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The photosynthetic cytochrome c550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre., This work was supported by the Spanish Ministry of Economy and Competitiveness (BIO2012-35271, BIO2015-64169-P, MAT2011-23861 and CTQ2015-64486-R) the Andalusian Government (PAIDI BIO-022) and the Aragón Government (Grupo consolidado B-18). All these grants were partially financed by the EU FEDER Program

Kinetic curves of reduction of the oxidized bacteriochlorophyll dimer by the proximal heme of cytochrome in seven mutated reaction centers from Rps. viridis was measured in the temperature range of 295-10 K under an ambient potential when two high-potential hemes were reduced. The data are analyzed in frames of a model which accounts for slow medium dynamics (the diffusion-reaction equation) and the quantum effect in the modes responsible for reorganization of the cofactors and the medium. The model reproduces the observed nonexponential kinetics down to the lowest temperature where the fast kinetic component still survives. The modeling results in determination of an average characteristic time for re-orientation of dipoles of the protein matrix as a function of temperature. At temperatures above the glass-transition temperature, this function shows Arrhenius behavior with the activation energy varying between the values of 0.5 eV for wild type Rps. viridis and 0 eV for Rps. sulfoviridis found previously. Possible reasons for disappearance of the fast kinetic component in some reaction centers under study at low temperatures are discussed.

The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) cytochrome b559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit cytochrome b559 like structure. The features of the new structure were determined by UV–Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane., This work was supported by Grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (AGL2011-23574, MAT2011-23861, BIO2012-35271, BFU2010-16297), the Aragón Regional Government (E33 and B18), and the Andalucía Regional Government (PADI BIO-022). All these Grants were partially financed by the EU FEDER Program.

Electron transfer (ET) in the reaction center from Rhodopseudomonas viridis has been studied experimentally by our group in the range of temperatures of 153–295 K. The kinetics of ET reaction from the proximal heme of cytochrome to the special pair was found to be non-exponential. The degree of non-exponentiality strongly depends on temperature, with increasing non-exponentiality at lower temperatures. Here, the experimental kinetic data for ET are analyzed in the frames of a theoretical Sumi–Marcus-type model, which allows establishing a connection between the observed kinetics and local structural dynamics of the protein in a close vicinity of the donor and acceptor sites. The phenomenological model subdivides the multi-time-scale dynamics of the protein into two groups: fast and slow. The division is determined by the rate of ET, which is dynamically controlled by the slow modes of the protein medium. The slow modes are described by a phenomenological collective coordinate , for which a diffusion type ...

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

The electron transfer from the heme of cytochrome c to the bacteriochlorophill dimer in reaction centers of photosynthetic purple bacteria Rps. sulfoviridis is studied by laser flash photolysis at 40–296 K in conditions where one, two, or three cytochrome hemes are chemically reduced. In the model used for the electron transfer kinetics, the protein relaxation is described with a temperature-independent oscillatory coordinate and a temperature-dependent diffusion coordinate, with the protein dielectric relaxation times widely distributed along the diffusion coordinate. It is found that all the protein complexes can be divided into proteins with fast (k et = 107 to 10–4 s–1) and slow (k et ≪ 100 s–1) electron transfer. These populations presumably differ by the protonation state of the functional group. The contribution of the oscillatory and diffusion coordinates alters, which severely affects k et. Parameters V ab, ΔG, λ, τ0, and β for these reactions are determined.

Kinetics of electron transfer from soluble cytochrome c2 to the tetraheme cytochrome c have been measured in isolated reaction centers and in membrane fragments of the photosynthetic purple bacterium Rhodopseudomonas viridis by time-resolved flash absorption spectroscopy. Absorbance changes kinetics in the region of cytochrome α-bands (540–560 nm) were measured at 21 °C under redox conditions where the two high-potential hemes (c-559 and c-556) of the tetraheme cytochrome were chemically reduced. After flash excitation, the heme c-559 donates an electron to the special pair of bacteriochlorophylls and is then re-reduced by heme c-556. The data show that oxidized heme c-556 is subsequently re-reduced by electron transfer from reduced cytochrome c2 present in the solution. The rate of this reaction has a non-linear dependence on the concentration of cytochrome c2, suggesting a (minimal) two-step mechanism involving the f ormation of a complex between cytochrome c2 and the reaction center, followed by intracomplex electron transfer. To explain the monophasic character of the reaction kinetics, we propose a collisional mechanism where the lifetime of the temporary complex is short compared to electron transfer. The limit of the halftime of the bimolecular process when extrapolated to high concentrations of cytochrome c2 is 60 ± 20 μs. There is a large ionic strength effect on the kinetics of electron transfer from cytochrome c2 to heme c-556. The pseudofirst-order rate constant decreases from 1.1 × 107 M-1 s-1 to 1.3 × 106 M-1 s-1 when the ionic strength is increased from 1 to 1000 mM. The maximum rate (1.1 × 107 M-1 s-1) was obtained at about 1 mM ionic strength. This dependence of the rate on ionic strength s uggests that attractive electrostatic interactions contribute to the binding of cytochrome c2 with the tetraheme cytochrome. On the basis of our data and of previous molecular modelling, it is proposed that cytochrome c2 docks close to the low-potential heme c-554 and reduces heme c-556 via c-554.

