Superoxide oxidase and reductase activity of cytochrome b559 in photosystem II

This study provides evidence for the superoxide oxidase and the superoxide reductase activity of cytochrome b₅₅₉ (cyt b₅₅₉) in PSII. It is reported that in Tris-treated PSII membranes upon illumination, both the intermediate potential (IP) and the reduced high potential (HP^red) forms of cyt b₅₅₉ exhibit superoxide scavenging activity and interconversion between IP and HP^red form. When Tris-treated PSII membranes were illuminated in the presence of spin trap EMPO, the formation of superoxide anion radical (O₂⁻) was observed, as confirmed by EPR spin-trapping spectroscopy. The observations that the addition of enzymatic (superoxide dismutase) and non-enzymatic (cytochrome c, α-tocopherol and Trolox) O₂⁻ scavengers prevented the light-induced conversion of IP ↔ HP^red cyt b₅₅₉ confirmed that IP and HP^red cyt b₅₅₉ are reduced and oxidized by O₂⁻, respectively. Redox changes in cyt b₅₅₉ by an exogenous source of O₂⁻ reconfirmed the superoxide oxidase and reductase activity of cyt b₅₅₉. Furthermore, the light-induced conversion of IP to HP^red form of cyt b₅₅₉ was completely inhibited at pH > 8 and by chemical modification of the imidazole ring of histidine residues using diethyl pyrocarbonate. We proposed that a change in the environment around the heme iron, induced by the protonation and deprotonation of His²² residue generates a favorable condition for the oxidation and reduction of O₂⁻, respectively.     

ESR spectroscopy demonstrates that cytochrome b559 remains low potential in Ca2+-reactivated,salt-washed PSII particles

Cytochrome b₅₅₉ in various Photosystem II preparations was studled by using low temperature ESR spectroscopy. This technique was used because it is able to distinguish high from low potential forms of the cytochrome owing to the g-value differences between these species. Moreover, by using low temperature irradiation to oxidize cyt b₅₅₉ we have avoided the use of redox mediators. Previous work (Ghanotakis DF., Topper J.N. and Yocum, C.F. (1984) Biochim. Biophys. Acta 767, 524–531) demonstrated that reduction and extraction of manganese of the oxygen evolving complex, which might be expected to alter the redox properties of cyt b₅₅₉, occurs when certain PSII preparations are exposed to reductants. The ESR data presented here show that a mixture of high potential and lower potential cyt b₅₅₉ species is observed in the oxygen evolving Photosystem II complex. Treatment of PSII membranes with 0.8 M Tris converts the high potential form(s) to those of lower potential. Exposure of the membranes to 2M NaCl shifts a significant amount of high potential cyt b₅₅₉ to lower potential form(s); addition of CaCl₂ reconstituted oxygen evolution activity but did not restore cyt b₅₅₉ to its high potential form(s).Abbreviations Chl chlorophyll - cyt cytochrome - DCBQ 2,5-dichloro-benzoquinone - DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone - ESR electron spin resonance - OEC oxygen evolving complex - PS photosystem Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Identification and characterization of a cytochrome b559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions

We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 μM DCMU. This mutant carries an R7L mutation on the α-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 Å PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lα cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lα cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lα cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the α-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress.     

Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

The functional role of cytochrome (cyt) b₅₅₉ in photosystem II (PSII) was investigated in H22Kα and Y18Sα cyt b₅₅₉ mutants of the cyanobacterium Synechocystis sp. PCC6803. H22Kα and Y18Sα cyt b₅₅₉ mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b₅₅₉ in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b₅₅₉ in H22Kα mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b₅₅₉ in Y18Sα mutant PSII core complexes was shifted to 561 nm. Y18Sα and H22Kα mutant PSII core complexes contained predominately the low potential form of cyt b₅₅₉. The findings lend support to the concept that the redox properties of cyt b₅₅₉ are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b₅₅₉ mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b₅₅₉ in protection of PSII under photoinhibition conditions in vivo.     

