTL PINAQGDTAV LSLKEIKKGQ QVFNAACAQC HALGVTKTNP DVNLSPEALA LATPPRDNIA M. aeruginosa LELNEKTLTI TLNDAGESVT LTSEQATEGQ KLFVANCTKC HLQGKTKTNN NVSLGLGDLA KAEPPRDNLL Synechocystis VELTESTRTI PLDEAGGTTT LTARQFTNGQ KIFVDTCTQC HLQGKTKTNN NVSLGLADLA GAEPRRDNVL C. paradoxa AALDEETRTV ALNST-ETVV LTPEQVKRGK RLFNSTCGIC HVGGITKTNP NVGLDSEALA LATPPRNNIE C. caldarium LELNEEARTV KLNSEGQSLT LNEEQIKKGK RLFNSHCGSC HVGGITKTNP NVGLDLESLN GANPPRNNIN Porphyra IELDEATRTV PLESSGRTVV LTPEQVKRGK RLFNNSCAIC HNGGITKTNP NVGLDPESLG LATPQRDNIE Gracilaria MELDEATRTV TLEESGKTIT LTPEQVKRGK RLFNNSCAQC HNGGITKTNP NIGLDPESLS GATPVRDNIR Fistulifera IELDEATRTV VADGSGKTIV LTPEQVKRGK RLFNATCGAC HVGGITKTNP NVGLDPEALS LATPRRDNIA P. tricornutum IDLDEATRTV VVDSSGKTIV LTPEQVKRGK RLFNATCGAC HVGGVTKTNP NVGLDPEALS LATPRRDNIA T. pseudonana IDLDEATRTV VTDSSGNTTV LTPEQVKRGK RLFNATCGAC HTGGITKTNP NVGLDPEALS LATPRRDNIS O. sinensis IDLDEATRTV VADSNGNTTV LTPEQVKRGK RLFNNTCGAC HVGGVTKTNP NVGLRPEGLS LATPRRDNAA F. vesiculosus IELDEATRTI PVTSDGKTTI LTPEQVKRGK RLFNSSCGQC HVGGVTKTNP NLGLDPEALS LATPARNNIN Chromera RSLTDSIRTV KLSENNDKAI ITPNELGRGK VLFAKTCSAC HTGGITKTNP NIGLALSTLK NAIPERDNVV Durinskia IDLDEATRTV VKDASGKTVV LTPEQVKRGK RLFNATCGAC HVGGITKTNP NVGLDPEALS LATPRRDNID Karlodinium LDLKENIRTV SFDTTDKIVV ITKTQIKRGK RLFTNACANC HVGGVTKPDP NIGLDMIALR FATPPKNNIV *: :. : .. :* * * * * **.: .:.* * * * ::. Table 3 80 90 100 110 120 130 T. elongatus GLVDYMKNPT TYDGEQEIAE VHPSLRSADI FPKMRNLTEK DLVAIAGHIL VEPKILGDKW GGGKVYY Synechococcus SLVDYLHNPT TYDGEREISE LHPSTKSTDI FPKMRNLTED DLVAISGHIL LQPKIVGTKW GGGKIYY Nostoc GLVDYMKNPT TYDGVEEISE IHPSLKSADI FTAMRNLTDK DLESIAGHIL LQPKILGDKW GGGKIYY A. variabilis GLVDYMKNPT TYDGVEEISE IHPSIKSADI FTAMRNLTDK DLESIAGHIL LQPKILGDKW GGGKIYY P. marinus GLVEFLKNPM SYDGEYSIAD THPGISSSDV YVQMRTLNDE DLRLIAGYIL TAEKVQGDQW GGGKIYF A. marina GLVDYMNNPT TYDGFDTISE VHPSTQSTDV FPLMRNLSDE DLFDIAGHIL IQPSVIGDQW GGGKANR A. maxima ALVDYVKNPT TYDGFIEIYE LHPSLKSADI FPKMRNLSED DLYNVAAYIL LQPKVRGEQW GGGKYLR M. aeruginosa ALIDYLEHPT SYDGEDDLSE LHPNVSRPDI FPELRNLTED DVYNVAAYML VAPRL-DERW GG-TIYF Synechocystis ALVEFLKNPK SYDGEDDYSE LHPNISRPDI YPEMRNYTED DIFDVAGYTL IAPKL-DERW GG-TIYF C. paradoxa SLVDYMKNPT SYDGSEEIYD IHPSIRSADA FPKMRNLTEE DLYDIAGHIL LSPKILPSQW GGGKIYY C. caldarium ALVEYMKDPK TYDGSESIAE IHPSIKSADI FPKMRDLSDD DLKVIAGHIL VQPKINSEKW GGGKIYY Porphyra ALVDYMKDPT SYDGAESIAE LHPSIKSAEI FPKMRNLTDE DLFTIAGHIL LQPKIVSEKW GGGKIYY Gracilaria NLIEYIKDPT SYDGATSIAE LHPSIKSAEI FPKMRNLTDE DLFAIAGHIL IQPKIAAEKW GGGKIYY Fistulifera SLVDYMKNPT TYDGLESIAE VHPSIKSADI YPRMRSVTEE DLTAMAGHIL LSPKVLSEKW GGGKIYY P. tricornutum GLVDFLKNPT TYDGLESIAE VHPSIKSADI YPRMRSVTDE DLTAMAGHIL