Isolated reaction centers of Rhodopseudomonas viridis with the two high-potential hemes reduced were illuminated at 5 K. Difference spectra show a bleaching of the heme c-556 alpha bands and a red shift of the Soret band. These effects are reversed by warming to around 80 K. They are not induced by near infra-red light absorbed by the chlorine pigments of the reaction centers and they are not associated with electron transfer from P to QA. It is concluded that, following direct excitation, heme c-556 becomes five-coordinated. We find no evidence of a significant photooxidation of heme c-559 under the same conditions.

Electron transfer from the proximal heme c -559 to the primary donor P has been studied in reaction centers of the photosynthetic bacterium Rhodopseudomonas viridis in which the tyrosine residue L162 was replaced by threonine. In the wild type, when the two high-potential hemes of the tetraheme cytochrome are reduced before flash excitation, a rapid electron transfer ( t 1/2 =190 ns) observed at ambient temperature disappears below 190 K. In the mutant, the reaction is partly maintained down to 8 K, leading to irreversible charge separation. The reaction rate is nearly temperature-independent between 294 K and 8 K ( t 1/2 ≈450 ns). The different behavior of wild type and mutant reaction centers is attributed to differences in a network of water molecules, the freezing of which may block structural reorganizations associated with cytochrome oxidation, in the wild type but not in the mutant.

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

Cytochrome c550 (cyt c550), which is one of the extrinsic proteins of photosystem II (PSII), is only present in cyanobacteria and red algae. Although this cytochrome has been reported to stabilize the binding of Ca2+ and Cl− ions, which are essential for activity of PSII, the specific function of heme is not yet clear. The reported negative values of the midpoint redox potential (E m) of cyt c550 (−300 mV in the soluble state and −80 mV when associated with PSII) appear to be incompatible with a redox function in PSII. It has been reported that the E m of QA in PSII-enriched membranes was affected by the presence of redox mediators at low ambient potentials. We have carried out new measurements of E m of cyt c550 associated to PSII changing the type and number of redox mediators used. We have determined that the E m of cyt c550 is about +200 mV in the absence of mediators or in the presence of a very limited number of mediators. Our results suggest that the highly reducing conditions reached in the presence of mediators, favor the reduction of a PSII component, most likely the Mn cluster, thereby inducing alterations in protein, the heme environment and consequently the E m of the heme. The new value of E m of cyt c550 opens the possibility of a redox function for this protein.

We performed spectroscopic and functional characterization on cyanobacterium Synechocystis PCC6803 with mutations of charged residues of the cytoplasmic side of cytochrome (Cyt) b 559 in photosystem II (PSII). All of the mutant cells grew photoautotrophically and assembled stable PSII. However, R7Eα, R17Eα and R17Lβ mutant cells grew significantly slower and were more susceptible to photoinhibition than wild-type cells. The adverse effects of the arginine mutations on the activity and the stability of PSII were in the following order (R17Lβ > R7Eα > R17Eα and R17Aα). All these arginine mutants exhibited normal period-four oscillation in oxygen yield. Thermoluminescence characteristics indicated a slight decrease in the stability of the S 3 Q B − /S 2 Q B − charge pairs in the R7Eα and R17Lβ mutant cells. R7Eα and R17Lβ PSII core complexes contained predominantly the low potential form of Cyt b 559 . EPR results indicated the displacement of one of the two axial ligands to the heme of Cyt b 559 in R7Eα and R17Lβ mutant reaction centers. Our results demonstrate that the electrostatic interactions between these arginine residues and the heme propionates of Cyt b 559 are important to the structure and redox properties of Cyt b 559 . In addition, the blue light-induced nonphotochemical quenching was significantly attenuated and its recovery was accelerated in the R7Lα and R17Lβ mutant cells. Furthermore, ultra performance liquid chromatography–mass spectrometry results showed that the PQ pool was more reduced in the R7Eα and R17Lβ mutant cells than wild-type cells in the dark. Our data support a functional role of Cyt b 559 in protection of PSII under photoinhibition conditions in vivo.