Site-directed mutagenesis on the heme axial-ligands of cytochrome b559 in photosystem II by using cyanobacteria Synechocystis PCC 6803

Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the α or the β subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kα mutant grew photoautotrophically, and accumulated stable PSII reaction centers (∼ 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Yβ mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers (∼ 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559α and β polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559β polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YβPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YβPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YβPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kα mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kα mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kα and H22YβPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII.     

Histidine residue 252 of the Photosystem II D1 polypeptide is involved in a light-induced cross-linking of the polypeptide with the α subunit of cytochrome b-559: study of a site-directed mutant of Synechocystis PCC 6803

Properties of the Photosystem II (PSII) complex were examined in the wild-type (control) strain of the cyanobacterium Synechocystis PCC 6803 and its site-directed mutant D1-His252Leu in which the histidine residue 252 of the D1 polypeptide was replaced by leucine. This mutation caused a severe blockage of electron transfer between the PSII electron acceptors Q_A and Q_B and largely inhibited PSII oxygen evolving activity. Strong illumination induced formation of a D1-cytochrome b-559 adduct in isolated, detergent-solubilized thylakoid membranes from the control but not the mutant strain. The light-induced generation of the adduct was suppressed after prior modification of thylakoid proteins either with the histidine modifier platinum-terpyridine-chloride or with primary amino group modifiers. Anaerobic conditions and the presence of radical scavengers also inhibited the appearance of the adduct. The data suggest that the D1-cytochrome adduct is the product of a reaction between the oxidized residue His²⁵² of the D1 polypeptide and the N-terminal amino group of the cytochrome α subunit. As the rate of the D1 degradation in the control and mutant strains is similar, formation of the adduct does not seem to represent a required intermediary step in the D1 degradation pathway.     

Flash-induced reduction of cytochrome b-559 by Q infB sup− in chloroplasts in the presence of protonophores

Flash-induced, fast (t _1/2 1 ms), reversible reduction of the high potential cytochrome b-559 (cyt b-559HP) was observed in chloroplasts in the presence of 2 M protonophore, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), CCCP (carbonylcyanide 3-chlorophenylhydrazone) or SF 6847 (2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)phenol). These protonophores promote autooxidation of cyt b-559HP in the dark (Arnon and Tang 1988, Proc Natl Acad Sci USA 85: 9524). No fast photoreduction could, however, be observed if the molecules were oxidized with ferricyanide in the absence of protonophores. This suggests that the molecules must be deprotonated to be capable for fast photoreduction.Photoreduction of cyt b-559HP was largely insensitive to DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), but was inhibited by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). With a train of flashes, no oscillation could be observed in the amplitudes of photoreduction. These data strongly suggest that cyt b-559HP is reduced by the semireduced secondary quinone acceptor (Q_B ^–) of Photosystem 2.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis - Ant 2p- 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - cyt- cyto-chrome - CCCP- carbonylcyanide 3-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl-6-iso-propyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimehtylurea - FCCP- carbonylcyanide p-trifluoromethoxyphenylhydrazone - FeCy- ferricyanide - HP- high potential form - HQ- hydroquinone - PQ- plastoquinone - PS 2- Photosystem 2 - SF 6847- 2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)-phenol     

His92 and His110 selectively affect different heme centers of adrenal cytochrome b561

Adrenal cytochrome b₅₆₁ (cyt b₅₆₁), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b₅₆₁ (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31-40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (b_H) peak were seen with mutation of His92; the largest changes in the low-potential (b_L) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g = 3.1 signal (b_H) but not the g = 3.7 signal (b_L). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the b_H transition; mutations in His110 produced the largest decreases in ΔA₅₆₁ for the b_L transition. These results indicate that His92 can be considered part of the b_H heme center, and His110 part of the b_L heme center, in adrenal cyt b₅₆₁.     

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.     

