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.     

Copper effect on cytochrome b of photosystem II under photoinhibitory conditions

Toxic Cu (II) effect on cytochrome b ₅₅₉ under aerobic photoinhibitory conditions was examined in two different photosystem II (PSII) membrane preparations active in oxygen evolution. The preparations differ in the content of cytochrome b ₅₅₉ redox potential forms. Difference absorption spectra showed that the presence of Cu (II) induced the oxidation of the high-potential form of cytochrome b ₅₅₉ in the dark. Addition of hydroquinone reduced the total oxidized high-potential form of cytochrome b ₅₅₉ present in Cu (II)-treated PSII membranes indicating that no conversion to the low-potential form took place. Spectroscopic determinations of cytochrome b ₅₅₉ during photoinhibitory treatment showed slower kinetics of Cu (II) effect on cytochrome b ₅₅₉ in comparison with the rapid loss of oxygen evolution activity in the same conditions. This result indicates that cytochrome b ₅₅₉ is affected after PSII centres 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 b ₅₅₉ was completely lost; however, the low-potential content was unaffected in these conditions. This loss did not involve cytochrome protein degradation. The 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 b ₅₅₉, respectively.     

Coupling between redox and acid-base energy by cytochrome b-564 in Baker's yeast mitochondria

Baker's yeast mitochondrial cytochrome b-564 is characterized by exhibiting both a labile pH-independent high-potential form (E'o, pH 7 = + 190 mV) and a stable pH-dependent (pKa = 6.8) low-potential form (E'o, pH 7 = + 70 mV). The different behavior of these two forms of cytochrome b-564 versus pH seems to be a decisive factor for transduction of redox energy into acid-base energy in oxidative phosphorylation site 2. Deenergizing treatments, such as ADP plus Pi, result in the conversion of all the mitochondrial cytochrome b-564 into its low-potential form, whereas energization with ATP specifically transforms the cytochrome into its high-potential form, the ATP effect being neutralized by the ATPase inhibitor oligomycin and by the uncoupler FCCP. Accordingly, a minimal model for coupling between redox energy and acid-base energy through an electronically energized and protonated ferricytochrome b-564 intermediate is proposed. The energy-transducing properties of mitochondrial cytochrome b-564 seems to be shared by chloroplast cytochrome b-559.     

Redox and acid-base characterization of cytochrome b-559 in photosystem II particles

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.     

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.     

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.     

Differential mechanism of light-induced and oxygen-dependent restoration of the high-potential form of cytochrome b559 in Tris-treated Photosystem II membranes

The effect of illumination and molecular oxygen on the redox and the redox potential changes of cytochrome b₅₅₉ (cyt b₅₅₉) has been studied in Tris-treated spinach photosystem II (PSII) membranes. It has been demonstrated that the illumination of Tris-treated PSII membranes induced the conversion of the intermediate-potential (IP) to the reduced high-potential (HP^Fe2+) form of cyt b₅₅₉, whereas the removal of molecular oxygen resulted in the conversion of the IP form to the oxidized high-potential (HP^Fe3+) form of cyt b₅₅₉. Light-induced conversion of cyt b₅₅₉ from the IP to the HP form was completely inhibited above pH 8 or by the modification of histidine ligand that prevents its protonation. Interestingly, no effect of high pH or histidine modification was observed during the conversion of the IP to the HP form of cyt b₅₅₉ after the removal of molecular oxygen. These results indicate that conversion from the IP to the HP form of cyt b₅₅₉ proceeds via different mechanisms. Under illumination, conversion of the IP to the HP form of cyt b₅₅₉ depends primarily on the protonation of the histidine residue, whereas under anaerobic conditions, the conversion of the IP to the HP form of cyt b₅₅₉ is driven by higher hydrophobicity of the environment around the heme iron resulting from the absence of molecular oxygen.     

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.     

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.     

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.     

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.     

