Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

“Reduced minus oxidized” difference extinction coefficients Δ? in the α-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1 ± 1.0 mM⁻¹ cm⁻¹ and 27.0 ± 1.0 mM⁻¹ cm⁻¹ were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from −250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E_m values at pH 6.5 of 244 ± 11 mV and −94 ± 21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the α-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q_B site and the microenvironment of the heme group of Cyt b559 are discussed.     

Redox properties of the photosystem II cytochromes b559 and c550 in the cyanobacterium Thermosynechococcus elongatus

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.     

Redox and spectral properties of cytochrome b559 in different preparations of photosystem II

A detailed analysis of the properties of cytochrome b(559) (Cyt b(559)) in photosystem II (PS II) preparations with different degrees of structural complexity is presented. It reveals that (i) D1-D2-Cyt b(559) complexes either in solubilized form or incorporated into liposomes contain only one type of Cyt b(559) with E(m) values of 60 +/- 5 and 100 +/- 10 mV, respectively, at pH 6.8; (ii) in oxygen-evolving solubilized PS II core complexes Cyt b(559) exists predominantly (>85%) as an LP form with an E(m,7) of 125 +/- 10 mV and a minor fraction with an E(m,7) of -150 +/- 15 mV; (iii) in oxygen-evolving PS II membrane fragments three different redox forms are discernible with E(m) values of 390 +/- 15 mV (HP form), 230 +/- 20 mV (IP form), and 105 +/- 25 mV (LP form) and relative amplitudes of 58, 24, and 18%, respectively, at pH 7.3; (iv) the E(m) values are almost pH-independent between pH 6 and 9.5 in all sample types except D1-D2-Cyt b(559) complexes incorporated into liposomes with a slope of -29 mV/pH unit, when the pH increases from 6 to 9.5 (IP and LP form in PS II membrane fragments possibly within a restricted range from pH 6.5 to 8); (v) at pH >8 the HP Cyt b(559) progressively converts to the IP form with increasing pH; (vi) the reduced-minus-oxidized optical difference spectra of Cyt b(559) are very similar in the lambda range of 360-700 nm for all types except for the HP form which exhibits pronounced differences in the Soret band; and (vii) PS II membrane fragments and core complexes are inferred to contain about two Cyt b(559) hemes per PS II. Possible implications of conformational changes near the heme group and spin state transitions of the iron are discussed.     

Spectroelectrochemistry of cytochrome b559 in the D1-D2-Cyt b559 complex from spinach

The redox potential of cytochrome b559 (Cyt b559) in the D1-D2-Cyt b559 complex from spinach has been determined to be +90+/-2mV vs. SHE at pH 6.0, by thin-layer cell spectroelectrochemistry for the first time. The redox potential, corresponding uniquely to the so-called "low-potential form", exhibited a sigmoidal pH-dependence from pH 4.0 to 9.0, ranging from +115 to +50mV. An analysis of the pH-dependence based on model equations suggests that two histidine residues coordinating to the heme iron in the protein subunits may exert electrostatic influence on the redox potential of Cyt b559.     

Analysis of the transformation effect in cytochrome b559 of photosystem II in terms of the model of the heme-quinone redox interaction

Transformation of three-component redox pattern of cytochrome (Cyt) b559 in PS II membrane fragments upon various treatments is manifested in decrease of the relative content (R) of the high potential (HP) redox form of Cyt b559 and concomitant increase in the fractions of the two lower potential forms. Redox titration of Cyt b559 in different types of PS II membrane preparations was performed and revealed that (1) alteration of redox titration curve of Cyt b559 upon treatment of a sample is not specific to the type of treatment; (2) each value of R_HP defines the individual shape of the redox titration curve; (3) population of Cyt b559 may exist in several stable forms with multicomponent redox pattern: three types of three-component redox pattern and one type of two-component redox pattern as well as in the form with a single E_m; (4) transformation of Cyt b559 proceeds as successive conversion between the stable forms with multicomponent redox pattern; (5) upon harsh treatments, Cyt b559 abruptly converts into the state with a single E_m which value is intermediate between the E_m values of the two lower potential forms. Analysis of the data using the model of Cyt b559-quinone redox interaction revealed that diminution of R_HP in a range from 80 to 10% reflects a shift in redox equilibrium between the heme group of Cyt b559 and the interacting quinone, due to a gradual decrease of 90?mV in E_m of the heme group at the virtually unchanged E_m of the quinone component.     