Characterisation of a H2O2-oxidisable cytochrome b-559 in intact chloroplasts with a new type of LED Array Spectrophotometer

Cytochrome (cyt) b-559 absorbance changes in intact chloroplasts were deconvoluted using a previously described LED-Array-Spectrophotometer (Klughammer et al. (1990), Photosynth Res 25: 317–327). When intact chloroplasts were isolated in the presence of ascorbate, approx. 15% of the total cyt b-559 could be transiently oxidised by 200 M H₂O₂ in the dark. This fraction displays low-potential properties, as it can be also oxidised by menadione in the presence of 5 mM ascorbate. Heat pretreatment increased the size of this fraction by a factor of 3–4. Low concentrations of cyanide (in the M range) prolonged the oxidation time while high concentrations suppressed the oxidation (I₅₀=1.5 mM KCN). The former KCN-effect relates to inhibition of ascorbate dependent H₂O₂-reduction which is catalysed by ascorbate peroxidase, whereas the latter effect reflects competition between H₂O₂ and CN^– for the same binding site at the cytochrome heme. In the light, much lower concentrations of H₂O₂ were required to obtain oxidation, the amplitude depending on light intensity and on the concentration of the added H₂O₂, but never exceeding approx. 15% of the total cyt b-559. In the light, but not in the dark, H₂O₂ also induced the transient oxidation of a cyt f fraction similar in size to the H₂O₂-oxidisable cyt b-559 fraction. In this case, H₂O₂ serves as an acceptor of Photosystem I in conjunction with the ascorbate peroxidase detoxification system. Light can also induce oxidation of a 15% cyt b-559 fraction without H₂O₂-addition, if nitrite is present as electron acceptor and the chloroplasts are depleted of ascorbate. It is concluded that light-induced cyt b-559 oxidation in vivo is likely to be restricted to the H₂O₂-oxidisable cyt b-559 LP fraction and is normally counteracted by ascorbate.Abbreviations APX ascorbate peroxidase - chl chlorophyll - cyt cytochrome - HP high potential - LP low potential - MDA monodehydroascorbate - PQ plastoquinone - PS I and PS II Photosystems I and II     

Restoration of the high potential form of cytochrome b-559 through the photoreactivation of Tris-inactivated oxygen-evolving center

Restoration of a high potential (HP) form of cytochrome b-559 (Cyt b-559) from a low potential (LP) form was the primary process in the reconstitution of O₂-evolving center during the photoreactivation of Tris-inactivated chloroplasts. In normal chloroplasts, about 0.5 to 0.7 mol of Cyt b-559 was present in the HP form per 400 chlorophyll molecules. However, the HP form was converted to the LP form when the O₂-evolving center was inactivated by 0.8 M alkaline Tris-washing (pH 9.1). The inactivation was reversible and both the Cyt b-559 HP form and the O₂-evolving activity were restored by incubating the inactivated chloroplasts with weak light, Mn²⁺, Ca²⁺ and an electron donor (photoreactivation). The recovery of the HP form preceded the recovery of O₂-evolving activity. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) did not inhibit the recovery of the HP form. Thus, the recovery of Cyt b-559 HP form was the primary reaction in the photoreactivation, which was stimulated by the light-induced redox reaction of the PS-II core center.Abbreviations ASC ascorbate - BSA bovine serum albumin - Chl chlorophyll - Cyt b-559 HP form high potential form of cytochrome b-559 - Cyt b-559 LP form low potential form of cytochrome b-559 - Cyt b-559 VLP form very low potential form of cytochrome b-559 - Cyt f cytochrome f - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - HQ hydroquinone - SHN chloroplast-preparation medium containing 0.4 M sucrose, 50 mM Hepes-Na (pH 7.8) and 20 mM NaCl - PS-II Photosystem II     

Photo-catalytic oxidation of a di-nuclear manganese centre in an engineered bacterioferritin ‘reaction centre’