Axial ligands of chloroplast cytochrome b-559: identification and requirement for a heme-cross-linked polypeptide structure

Optical, resonance Raman, and electron paramagnetic resonance spectroscopies have been used to characterize the ligands and spin state of the chloroplast cytochrome b-559. The protein was isolated from both maize and spinach in a low-potential form. The spectroscopic data indicate that the heme iron in both ferric and ferrous cytochrome b-559 is in its low-spin state and ligated in its fifth and sixth coordination positions by histidine nitrogens. Electron paramagnetic resonance data for the purified spinach cytochrome are in good agreement with those determined by Bergstr?m and V?nng?rd [Bergstr?m, J., & V?nng?rd, T. (1982) Biochim. Biophys. Acta 682, 452-456] for a low-potential membrane-bound form of cytochrome b-559. The g values of high-potential cytochrome b-559 are shifted from those of its low-potential forms; this shift is interpreted as arising from a deviation of the planes of the two axial histidine imidazole rings from a parallel orientation. The model is consistent with the physical data and may also account for the facility with which cytochrome b-559 can be converted between low- and high-potential forms. Recent biochemical and molecular biological data [Widger, W. R., Cramer, W. A., Hermodson, M., Meyer, D., & Gullifor, M. (1984) J. Biol. Chem. 259, 3870-3876; Herrmann, R. G., Alt, J., Schiller, D., Cramer, W. A., & Widger, W. R. (1984) FEBS Lett. 179, 239-244] have shown that two polypeptides, one with 83 residues and a second with 39 residues, most likely constitute the protein of the cytochrome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Composition and function of cytochrome b559 in reaction centers of photosystem II of green plants

A review of a recent study of the spectral and thermodynamic properties of cytochrome b559 as well as of the electron transfer between b559 and photosystem II reaction center cofactors in isolated D1/D2/cytochrome b559 complex RC-2 is presented. Attention is paid to the existence of intermediary-potential (IP, +150 mV) and extra-low-potential (XLP, –45 mV) hemes located close to the acceptor (quinone) and donor (P680) sides of the reaction center cofactors, respectively. These hemes found in isolated RC-2 probably correspond to the high-potential and low-potential hemes in chloroplasts, respectively. The above location of the hemes is believed to allow the photoreduction of the XLP heme and photooxidation of the IP heme. The electron transfer between the two hemes is discussed in terms of the cyclic electron flow and possible involvement in water splitting.     

Reduction potentials of Rieske clusters: importance of the coupling between oxidation state and histidine protonation state

Rieske [2Fe-2S] clusters can be classified into two groups, depending on their reduction potentials. Typical high-potential Rieske proteins have pH-dependent reduction potentials between +350 and +150 mV at pH 7, and low-potential Rieske proteins have pH-independent potentials of around -150 mV at pH 7. The pH dependence of the former group is attributed to coupled deprotonation of the two histidine ligands. Protein-film voltammetry has been used to compare three Rieske proteins: the high-potential Rieske proteins from Rhodobacter sphaeroides (RsRp) and Thermus thermophilus (TtRp) and the low-potential Rieske ferredoxin from Burkholderia sp. strain LB400 (BphF). RsRp and TtRp differ because there is a cluster to serine hydrogen bond in RsRp, which raises its potential by 140 mV. BphF lacks five hydrogen bonds to the cluster and an adjacent disulfide bond. Voltammetry measurements between pH 3 and 14 reveal that all the proteins, including BphF, have pH-dependent reduction potentials with remarkably similar overall profiles. Relative to RsRp and TtRp, the potential versus pH curve of BphF is shifted to lower potential and higher pH, and the pK(a) values of the histidine ligands of the oxidized and reduced cluster are closer together. Therefore, in addition to simple electrostatic effects on E and pK(a), the reduction potentials of Rieske clusters are determined by the degree of coupling between cluster oxidation state and histidine protonation state. Implications for the mechanism of quinol oxidation at the Q(O) site of the cytochrome bc(1) and b(6)f complexes are discussed.     

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.     

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.     

pH-dependent photoreactions of the high- and low-potential forms of cytochrome b559 in spinach PS II-enriched membranes

