An ascorbate-reducible cytochrome b561 is localized in macrophage lysosomes

Cytochromes b561 (Cyts b561) are a family of intrinsic membrane proteins involved in ascorbate-mediated transmembrane electron transport. The chromaffin granule Cyt b561 (CGCytb) is believed to transport electrons donated by extravesicular ascorbate (ASC) across the membrane to intravesicular monodehydroascorbate (MDA) supporting catecholamine synthesis in neuroendocrine tissues. Another isoform, the duodenal Cyt b561 (Dcytb), was reported to have ferric reductase activity, possibly facilitating intestinal iron uptake. Herein, a new Cyt b561 homologue, LCytb (for lysosomal Cytb561) was found expressed in the late endosomal-lysosomal membrane. LCytb shared high sequence similarity with CGCytb (45% identity) and Dcytb (42% identity). Moreover, four heme-coordinating His residues, and putative ASC and MDA binding sites were highly conserved. Recombinant LCytb exhibited an ASC-reducible b-type Cyt absorbance spectrum with alpha-band maximum at 561 nm in the spectrum of the reduced protein. Northern blots and Western blots revealed that LCytb was predominantly expressed in lung, spleen, thymus, testis and placenta. In situ hybridization and immunofluorescence studies further demonstrated that the protein was expressed in the alveolar macrophages of the lung, in the white pulp of the spleen, widespread in the thymus, and in the Sertoli cells of the testis. Sequence analysis indicated the presence of a (DE)XXXL(LI)-type signal in the C-terminal of the protein, predicting a late endosomal-lysosomal subcellular localization. This localization was confirmed by double labeling experiments in RAW264.7 and 293 cells, stably transfected with LCytb.     

Human erythrocyte membranes contain a cytochrome b561 that may be involved in extracellular ascorbate recycling

Human erythrocytes contain an unidentified plasma membrane redox system that can reduce extracellular monodehydroascorbate by using intracellular ascorbate (Asc) as an electron donor. Here we show that human erythrocyte membranes contain a cytochrome b(561) (Cyt b(561)) and hypothesize that it may be responsible for this activity. Of three evolutionarily closely related Cyts b(561), immunoblots of human erythrocyte membranes showed only the duodenal cytochrome b(561) (DCytb) isoform. DCytb was also found in guinea pig erythrocyte membranes but not in erythrocyte membranes from the mouse or rat. Mouse erythrocytes lost a majority of the DCytb in the late erythroblast stage during erythropoiesis. Absorption spectroscopy showed that human erythrocyte membranes contain an Asc-reducible b-type Cyt having the same spectral characteristics as recombinant DCytb and biphasic reduction kinetics, similar to those of the chromaffin granule Cyt b(561). In contrast, mouse erythrocytes did not exhibit Asc-reducible b-type Cyt activity. Furthermore, in contrast to mouse erythrocytes, human erythrocytes much more effectively preserved extracellular Asc and transferred electrons from intracellular Asc to extracellular ferricyanide. These results suggest that the DCytb present in human erythrocytes may contribute to their ability to reduce extracellular monodehydroascorbate.     

Heterologous expression and site-directed mutagenesis of an ascorbate-reducible cytochrome b561

Cytochromes b561 (Cyts b561) are ubiquitous membrane proteins catalyzing ascorbate-mediated trans-membrane electron transfer. A heterologous expression system in Saccharomyces cerevisiae was developed to study their structure-function relationship. Recombinant mouse chromaffin granule Cyt b561 (CGCytb) shows spectral characteristics, ascorbate reducibility, and redox potentials identical to that of the native bovine protein. Moreover, the reconstituted recombinant protein mediated trans-membrane electron transport with kinetic characteristics similar to that of bovine CGCytb. Site-directed mutant analysis supports the presence of two hemes coordinated by the highly conserved His pairs H52/H120 and H86/H159. Reduction of CGCytb by ascorbate showed biphasic kinetics (Kd1: 0.016 +/- 0.005 mM, Kd2: 1.24 +/- 0.19 mM). Mutation of a well-conserved Arg residue (R72) abolished high affinity CGCytb reduction by ascorbate, indicating that this residue may be critical for substrate binding. On the other hand, mutation of a Lys previously suggested to play a role in ascorbate binding (K83), did not affect the ascorbate-mediated reduction of the protein.     