Characterization of the multiple forms of cytochrome b559 in photosystem II

Cytochrome b559 is an essential component of the photosystem II (PSII) protein complex. Its function, which has long been an unsolved puzzle, is likely to be related to the unique ability of PSII to oxidize water. We have used EPR spectroscopy and spectrophotometric redox titrations to probe the structure of cytochrome b559 in PSII samples that have been treated to remove specific components of the complex. The results of these experiments indicate that the low-temperature photooxidation of cytochrome b559 does not require the presence of the 17-, 23-, or 33-kDa extrinsic polypeptides or the Mn complex (the active site in water oxidation). We observe a shift in the g value of the EPR signal of cytochrome b559 upon warming a low-temperature photooxidized sample, which presumably reflects a change in conformation to accommodate the oxidized state. At least three redox forms of cytochrome b559 are observed. Untreated PSII membranes contain one high-potential (375 mV) and one intermediate-potential (230 mV) cytochrome b559 per PSII. Thylakoid membranes also appear to contain one high-potential and one intermediate-potential cytochrome b559 per PSII, although this measurement is more difficult due to interference from other cytochromes. Removal of the 17- and 23-kDa extrinsic polypeptides from PSII membranes shifts the composition to one intermediate-potential (170 mV) and one low-potential (5 mV) cytochrome b559. This large decrease in potential is accompanied by a very small g-value change (0.04 at gz), indicating that it is the environment and not the ligand field of the heme which changes significantly upon the removal of the 17- and 23-kDa polypeptides.     

Molecular changes following oxidoreduction of cytochrome b559 characterized by Fourier transform infrared difference spectroscopy and electron paramagnetic resonance: photooxidation in photosystem II and electrochemistry of isolated cytochrome b559 and iron protoporphyrin IX-bisimidazole model compounds.

The vibrational infrared absorption changes associated with the oxidation of cytochrome b559 (Cyt b559) have been characterized. In photosystem II (PS II) enriched membranes, low-potential (LP) and high-potential (HP) Cyt b559 were investigated by light-induced FTIR difference spectroscopy. The redox transition of isolated Cyt b559 is characterized by protein electrochemistry. On the basis of a model of the assembly of Cyt b559 with the two axial Fe ligands being histidine residues of two distinct polypeptides, each forming a transmembrane alpha-helix [Cramer, W.A., Theg, S.M., & Widger, W.R. (1986) Photosynth. Res. 10, 393-403], the bisimidazole and bismethylimidazole complexes of Fe protoporphyrin IX were electrochemically oxidized and reduced to detect the IR oxidation markers of the heme and its two axial ligands. Major bands at 1674/1553, 1535, and 1240 cm-1 are tentatively assigned to nu 37 (CaCm), nu 38-(CbCb) and delta (CmH) modes, respectively; other bands at 1626, 1613, 1455, 1415, and 1337 cm-1 are assigned to porphyrin skeletal and vinyl modes. Modes at 1103 and 1075/1066 cm-1 are assigned to the 4-methylimidazole and imidazole ligands, respectively. For the isolated Cyt b559, it is shown that both the heme (at 1556-1535, 1337, and 1239 cm-1), the histidine ligands at 1104 cm-1 and the protein (between 1600 and 1700 cm-1 and at 1545 cm-1) are affected by the charge stabilization. The excellent agreement between model compounds and isolated Cyt b559 reinforces the validity of the model of a heme iron coordinated to two histidine residues for Cyt b559. A differential signal at 1656/1641 cm-1 is assigned to peptide C = O mode(s). We speculate that this signal reflects the change in strength of a hydrogen bond formed between the histidine ligand(s) and the polypeptide backbone upon oxidoreduction of the cytochrome. In PS II membranes, the signals characteristic of Cyt b559 photooxidation are found at 1660/1652 and 1625 cm-1, for both the high- and low-potential forms. The differences observed in the amplitude of the 1660/1652-cm-1 band, at 1700 and 1530-1510 cm-1 in the light-induced FTIR difference spectra of Cyt b559 HP and LP, show that the mechanisms of heme oxidation in vivo imply different molecular processes for the two forms Cyt b559 HP and LP.     