LQPKIVTEKW GGGKIYY T. pseudonana ALVDYLKNPT TYDGLESIAE IHPSIKSADI YPRMRSLTDE DLYSIAGHIM LQPKIVAEKW GGGKIYF O. sinensis ALVDYLKNPT SYDGLESIAE IHPSIKSGDI YPRMRSLTDE DLFSIAGHIL LQPKIVTEKW GGGKIYY F. vesiculosus ALVDYMKNPT TYDGLESIAE IHPSIKSANI FTRMRSLDEK DLVDIAGHIL LQPKIVSEKW GGGKIYY Chromera NLVQYMKYPT AYDGTFTLNE THPNTTFAVF FPSMRTLTEK DLYSIAGYIL VQAQVLGEKW GGGKVYY Durinskia ALIDYLKNPT SYDGLDSIAE VHPSIKSADL YPRMRSVTDD DLYAMAGHIL LQPKIVTEKW GGGKIYY Karlodinium NLVAYFKDPI TYDGLYSISE LHPSIKGADL FPKMRFLTDE DLFSVAGYIL YQYNILGDRW GGGKVYY *: :.. * :*** : **. : :* :. *: ::.: : : :* ** . Complete names of the organisms included in the table are: T. elongatus, Thermosynechococcus elongatus (gi_58177112); Synechococcus, Synechococcus sp. PCC 6301 (BAD80275); Nostoc, Nostoc sp. PCC 7120 (BAB77783); A. variabilis, Anabaena variabilis (ABA22359); P. marinus, Prochlorococcus marinus (YP_001016548); Acaryochoris marina, A. marina (YP_001518187); A. maxima, Arthrospira maxima (ZP_03274665); M. aeruginosa, Microcystis aeruginosa (YP_001656196); Synechocystis, Synechocystis sp. PCC 6803 (BAA18512); C. paradoxa, Cyanophora paradoxa (NP_043260); C. caldarium, Cyanidium caldarium (NP_045156); Porphyra, Porphyra purpurea (AAC08085); Gracilaria, Gracilaria tenuistipitata var. liui (YP_063518); Fistulifera, Fistulifera sp. JPCC DA0580 (YP_004376577); P. tricornutum, Phaeodactylum tricornutum (YP_874401); T. pseudonana, Thalassiosira pseudonana (YP_87449); O. sinensis, Odontella sinensis (NP_043700); F. vesiculosus, Fucus vesiculosus (CAX12410); Chromera, Chromera velia (ADJ66524); Durinskia, Durinskia baltica (YP_003734993); Karlodinium, Karlodinium veneficum (AEJ72991). KEY (each species name is followed by its data base Accession Numbers in parentheses). Conserved Cys and His residues are in black bold and red bold, respectively. Identical, strongly and weakly similar residues were indicated by (*), (:) and (.), respectively. Table 4. Summary of functions proposed for cytochrome c550 Form Function Ref. Soluble form Anaerobic disposal of electron from carbohydrate reserves [21] Anaerobic fermentation [21, 22] Removing excess electrons in anaerobic grown cells [86] Hydrogenase reactions [54] Nitrate reduction [14] Endogenous cofactor for cyclic phosphorylation [11] Detoxifying reactive oxygen species [51] Bound form Enhacing O2 evolution by facilitating the binding of [11, 33] another extrinsic proteins (12 kD) Acting as a barrier to reductive attack on the Mn4Ca cluster [52] Electron acceptor to remove excess electron under [28] conditions where photosynthesis is not maximal Electron donor to the Mn4Ca cluster in a protective cycle [18] Table 4