El pdf del artículo es la versión pre-print., A study of the in vitro reconstitution of sugar beet cytochrome b 559 of the photosystem II is described. Both α and β cytochrome subunits were first cloned and expressed in Escherichia coli. In vitro reconstitution of this cytochrome was carried out with partially purified recombinant subunits from inclusion bodies. Reconstitution with commercial heme of both (αα) and (ββ) homodimers and (αβ) heterodimer was possible, the latter being more efficient. The absorption spectra of these reconstituted samples were similar to that of the native heterodimer cytochrome b 559 form. As shown by electron paramagnetic resonance and potentiometry, most of the reconstituted cytochrome corresponded to a low spin form with a midpoint redox potential +36 mV, similar to that from the native purified cytochrome b 559. Furthermore, during the expression of sugar beet and Synechocystis sp. PCC 6803 cytochrome b 559 subunits, part of the protein subunits were incorporated into the host bacterial inner membrane, but only in the case of the β subunit from the cyanobacterium the formation of a cytochrome b 559-like structure with the bacterial endogenous heme was observed. The reason for that surprising result is unknown. This in vivo formed (ββ) homodimer cytochrome b 559-like structure showed similar absorption and electron paramagnetic resonance spectral properties as the native purified cytochrome b 559. A higher midpoint redox potential (+126 mV) was detected in the in vivo formed protein compared to the in vitro reconstituted form, most likely due to a more hydrophobic environment imposed by the lipid membrane surrounding the heme., This work was supported by Grants AGL2008-00377, MAT2008-03461, and BFU2007-68107-C02-01 from the Spanish Ministry of Science and Innovation (MICINN), PADI CVI-261 from the Andalusia Regional Government, and DGA-GC E33 and DGA-GE B18 from Aragon Regional Government. All these Grants were partially financed by the EU FEDER Program. M. A. Luján would like to thank the FPI Fellowship Program of the MICINN for financial support.

Cytochrome c550 (cyt c550) is a membrane component of the PSII complex in cyanobacteria and some eukaryotic algae, such as red and brown algae. Cyt c550 presents a bis-histidine heme coordination which is very unusual for monoheme c-type cytochromes. In PSII, the cyt c550 with the other extrinsic proteins stabilizes the binding of Cl¿ and Ca2+ ions to the oxygen evolving complex and protects the Mn4Ca cluster from attack by bulk reductants. The role (if there is one) of the heme of the cyt c550 is unknown. The low midpoint redox potential (Em) of the purified soluble form (from ¿250 to ¿314 mV) is incompatible with a redox function in PSII. However, more positive values for the Em have been obtained for the cyt c550 bound to the PSII. A very recent work has shown an Em value of +200 mV. These data open the possibility of a redox function for this protein in electron transfer in PSII. Despite the long distance (22 Å) between cyt c550 and the nearest redox cofactor (Mn4Ca cluster), an electron transfer reaction between these components is possible. Some kind of protective cycle involving a soluble redox component in the lumen has also been proposed. The aim of this article is to review previous studies done on cyt c550 and to consider its function in the light of the new results obtained in recent years. The emphasis is on the physical properties of the heme and its redox properties. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial

This research was originally published in Journal of Biological Chemistry. Fernando Guerrero, Arezki Sedoud, Diana Kirilovsky, A. William Rutherford, José M. Ortega, and Mercedes Roncel. A High Redox Potential Form of Cytochrome c550 in Photosystem II from Thermosynechococcus elongatus. Journal of Biological Chemistry. 2011. Vol 286: 5985-5994. © the American Society for Biochemistry and Molecular Biology, Cytochrome c550 (cyt c550) is a component of photosystem II (PSII) from cyanobacteria, red algae, and some other eukaryotic algae. Its physiological role remains unclear. In the present work, measurements of the midpoint redox potential (Em) were performed using intact PSII core complexes preparations from a histidine-tagged PSII mutant strain of the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus. When redox titrations were done in the absence of redox mediators, an Em value of +200 mV was obtained for cyt c550. This value is ∼300 mV more positive than that previously measured in the presence of mediators (Em = −80 mV). The shift from the high potential form (Em = +200 mV) to the low potential form (Em = −80 mV) of cyt c550 is attributed to conformational changes, triggered by the reduction of a component of PSII that is sequestered and out of equilibrium with the medium, most likely the Mn4Ca cluster. This reduction can occur when reduced low potential redox mediators are present or under highly reducing conditions even in the absence of mediators. Based on these observations, it is suggested that the Em of +200 mV obtained without mediators could be the physiological redox potential of the cyt c550 in PSII. This value opens the possibility of a redox function for cyt c550 in PSII., This work was supported by grants from the Ministry of Education and Culture of Spain (BFU2007- 68107-C02-01/BMC) and Andalusia Government (PAI CVI-261). The work done in France was supported by the EU/Energy Network project SOLAR-H2 (FP7 contract 212508).

A soluble monoheme c-type cytochrome (cytochrome c6) has been isolated from the green alga Monoraphidium braunii. It has a molecular mass of 9.3 kDa, an isoelectric point of 3.6 and a reduction potential of 358 mV at pH 7. The determined amino acid sequence allows its classification as a class-I c-type cytochrome. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using 1H-NMR, ultraviolet/visible, EPR and Mossbauer spectroscopies. The pH equilibria are complex, several pKa values and pH-dependent forms being observed. The amino acid sequence, the reduction-potential value and the visible and NMR spectroscopies data in the pH range 4-9 indicate that the heme iron has a methionine-histidine axial coordination. However, the EPR and Mossbauer data obtained for the ferricytochrome show that in this pH range two distinct forms are present: form I, gz = 3.27, gy = 2.05 and gx = 1.05; form II, gz = 2.95, gy = 2.29 and gx = 1.43. While form I has crystal-field parameters typical of a methionine-histidine coordination, those associated with form II would suggest a histidine-histidine axial ligation. This possibility was extensively analyzed by spectroscopic methods and by chemical modification of a histidine residue. It was concluded that form II actually corresponds to an unusual type of methionine-histidine axial coordination. Straightforward examples of this type of coordination have recently been found in other c-type hemeproteins [Teixeira, M., Campos, A. P., Aguiar, A. P., Costa, H. S., Santos, H., Turner, D. L. & Xavier, A. V. (1993) FEBS Lett. 317, 233-236], corroborating our proposal. Since both forms, with very distinct crystal-field parameters, are shown to have the same reduction potential, it may be concluded that the axial and rhombic distortions of the heme-iron ligand field cannot be directly correlated with the heme-reduction potential. The pH-dependence studies have also shown that the form I and form II are interconvertible, with pKa approximately 5. To establish a possible physiological significance for this process, in particular for the interaction of the cytochrome with the membrane-bound electron-transfer complexes b6f and photosystem I, the effect of surfactants on the spectroscopic characteristics of cytochrome c6 has been studied.

Kinetics of electron transfer from the bound tetraheme cytochrome c to the primary donor (P) have been measured in isolated reaction centers of the purple bacterium Rhodopseudomonas viridis by time-resolved flash absorption spectroscopy. The influence of two major parameters has been studied: temperature (7-305 K) and the redox state of the cytochrome. Most experiments were done with one heme (c-559), two hemes (c-559 and c-556), or three hemes (c-559, c-556, and c-552) poised in a reduced state before the flash. Measurements were done at 1283 nm in the absorption band of P+, and in the region of cytochrome alpha-bands. At room temperature, c-559 donates an electron to P+ with a half-time of 115, 190, or 230 ns (with three, two, or one heme reduced, respectively) and is then eventually rereduced by c-556 (t1/2 = 1.7 microseconds) or by c-552 (in less than 40 ns). The kinetics also include a minor microsecond phase of P+ reduction. At decreasing temperatures, the polyphasic character of P+ rereduction is accentuated. Fast phases (115 ns-10 microseconds) are slightly slowed down, following Arrhenius behavior with a weak activation energy (3.6-8.6 kJ.mol-1), until they become temperature-independent. Their extent decreases rather sharply, at temperatures which vary according to the redox poising: 250, 210, or 80 K when one, two, or three hemes are reduced, respectively. In the last case, P+ can still be reduced at low temperature, apparently directly by c-552 (t1/2 = 1.1 ms, nearly temperature-independent).(ABSTRACT TRUNCATED AT 250 WORDS)