Two reaction pathways for transformation of high potential cytochrome b559 of PS II into the intermediate potential form

This study describes an analysis of different treatments that influence the relative content and the midpoint potential of HP Cyt b559 in PS II membrane fragments from higher plants. Two basically different types of irreversible modification effects are distinguished: the HP form of Cyt b559 is either predominantly affected when the heme group is oxidized ("O-type" effects) or when it is reduced ("R-type" effects). Transformation of HP Cyt b559 to lower potential redox forms (IP and LP forms) by the "O-type" mechanism is induced by high pH and detergent treatments. In this case the effects consist of a gradual decrease in the relative content of HP Cyt b559 while its midpoint potential remains unaffected. Transformation of HP Cyt b559 via an "R-type" mechanism is caused by a number of exogenous compounds denoted L: herbicides, ADRY reagents and tetraphenylboron. These compounds are postulated to bind to the PS II complex at a quinone binding site designated as Q(C) which interacts with Cyt b559 and is clearly not the Q(B) site. Binding of compounds L to the Q(C) site when HP Cyt b559 is oxidized gives rise to a gradual decrease in the E(m) of HP Cyt b559 with increasing concentration of L (up to 10 K(ox)(L) values) while the relative content of HP Cyt b559 is unaffected. Higher concentrations of compounds L required for their binding to Q(C) site when HP Cyt b559 is reduced (described by K(red)(L)) induce a conversion of HP Cyt b559 to lower potential redox forms ("R-type" transformation). Two reaction pathways for transitions of Cyt b559 between the different protein conformations that are responsible for the HP and IP/LP redox forms are proposed and new insights into the functional regulation of Cyt b559 via the Q(C) site are discussed.     

Effect of dehydration on light-induced reactions in photosystem II: photoreactions of cytochrome b559

The effect of dehydration on the reaction pattern of photosystem II (PS II) has been studied by measuring and analyzing spectral changes induced by continuous wavelength illumination in films of untreated and hydroxylamine-washed PS II membrane fragments dehydrated to different levels. The obtained data revealed (i) the extent of light-induced formation of about one Q(A)(-*)per 230 chlorophylls (Chl) remains virtually invariant to dehydration down to the lowest values of relative humidity (6-8% RH); (ii) a decrease of the RH to 30% leads to severe blockage of the electron transfer from Q(A)(-*) to Q(B) and the progressive replacement of water oxidation by photooxidation of high potential (HP) cytochrome (Cyt) b559 in untreated PS II samples or accessory Chl and carotenoid (Car) molecules in samples with preoxidized Cyt b559; (iii) photooxidation of Cyt b559 is followed by its photoreduction, concomitant with photooxidation of Chl and Car; (iv) in dry samples with preoxidized Cyt b559, not more than a half of total Cyt b559 can be photochemically reduced, independent of the extent of Cyt b559 in the HP form; (v) at low RH values, Cyt b559 photoreduction in samples with preoxidized heme groups and photoaccumulation of Q(A)(-*) take place with biphasic kinetics with similar rate constants for both processes; (vi) Cyt b559 photoreduction in dry samples is DCMU insensitive, while the dark rereduction of photooxidized Cyt b559 is inhibited by DCMU; (vii) fast and slow kinetic phases of Cyt b559 photoreduction dramatically differ in their dependencies on the intensity of CW illumination and are associated with electron donation to Cyt b559 from Q(A)(-*) and pheophytin(-*), respectively. The pathways of light-induced electron transfer in PS II involving Cyt b559 are discussed.     

Restoration of the high-potential form of cytochrome b559 of photosystem II occurs via a two-step mechanism under illumination in the presence of manganese ions