Photosynthesis involves the conversion of light into chemical energy through a series of electron transfer reactions within membrane-bound pigment/protein complexes. The Photosystem II (PSII) complex in plants, algae and cyanobacteria catalyse the oxidation of water to molecular O₂. The complexity of PSII has thus far limited attempts to chemically replicate its function. Here we introduce a reverse engineering approach to build a simple, light-driven photo-catalyst based on the organization and function of the donor side of the PSII reaction centre. We have used bacterioferritin (BFR) (cytochrome b1) from Escherichia coli as the protein scaffold since it has several, inherently useful design features for engineering light-driven electron transport. Among these are: (i.) a di-iron binding site; (ii.) a potentially redox-active tyrosine residue; and (iii.) the ability to dimerise and form an inter-protein heme binding pocket within electron tunnelling distance of the di-iron binding site. Upon replacing the heme with the photoactive zinc-chlorin e₆ (ZnCe₆) molecule and the di-iron binding site with two manganese ions, we show that the two Mn ions bind as a weakly coupled di-nuclear Mn₂^II,II centre, and that ZnCe₆ binds in stoichiometric amounts of 1:2 with respect to the dimeric form of BFR. Upon illumination the bound ZnCe₆ initiates electron transfer, followed by oxidation of the di-nuclear Mn centre possibly via one of the inherent tyrosine residues in the vicinity of the Mn cluster. The light dependent loss of the Mn^II EPR signals and the formation of low field parallel mode Mn EPR signals are attributed to the formation of Mn^III species. The formation of the Mn^III is concomitant with consumption of oxygen. Our model is the first artificial reaction centre developed for the photo-catalytic oxidation of a di-metal site within a protein matrix which potentially mimics water oxidation centre (WOC) photo-assembly.     

The role of the high potential form of the cytochrome b559: Study of Thermosynechococcus elongatus mutants

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 Q_B). 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 Q_B 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.     

Mutation of Residue Arginine^18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the α-subunit of cytochrome bsss （Cyt bsss） except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem （PS） Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSll complexes. In the present study, two arginine18 （R18） mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid （E） and glycine （G）. The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type （WT） levels and that, after irradiation at high light intensity, oxygen evolution for the PSll of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSll （Fv/Fm） of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the α-subunit of Cyt bsss and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt bss9 maintain the structure of the PSll complex and its activity, although it is not directly bound to the heme group.     

PCR-RFLP analysis of mitochondrial DNA cytochrome b gene among Haruan (Channa striatus) in Malaysia

Haruan (Channa striatus) is in great demand in the Malaysian domestic fish market. In the present study, mtDNA cyt b was used to investigate genetic variation of C. striatus among populations in Peninsular Malaysia. The overall population of C. striatus demonstrated a high level of haplotype diversity (h) and a low-to-moderate level of nucleotide diversity (π). Analysis of molecular variance (AMOVA) results showed a significantly different genetic differentiation among 6 populations (F_ST = 0.37566, P = 0.01). Gene flow (Nm) was high and ranged from 0.32469 to infinity (∞). No significant relationship between genetic distance and geographic distance was detected. A UPGMA tree based on the distance matrix of net interpopulation nucleotide divergence (d_A) and haplotype network of mtDNA cyt b revealed that C. striatus is divided into 2 major clades. The neutrality and mismatch distribution tests for all populations suggested that C. striatus in the study areas had undergone population expansion. The estimated time of population expansion in the mtDNA cyt b of C. striatus populations occurred 0.72-6.19 million years ago. Genetic diversity of mtDNA cyt b and population structure among Haruan populations in Peninsular Malaysia will be useful in fisheries management for standardization for Good Agriculture Practices (GAP) in fish-farming technology, as well as providing the basis for Good Manufacturing Practices (GMP).     

Spectral and kinetic resolution of the bc1 complex components in situ: A simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc₁ complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c_tot (cyt c₁ + cyt c₂), cyt b_L, cyt b_H, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c₁ and c₂ is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c₁, c₂, b_L, and b_H) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc₁ complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc₁ complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.     

Multiple step assembly of the transmembrane cytochrome b6

We have analyzed the role of individual heme-ligating histidine residues for assembly of holo-cytochrome b₆, and we show that the two hemes b_L and b_H bind in two subsequent steps to the apo-protein. Binding of the low-potential heme b_L is a prerequisite for binding the high-potential heme b_H. After substitution of His86, which serves as an axial ligand for heme b_L, the apo-protein did not bind heme, while substitution of the heme b_L-ligating residue His187 still allowed binding of both hemes. Similarly, after replacement of His202, one axial ligand to heme b_H, binding of only heme b_L was observed, whereas replacement of His100, the other heme b_H ligand, resulted in binding of both hemes. These data indicate sequential heme binding during formation of the holo-cytochrome, and the two histidine residues, which serve as axial ligands to the same heme molecule (heme b_L or heme b_H), have different importance during heme binding and cytochrome assembly. Furthermore, determination of the heme midpoint potentials of the various cytochrome b₆ variants indicates a cooperative adjustment of the heme midpoint potentials in cytochrome b₆.     