Partial cDNAs corresponding to the GapA, GapC and GapN genes that encode the three different glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) of the green microalga Scenedesmus vacuolatus SAG 211-8b have been cloned and characterized. Northern blot experiments, as well as immunoblots and activity measurements, demonstrate a differential regulation by sugars of the components of the algal Gap gene system. Addition of glucose or other metabolizable sugars to photoautotrophic cultures promoted a drastic repression of the GapA gene and depletion to negligible levels of the corresponding GAPDHA, a chloroplastic protein involved in photosynthetic CO2 assimilation. By contrast, expression of the GapC and GapN genes encoding their cytosolic counterparts involved in glycolysis was enhanced. However, no down-regulation of the GapA gene by glucose took place in the dark, indicating that the observed effect is associated with sugar assimilation in the light. Likewise, glucose promoted in illuminated algal cultures a severe decrease of photosystem II functionality, estimated by O2 evolution activity, thermoluminescence emission and D1 protein level, while again, no effect was observed in the dark. On the basis of the correlation found between photosystem II performance and sugar transcriptional regulation of the GapA gene, a scenario of sugar-mediated regulation of photosynthetic metabolism in microalgae is proposed that will help to explain the so-called glucose bleaching effect in photosynthetic eukaryotes.

The definitive version is available at www.blackwell-synergy.com, Toxic Cu(II) effect on Cytochrome b559 under aerobic photoinhibitory conditions was examined in two different PSII membrane preparations active in oxygen evolution. The preparations differ in the content of Cytochrome b559 redox potential forms. Difference absorption spectra showed that the presence of Cu(II) induced the oxidation of the high-potential form of Cytochrome b559 in the dark. Addition of hydroquinone reduced the total oxidised high-potential form of Cytochrome b559 present in Cu(II)-treated PSII membranes indicating that no conversion to the low-potential form took place. Spectroscopic determinations of Cytochrome b559 during photoinhibitory treatment showed slower kinetics of Cu(II) effect on Cytochrome b559 as compared to the rapid loss of oxygen evolution activity in the same conditions. This result indicates that Cytochrome b559 is affected after PSII centers are photoinhibited. The high-potential form was more sensitive to toxic Cu(II) action than the low-potential form under illumination at pH 6.0. The content of the high-potential form of Cytochrome b559 was completely lost, however the low-potential content was unaffected in these conditions. This loss did not involve cytochrome protein degradation. Results are discussed in terms of different binding properties of the heme iron to the protonated or unprotonated histidine ligand in the high-potential and low-potential forms of Cytochrome b559, respectively., M. Bernal was recipient of an I3P Programme fellowship from Consejo Superior de Investigaciones Científicas. This work was supported by the Dirección General de Investigación (Grant BMC2002-00031) to R.P. and Gobierno de Aragón (Grant P015/2001) to I.Y., and it has been done within GC DGA 2002 Program of Gobierno de Aragón.

Redox properties of cytochrome b559 (Cyt b559) and cytochrome c550 (Cyt c550) have been studied by using highly stable photosystem II (PSII) core complex preparations from a mutant strain of the thermophilic cyanobacterium Thermosynechococcus elongatus with a histidine tag on the CP43 protein of PSII. Two different redox potential forms for Cyt b559 are found in these preparations, with a midpoint redox potential ( E'(m)) of +390 mV in about half of the centers and +275 mV in the other half. The high-potential form, whose E'(m)is pH independent, can be converted into the lower potential form by Tris washing, mild heating or alkaline pH incubation. The E'(m) of the low-potential form is significantly higher than that found in other photosynthetic organisms and is not affected by pH. The possibility that the heme of Cyt b559 in T. elongatus is in a more hydrophobic environment is discussed. Cyt c550 has a higher E'(m)when bound to the PSII core (-80 mV at pH 6.0) than after its extraction from the complex (-240 mV at pH 6.0). The E'(m) of Cyt c550 bound to PSII is pH independent, while in the purified state an increase of about 58 mV/pH unit is observed when the pH decreases below pH 9.0. Thus, Cyt c550 seems to have a single protonateable group which influences the redox properties of the heme. From these electrochemical measurements and from EPR controls it is proposed that important changes in the solvent accessibility to the heme and in the acid-base properties of that protonateable group could occur upon the release of Cyt c550 from PSII.