Spinach photosystem II membranes that had been depleted of the Mn cluster contained four forms of cytochrome (Cyt) b559, namely, high-potential (HP), HP', intermediate-potential (IP) and low-potential (LP) forms that exhibited the redox potentials of +400, +310, +170 and +35 mV, respectively, in potentiometric titration. When the membranes were illuminated with flashing light in the presence of 0.1 mM Mn2+, the IP form was converted to the HP' form by two flashes and then the HP' form was converted to the HP form by an additional flash. The quantum efficiency of the first conversion appeared to be quite high since the conversion was almost complete after two flashes. By contrast, the second conversion proceeded with low quantum efficiency and 40 flashes were required for completion. The effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) suggested that the first conversion did not require electron transfer from QA to QB while the second conversion had an absolute requirement for it. It was also suggested that the first conversion involved the reduction of the heme of Cyt b559, probably by QA-, and we propose that direct reduction by QA- induces a shift in the redox potential of the heme. The second conversion was also accompanied by the reduction of heme but it appeared that this conversion did not necessarily involve the reduction. The effects of DCMU on the reduction of heme suggested that the heme became reducible by QB- after the first conversion had been completed. This observation implies that the efficiency of electron transfer from QA to QB increased upon the conversion of the IP form to the HP' form, and we propose that restoration of the high-potential forms of Cyt b559 itself acts to make the acceptor side of photosystem II functional.     

Oxygen-induced changes in the redox state of the cytochrome b559 in photosystem II depend on the integrity of the Mn cluster

The effect of oxygen and anaerobiosis on the redox properties of Cyt b ₅₅₉ was investigated in PSII preparations from spinach with different degree of disintegration of the donor side. Comparative studies were performed on intact PSII membranes and PSII membranes that were deprived of the 18-kDa peripheral subunit (0.25 NaCl washed), the 18- and 24-kDa peripheral subunits (1 M NaCl washed), the 18-, 24- and 33-kDa peripheral subunits (1.2 M CaCl₂ washed), Cl depleted and after complete depletion of the Mn cluster (Tris washed). In active PSII centers, about 75% of Cyt b ₅₅₉ was found in the high-potential form and the rest in the intermediate potential form. With decomposition of the donor side, the intermediate potential form started to dominate, reaching more than 90% after Tris treatment. The oxygen-dependent conversion of the intermediate potential form of Cyt b ₅₅₉ into the low-potential and high-potential forms was only observed after treatments that directly affect the Mn cluster. In PSII membranes, deprived of all three extrinsic subunits (CaCl₂ treatment), 21% of the intermediate potential form was converted into the low-potential form and 14% into the high-potential form by the removal of oxygen. In Tris-washed PSII membranes, completely lacking the Mn cluster, this conversion amounted to 60 and 33%, respectively. In intact PSII membranes, the oxygen-dependent conversion did not occur. The possible physiological role of this oxygen-dependent behavior of the Cyt b ₅₅₉ redox forms during the assembly/photoactivation cycle of PSII is discussed.     

The position of cytochrome b(559) relative to Q(A) in photosystem II studied by electron-electron double resonance (ELDOR)

The electron-electron double resonance (ELDOR) method was applied to measure the dipole interaction between cytochrome (Cyt) b(+)(559) and the primary acceptor quinone (Q(-)(A)), observed at g=2.0045 with the peak to peak width of about 9 G, in Photosystem II (PS II) in which the non-heme Fe(2+) was substituted by Zn(2+). The paramagnetic centers of Cyt b(+)(559)Y(D)Q(-)(A) were trapped by illumination at 273 K for 8 min, followed by dark adaptation for 3 min and freezing into 77 K. The distance between the pair Cyt b(+)(559)-Q(-)(A) was estimated from the dipole interaction constant fitted to the observed ELDOR time profile to be 40+/-1 A. In the membrane oriented PS II particles the angle between the vector from Q(A) to Cyt b(559) and the membrane normal was determined to be 80+/-5 degrees. The position of Cyt b(559) relative to Q(A) suggests that the heme plane is located on the stromal side of the thylakoid membrane. ELDOR was not observed for Cyt b(+)(559) Y(D) spin pair, suggesting the distance between them is more than 50 A.     

Evidence for a novel quinone-binding site in the photosystem II (PS II) complex that regulates the redox potential of cytochrome b559