Cytochrome b559 (Cyt b559) is a well-known intrinsic component of Photosystem II (PS II) reaction center in all photosynthetic oxygen-evolving organisms, but its physiological role remains unclear. This work reports the response of the two redox forms of Cyt b559 (i.e. the high- (HP) and low-potential (LP) forms) to inhibition of the donor or acceptor side of PS II. The photooxidation of HP Cyt b559 induced by red light at room temperature was pH-dependent under conditions in which electron flow from water was diminished. This photooxidation was observed only at pH values higher than 7.5. However, in the presence of 1 M CCCP, a limited oxidation of HP Cyt b559 was observed at acidic pH, At pH 8.5 and in the presence of the protonophore, this photooxidation of the HP form was accompanied by its partial transformation into the LP form. On the other hand, a partial photoreduction of LP Cyt b559 was induced by red light under aerobic conditions when electron transfer through the primary quinone acceptor Q_A was impaired by strong irradiation in the presence of DCMU. This photoreduction was enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreduction and photooxidation of Cyt b559 is described under inhibitory conditions using the same kind of membrane preparations. A model accommodating these findings is proposed.Abbreviations CCCP carbonylcyanide 3-chlorophenylhydrazone - Cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - DCMU dichlorophenyldimethylurea - E _m midpoint redox potential - HP and LP high- and low-potential forms of Cyt b559 - P680 primary donor - I_A acceptor side inhibition - I_D donor side inhibition - Pheo pheophytin - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Electron transfer from HiPIP to the photooxidized tetraheme cytochrome subunit of Allochromatium vinosum reaction center: new insights from site-directed mutagenesis and computational studies

The kinetics of electron transfer from reduced high-potential iron-sulfur protein (HiPIP) to the photooxidized tetraheme cytochrome c subunit (THC) bound to the photosynthetic reaction center (RC) from the purple sulfur bacterium Allochromatium vinosum were studied under controlled redox conditions by flash absorption spectroscopy. At ambient redox potential Eh = +200 mV, where only the high-potential (HP) hemes of the THC are reduced, the electron transfer from HiPIP to photooxidized HP heme(s) follows second-order kinetics with rate constant k = (4.2 +/- 0.2) 10(5) M(-1) s(-1) at low ionic strength. Upon increasing the ionic strength, k increases by a maximum factor of ca. 2 at 640 mM KCl. The role of Phe48, which lies on the external surface of HiPIP close to the [Fe4S4] cluster and presumably on the electron transfer pathway to cytochrome heme(s), was investigated by site-directed mutagenesis. Substitution of Phe48 with arginine, aspartate, and histidine completely prevents electron donation. Conversely, electron transfer is still observed upon substitution of Phe48 with tyrosine and tryptophan, although the rate is decreased by more than 1 order of magnitude. These results suggest that Phe48 is located on a key protein surface patch essential for efficient electron transfer, and that the presence of an aromatic hydrophobic residue on the putative electron-transfer pathway plays a critical role. This conclusion was supported by protein docking calculations, resulting in a structural model for the HiPIP-THC complex, which involves a docking site close to the LP heme farthest from the bacteriochlorophyll special pair.     

Interconversion of low- and high-potential forms of cytochrome b(559) in Tris-washed photosystem II membranes under aerobic and anaerobic conditions.

In this study, the reversible conversion between the high- (HP) and low-potential (LP) forms of Cytb(559) has been analyzed in Tris-washed photosystem II (PSII) enriched membranes. These samples are deprived of the Mn cluster of the water-oxidizing complex (WOC) and the extrinsic regulatory proteins. The results obtained by application of optical and EPR spectroscopy reveal that (i) under aerobic conditions, the vast majority of Cytb(559) exhibits a low midpoint potential, (ii) after removal of O(2) in the dark, a fraction of Cytb(559) is converted to the high-potential form which reaches level of about 25% of the total Cytb(559), (iii) a similar dark transformation of LP --> HP Cytb(559) occurs under reducing conditions (8 mM hydroquinone), (iv) under anaerobic conditions and in the presence of 8 mM hydroquinone, about 60% of the Cytb(559) attains the HP form, (v) the interconversion is reversible with the re-establishment of aerobic conditions, and (vi) aerobic and oxidizing conditions (2 mM ferricyanide or 0.5 mM potassium iridate) induce a decrease of the amount of the HP form, also showing that the conversion is reversible. This reversible interconversion between LP and HP Cytb(559) is not observed in PSII membrane fragments with an intact WOC. On the basis of these findings, the possibility is discussed that the O(2)-dependent conversion of Cytb(559) in PSII complexes lacking a functionally competent WOC is related to a protective role of Cytb(559) in photoinhibition and/or that it is involved in the regulation of the assembly of a competent water-oxidizing complex in PSII.     

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.