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

Electron transfer from the tetraheme cytochrome c to the special pair of bacteriochlorophylls (P) has been studied by flash absorption spectroscopy in reaction centers isolated from seven strains of the photosynthetic purple bacterium Rhodopseudomonas viridis, where the residue L162, located between the proximal heme c-559 and P, is Y (wild type), F, W, G, M, T, or L. Measurements were performed between 294 K and 8 K, under redox conditions in which the two high-potential hemes of the cytochrome were chemically reduced. At room temperature, the kinetics of P+ reduction include two phases in all of the strains: a dominant very fast phase (VF), and a minor fast phase (F). The VF phase has the following t(1/2): 90 ns (M), 130 ns (W), 135 ns (F), 189 ns (Y; wild type), 200 ns (G), 390 ns (L), and 430 ns (T). These data show that electron transfer is fast whatever the nature of the amino acid at position L162. The amplitudes of both phases decrease suddenly around 200 K in Y, F, and W. The effect of temperature on the extent of fast phases is different in mutants G, M, L, and T, in which electron transfer from c-559 to P+ takes place at cryogenic temperatures in a substantial fraction of the reaction centers (T, 48%; G, 38%; L, 23%, at 40 K; and M, 28%, at 60 K), producing a stable charge separated state. In these nonaromatic mutants the rate of VF electron transfer from cytochrome to P+ is nearly temperature-independent between 294 K and 8 K, remaining very fast at very low temperatures (123 ns at 60 K for M; 251 ns at 40 K for L; 190 ns at 8 K for G, and 458 ns at 8 K for T). In all cases, a decrease in amplitudes of the fast phases is paralleled by an increase in very slow reduction of P+, presumably by back-reaction with Q(A)-. The significance of these results is discussed in relation to electron transfer theories and to freezing at low temperatures of cytochrome structural reorganization.

Cytochrome b559 (Cyt b559) is a well-known intrinsic component of the photosystem II (PSH) reaction center (RC) in all oxygen-evolving photosynthetic organisms. Although neither its structure nor its organization in PSII are known, it has been proposed that Cyt b559 is a heme-bridged protein heterodimer with two subunits, a and [3, of 9 and 4 kDa, respectively [1]. An important characteristic of this cytochrome is that it may exhibit different redox potential forms. Usually, Cyt b559 is present in two interconvertible redox forms: a pH-independent high-potential (HP) form (E ‘m = +380 mV) and a pH-dependent low-potential (LP) form (E ’ m = +140 mV, pH > 7.6) [2]. The HP form is labile and easily converted to the LP form by a variety of treatments which alter the structure of the membrane.

The photosynthetic reaction center (RC) of purple bacteria is a large membrane protein which performs the primary steps of photo-induced electron transfer. After light excitation, a dimer of bacteriochlorophyll molecules (P) transfers an electron to an acceptor, and the oxidized species P+ is then re-reduced by a secondary electron donor, a c-type cytochrome, in order to be reactivated. In some purple bacteria this secondary donor is a soluble c 2 cytochrome, but in most cases it is a RC-bound tetraheme cytochrome c. The best studied example is Rhodopseudomonas (Rps.) viridis, in which the tridimensional structure of the RC has been solved with a high atomic resolution (1). Some of the electron transfer reactions from c-type cytochromes to P+ in the RC of purple bacteria occur even at cryogenic temperatures. Experimental observations of the effects of temperature on the rate and the yield of the cytochrome oxidation have contributed a lot to the development of current electron transfer theories (2). However, several of these observations, such as the extent of photooxidation of heme c556 at cryogenic temperature in Rps. viridis are still controversial (for a review see (3)). In order to understand some of these effects we have investigated the effect of flash and continuous illumination at low temperature on cytochrome hemes in RC isolated from Rps. viridis.

In purple photosynthetic bacteria the electron donor to the special pair, after its oxidation by a light-induced reaction, is a c-type cytochrome: either a soluble monoheme cytochrome which forms a transitory complex with the reaction center, or a tetraheme cytochrome which remains permanently bound to the reaction center. The effects of low temperatures on electron transfer in the complex are presented and discussed. They provide estimates for the reorganization energy. The most prominent effect of low temperature is that a dominant fast phase of electron transfer becomes impossible at a temperature of around 250 K (monoheme cytochrome) or located between 250 K and 80 K according to the redox state (tetraheme cytochrome). This inhibition is attributed to a freezing-like transition of pools of water molecules which blocks structural changes of the protein which are normally associated with the cytochrome oxidation.