The present study provides a thorough analysis of effects on the redox properties of cytochrome (Cyt) b559 induced by two photosystem II (PS II) herbicides [3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,4-dinitro-6-sec-butylphenol (dinoseb)], an acceleration of the deactivation reactions of system Y (ADRY) agent carbonylcyanide-m-chlorophenylhydrazone (CCCP), and the lipophilic PS II electron-donor tetraphenylboron (TPB) in PS II membrane fragments from higher plants. The obtained results revealed that (1) all four compounds selectively affected the midpoint potential (E(m)) of the high potential (HP) form of Cyt b559 without any measurable changes of the E(m) values of the intermediate potential (IP) and low potential (LP) forms; (2) the control values from +390 to +400 mV for HP Cyt b559 gradually decreased with increasing concentrations of DCMU, dinoseb, CCCP, and TPB; (3) in the presence of high TPB concentrations, a saturation of the E(m) decrease was obtained at a level of about +240 mV, whereas no saturation was observed for the other compounds at the highest concentrations used in this study; (4) the effect of the phenolic herbicide dinoseb on the E(m) is independent of the occupancy of the Q(B)-binding site by DCMU; (5) at high concentrations of TPB or dinoseb, an additional slow and irreversible transformation of HP Cyt b559 into IP Cyt b559 or a mixture of the IP and LP Cyt b559 is observed; and (6) the compounds stimulate autoxidation of HP Cyt b559 under aerobic conditions. These findings lead to the conclusion that a binding site Q(C) exists for the studied substances that is close to Cyt b559 and different from the Q(B) site. On the basis of the results of the present study and former experiments on the effect of PQ extraction and reconstitution on HP Cyt b559 [Cox, R. P., and Bendall, D. S. (1974) The functions of plastoquinone and beta-carotene in photosystem II of chloroplasts, Biochim. Biophys. Acta 347, 49-59], it is postulated that the binding of a plastoquinone (PQ) molecule to Q(C) is crucial for establishing the HP form of Cyt b559. On the other hand, the binding of plastoquinol (PQH2) to Q(C) is assumed to cause a marked decrease of E(m), thus, giving rise to a PQH2 oxidase function of Cyt b559. The possible physiological role of the Q(C) site as a regulator of the reactivity of Cyt b559 is discussed.     

Characterization of the secondary electron-transfer pathway intermediates of photosystem II containing low-potential cytochrome b 559

Beta-carotene (Car) and chlorophyll (Chl) function as secondary electron donors in photosystem II (PS II) under conditions, such as low temperature, when electron donation from the O(2)-evolving complex is inhibited. In prior studies of the formation and decay of Car(*+) and Chl(*+) species at low temperatures, cytochrome b(559) (Cyt b(559)) was chemically oxidized prior to freezing the sample. In this study, the photochemical formation of Car(*+) and Chl(*+) is characterized at low temperature in O(2)-evolving Synechocystis PS II treated with ascorbate to reduce most of the Cyt b(559). Not all of the Cyt b(559) is reduced by ascorbate; the remainder of the PS II reaction centers, containing oxidized low-potential Cyt b(559), give rise to Car(*+) and Chl(*+) species after illumination at low temperature that are characterized by near-IR spectroscopy. These data are compared to the measurements on ferricyanide-treated O(2)-evolving Synechocystis PS II in which the Car(*+) and Chl(*+) species are generated in PS II centers containing mostly high- and intermediate-potential Cyt b(559). Spectral differences observed in the ascorbate-reduced PS II samples include decreased intensity of the Chl(*+) and Car(*+) absorbance peaks, shifts in the Car(*+) absorbance maxima, and lack of formation of a 750 nm species that is assigned to a Car neutral radical. These results suggest that different spectral forms of Car are oxidized in PS II samples containing different redox forms of Cyt b(559), which implies that different secondary electron donors are favored depending on the redox form of Cytb(559) in PS II.     

Two reaction pathways for transformation of high potential cytochrome b559 of PS II into the intermediate potential form

This study describes an analysis of different treatments that influence the relative content and the midpoint potential of HP Cyt b559 in PS II membrane fragments from higher plants. Two basically different types of irreversible modification effects are distinguished: the HP form of Cyt b559 is either predominantly affected when the heme group is oxidized (“O-type” effects) or when it is reduced (“R-type” effects). Transformation of HP Cyt b559 to lower potential redox forms (IP and LP forms) by the “O-type” mechanism is induced by high pH and detergent treatments. In this case the effects consist of a gradual decrease in the relative content of HP Cyt b559 while its midpoint potential remains unaffected. Transformation of HP Cyt b559 via an “R-type” mechanism is caused by a number of exogenous compounds denoted L: herbicides, ADRY reagents and tetraphenylboron. These compounds are postulated to bind to the PS II complex at a quinone binding site designated as Q_C which interacts with Cyt b559 and is clearly not the Q_B site. Binding of compounds L to the Q_C site when HP Cyt b559 is oxidized gives rise to a gradual decrease in the E_m of HP Cyt b559 with increasing concentration of L (up to 10 K_ox(L) values) while the relative content of HP Cyt b559 is unaffected. Higher concentrations of compounds L required for their binding to Q_C site when HP Cyt b559 is reduced (described by K_red(L)) induce a conversion of HP Cyt b559 to lower potential redox forms (“R-type” transformation). Two reaction pathways for transitions of Cyt b559 between the different protein conformations that are responsible for the HP and IP/LP redox forms are proposed and new insights into the functional regulation of Cyt b559 via the Q_C site are discussed.     