High-light induced singlet oxygen formation in cytochrome b6f complex from Bryopsis corticulans as detected by EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen (¹O₂*) in the intact and the Rieske-depleted cytochrome b₆f complexes (Cyt b₆f) from Bryopsis corticulans, as well as in the isolated Rieske Fe–S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b₆f can be bleached by light-induced ¹O₂*, and that the ¹O₂* production can be promoted by D₂O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, β-carotene and glutathione. Under similar experimental conditions, ¹O₂* was also detected in the Rieske-depleted Cyt b₆f complex, but not in the isolated Rieske Fe–S protein. The results prove that Chl a cofactor, rather than Rieske Fe–S protein, is the specific site of ¹O₂* formation, a conclusion which draws further support from the generation of ¹O₂* with selective excitation of Chl a using monocolor red light.     

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.     

pH sensitivity of the redox state of cytochrome b559 may regulate its function as a protectant against donor and acceptor side photoinhibition

A series of experiments have been conducted with isolated reaction centers of photosystem two (PS II) with the aim to elucidate the functional role of cytochrome (Cyt b ₅₅₉). At pH 6.5 it was found that Cyt b ₅₅₉ was reversibly photoreduced by red actinic light when Mn²⁺ was present as an electron donor while at pH 8.5 a photo-oxidation was observed under the same lighting conditions, which was dark reversible in the presence of hydroquinone. These pH dependent light induced changes were measured under anaerobic conditions and correlated with changes in the relative levels of high (HP) and low (LP) potential forms of the cytochrome. At pH 6.5 the cytochrome was mainly in its LP form while at pH 8.5 a significant proportion was converted to the HP form as detected by dark titrations with hydroquinone. This pH dependent difference in the levels of HP and LP Cyt b ₅₅₉ was also detected when bright white light was used to monitor the level of the LP form using a novel reaction involving direct electron donation from the flavin of glucose oxidase (present in the medium and used together with glucose and catalase as an oxygen trap). The results suggest that PS II directly oxidises and reduces the HP and LP forms, respectively and that the extent of these photo-reactions is dependent on the relative levels of the two forms, which are in turn governed by the pH. This conclusion is interpreted in terms of the model presented previously (Barber J and De Las Rivas J (1993) Proc Natl Acad Sci USA 90: 10942–10946) whereby the pH induced effect is considered as a possible mechanism by which interconversion of LP and HP forms of Cyt b ₅₅₉ is achieved. In agreement with this was the finding that as the extent of photo-oxidisable HPCyt b ₅₅₉ increases, with increasing pH, the rate of irreversible photo-oxidation of -carotene decreases, a result expected if the HP form protects against donor side photoinhibition.Abbreviations -car -carotene - CCCP carbonylcyanide m-chloro-phenylhydrazone - Chl chlorophyll - Cyt b ₅₅₉ cytochrome b ₅₅₉ - HPCyt b ₅₅₉ high potential form of cytochrome b ₅₅₉ which is reducible by hydroquinone - LPCyt b ₅₅₉ low potential form of cytochrome b ₅₅₉ which is non-reducible by hydroquinone - D1 and D2 products of the psbA and psbD genes, respectively - LHC II light-harvesting chlorophyll protein complex associated with PS II - Mes 2-(N-morpholino) ethanesulphonic acid - P680 primary electron donor of PS II - Pheo pheophytin - PQ plastoquinone - PS II Photosystem II - Q_A first stable quinone electron acceptor of PS II - Q_B second stable quinone electron acceptor of PS II - RC reaction center - SDS sodium dodecyl sulphate - SiMo silicomolybdate - Tris tris(hydroxymethyl) amino methane - Y_Z and Y_D tyrosine residues 161 in D1 and D2 proteins of the PS II RC which act as secondary electron donors to P680