The kinetics of electron transfer from the third highest potential heme (c-522, Em = +20 mV) to the primary donor (P-960) have been measured by flash absorption spectroscopy in isolated reaction centers of Rhodopseudomonas viridis between 300 K and 7 K. The data are analyzed on the basis of three exponential components with a very fast phase (? 1 2 = 120 ns) dominating at high temperature and a very slow one (? 1 2 = 1.2 ms) at low temperature. This multiphasic behavior is interpreted in terms of the existence of three states with a temperature-dependent population and a very limited effect of the temperature on the kinetics for each state.

Selective light-induced redox changes of cytochrome b-559 in relation to cytochrome f have been investigated at 20°C in fresh spinach thylakoids under physiological conditions and in the presence of specific inhibitors and uncouplers. Light absorbed by PS II (650 nm) promotes the sequential reduction of cytochrome b-559 and cytochrome f, whereas light absorbed by PS I (720 nm) brings about the oxidation of cytochrome f and, by way of this, the oxidation of cytochrome b-559. Either DCMU or DBMIB completely blocks the reduction of the two cytochromes by PS II light, but not the oxidation of cytochrome f by PS I light. On the other hand, cyanide as well as polylysine inhibits almost totally the oxidation of both cytochromes f by PSI light. From these and previous results, it is concluded that photoreduction of cytochrome b-559 by PS II occurs through the plastoquinone pool and that its photooxidation by PS I involves both plastocyanin and cytochrome f. It thus appears that cytochrome b-559 is located between plastoquinone and cytochrome f in noncyclic electron transport in chloroplasts.

Cytochrome b-564 in isolated complex III from baker's yeast mitochondria exhibits the midpoint redox potential proper to the low-potential couple (+60 mV, pH 7.2). Incorporation of the complex into liposomes promotes total conversion to the high-potential couple (+170 mV, pH 7.2). The reconstituted system shows electrogenic proton translocation, which is inhibited by the uncoupler CCCP. Deenergizing treatments result, moreover, in reversal of the redox potential change. These results support our previous proposal that cytochrome b-564 acts as a transducer of redox energy into acid-base energy in the complex III region of the respiratory chain.

Cytochrome b-559. Spinach Thylakoids. PS II Particles. High and Low Potential. Interconvertible Forms Cytochrome b-559 has been purified from both spinach (Spinacia oleracea L.) thylakoids and photosystem II particles in order to compare some of its more polemic molecular properties. In the two cases, cytochrome 6-559 (which is initially present in its reduced high-potential form and in its oxidized low-potential form) is purified as a single species by preparative disc electrophoresis in Triton-containing polyacrylamide gel, the isolated protein exhibiting similar molecular mass and almost identical low midpoint redox potential and absorption spectra. Moreover, sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified cytochrom e b-559 reveals, again in both cases, only one polypeptide band of similar molecular mass. Integration o f the isolated low-potential form into liposomes partly restores the high-potential form. These data corroborate our previous results and indicate that there exists in thylakoids only a cytochrome b -559 molecular species tightly bound to PS II and that the two naturally occurring low-and high-potential couples are physiological and correspond to interconvertible states of the genuine hem e protein.

The redox and acid/base states and midpoint potentials of cytochrome b-559 have been determined in oxygen-evolving photosystem II (PS II) particles at room temperature in the pH range from 6.5 to 8.5. At pH 7.5 the fresh PS II particles present about 2/3 of their cytochrome b-559 in its reduced and protonated (non-auto-oxidizable) high-potential form and about 1/3 in its oxidized and non-protonated low-potential form. Potentiometric reductive titration shows that the protonated high-potential couple is pH-independent (E'0, + 380 mV), whereas the low-potential couple is non-protonated and pH-independent above pH 7.6 (E'0, pH greater than 7.6, + 140 mV), but becomes pH-dependent below this pH, with a slope of -72 mV/pH unit. Moreover, evidence is presented that in PS II particles cytochrome b-559 can cycle, according to its established redox and acid/base properties, as an energy transducer at two alternate midpoint potentials and at two alternate pKa values. Red light absorbed by PS II induces reduction of cytochrome b-559 in these particles at room temperature, the reaction being completely blocked by dichlorophenyldimethylurea.