Photosynthetic cytochrome c550

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 Ca(2+) ions to the oxygen evolving complex and protects the Mn(4)Ca 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 (E(m)) of the purified soluble form (from -250 to -314mV) 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 E(m) value of +200mV. These data open the possibility of a redox function for this protein in electron transfer in PSII. Despite the long distance (22?) between cyt c550 and the nearest redox cofactor (Mn(4)Ca 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.     

Site directed mutagenesis of the heme axial ligands of cytochrome b559 affects the stability of the photosystem II complex.

Cytochrome (cyt) b559, an integral membrane protein, is an essential component of the photosystem II (PSII) complex in the thylakoid membranes of oxygenic photosynthetic organisms. Cyt b559 has two subunits, alpha and beta, each with one predicted membrane spanning alpha-helical domain. The heme cofactor of this cytochrome is coordinated between two histidine residues. Each of the two subunit polypeptides of cyt b559 has one His residue. To investigate the influence of these His residues on the structure of cyt b559 and the PSII complex, we used a site directed mutagenesis approach to replace each His residue with a Leu residue. Introduction of these missense mutations in the transformable unicellular cyanobacterium, Synechocystis 6803, resulted in complete loss of PSII activity. Northern blot analysis showed that these mutations did not affect the stability of the polycistronic mRNA that encompasses both the psbE and the psbF genes, encoding the alpha and the beta subunits, respectively. Moreover, both of the single His mutants showed the presence of the alpha subunit which was 1.5 kd smaller than the same polypeptide in wild type cells. A secondary effect of such a structural change was that D1 and D2, two proteins that form the catalytic core (reaction center) of PSII, were also destabilized. Our results demonstrate that proper axial coordination of the heme cofactor in cyt b559 is important for the structural integrity of the reaction center of PSII.     

细胞色素 b559与PSⅡ光抑制的光保护机制

Cyt b559是由两条多肽,即α_、β_两个亚基组成的一种血红蛋白,是光系统Ⅱ（PSⅡ）蛋白复合体必不可少的组分。简要介绍了Cyt b559的分子组成及其氧化还原特性。重点阐述了在光抑制条件下Cyt b559对PSⅡ反应中心的可能保护机制和由Cyt b559参与的围绕PSⅡ的循环电子传递。     

Reconstitution, spectroscopy, and redox properties of the photosynthetic recombinant cytochrome b 559 from higher plants

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.     

Effects of Light Stress on Redox Potential Forms of Cyt b-559 in Photosystem II Membranes Depleted of Water-Oxidizing Complex

Effects of photoinhibition on the redox properties of Cyt b-559were studied with NH₂OH treated PSII membranes, which are depletedof the water-oxidizing complex. The membranes contained threeredox forms (HP-, IP- and LP-forms) of Cyt b-559, with E_m valuesof +435, +237 and +45 mV, respectively. A novel intermediate-potentialform of Cyt b-559 was generated during photoinhibition on thedonor side of PSII: photoinhibitory illumination (7,000 µEm^–2 s^–1) for 1 min induced a 30% decrease in thelevel of the HP-form, with concomitant generation of the intermediate-potential(IP-) form whose E_m value was about +350mV. Prolonged illumination(10 min) resulted in complete loss of the HP-form and an apparentincrease in the level of the IPform. After further photoinhibitorytreatment (60 min), complete loss of the IP'-form was observedand levels of the IP- and LP-forms each increased to about 50%of the total amount of Cyt b-559. Kinetic analysis of thesedata led to the conclusion that the HP-form is converted tothe LP-form via two intermediate-potential forms (IP' and IP),and that IP'-form appears only at the early phase of photoinhibition. (Received March 30, 1994; Accepted February 27, 1995)