An Arabidopsis cytochrome b561 with trans-membrane ferrireductase capability

Ascorbate-reducible cytochromes b561 (Cyts-b561) are a class of intrinsic trans-membrane proteins. Tonoplast Cyt-b561 (TCytb), one of the four Cyt-b561 isoforms in Arabidopsis was localized to the tonoplast. We demonstrate here that the optical spectra, EPR spectra and redox potentials of recombinant TCytb are similar to those of the well characterized bovine chromaffin granule Cyt-b561. We provide evidence for the reduction of ferric-chelates by the reduced TCytb. It is also shown that TCytb is capable of trans-membrane electron transport from intracellular ascorbate to extracellular ferric-chelates in yeast cells.     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b(561) enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b(561) proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b(561)-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b(561) paralogs from Arabidopsis thaliana (Acytb(561)-A, Acytb(561)-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb(561)-A resembles the best characterised member of the CYBASC family, the cytochrome b(561) from adrenomedullary chromaffin vesicles, and that Acytb(561)-B is atypical compared to other CYBASC proteins. Haem oxidation-reduction midpoint potential (E(M)) values were found to be fully consistent with ascorbate oxidation activities and Fe(3+)-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b(561) from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E(M) values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E(M) values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe(3+)-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E(M) values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b(561) paralogs exist as homodimers.     

Chromaffin granule membranes contain at least three heme centers: direct evidence from EPR and absorption spectroscopy

Low-temperature electron paramagnetic resonance (EPR) spectroscopy, circular dichroism and two-component redox titration have previously provided evidence for two different ascorbate-reducible heme centers in cytochrome b(561) present in chromaffin granule membranes. These species have now been observed by room and liquid nitrogen temperature absorption spectroscopy. The visualization of these heme centers becomes possible as a consequence of utilizing chromaffin granule membranes prepared by a mild procedure. Additionally, a new redox center, not reducible by ascorbate, was discovered by both EPR and absorption spectroscopy. It constitutes about 15% of the heme absorbance of chromaffin membranes at 561 nm and has EPR characteristics of a well-organized highly axial low-spin heme center (thus making it unlikely that it is a denatured species). This species is either an alternative form of one of the hemes of cytochrome b(561) that has a very low redox potential or a b-type cytochrome distinct from b(561).     

Photosynthetic membrane development in Rhodopseudomonas sphaeroides. Spectral and kinetic characterization of redox components of light-driven electron flow in apparent photosynthetic membrane growth initiation sites

The kinetics of light-driven electron flow and the nature of redox centers at apparent photosynthetic membrane growth initiation sites in Rhodopseudomans sphaeroides were compared to those of intracytoplasmic photosynthetic membranes. In sucrose gradients, these membrane growth sites sediment more slowly than intracytoplasmic membrane-derived chromatophores and form an upper pigmented band. Cytochromes c1, c2, b561, and b566 were demonstrated in the upper fraction by redox potentiometry; c-type cytochromes were also detected electrophoretically. Signals characteristic of light-induced reaction center bacteriochlorophyll triplet and photooxidized reaction center bacteriochlorophyll dimer states were observed by EPR spectroscopy but the Rieske iron-sulfur signal of the ubiquinol-cytochrome c2 oxidoreductase was present at a 3-fold reduced level on a reaction center basis in comparison to chromatophores. Flash-induced absorbance measurements of the upper pigmented fraction demonstrated reaction center primary and secondary semiquinone anion acceptor signals, but cytochrome b561 photoreduction and cytochrome c1/c2 reactions occurred at slow rates. This fraction was enriched approximately 2- and 4-fold in total b- and c-type cytochromes, respectively, per reaction center over chromatophores, but photoreducible b-type cytochrome was lower. Measurements of respiratory activity indicated a 1.6-fold higher level of succinate-cytochrome c oxidoreductase/reaction center than in chromatophores, but the apparent turnover rates in both preparations were low. Overall, the results suggest that complete cycles of rapid, light-driven electron flow do not occur merely by introduction of newly synthesized reaction centers into respiratory membrane, but that subsequent synthesis and assembly of appropriate components of the ubiquinol-cytochrome c2 oxidoreductase is required.     

Development of a bacterial system for high yield expression of fully functional adrenal cytochrome b561

Adrenal cytochrome b561 (cyt b561) is the prototypical member of an emerging family of proteins that are distributed widely in vertebrate, invertebrate and plant tissues. The adrenal cytochrome is an integral membrane protein with two b-type hemes and six predicted transmembrane helices. Adrenal cyt b561 is involved in catecholamine biosynthesis, shuttling reducing equivalents derived from ascorbate. We have developed an Escherichia coli system for expression, solubilization and purification of the adrenal cytochrome. The spectroscopic and redox properties of the purified recombinant protein expressed in this prokaryotic system confirm that the cytochrome retains a native, fully functional form over a wide pH range. Mass spectral analysis shows that the N-terminal signal peptide is intact. The new bacterial expression system for cyt b561 offers a sixfold improvement in yield and other substantial advantages over existing insect and yeast cell systems for producing the recombinant cytochrome for structure-function studies.     

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.     

Analysis of an Arabidopsis thaliana protein family, structurally related to cytochromes b561 and potentially involved in catecholamine biochemistry in plants

Cytochromes b561 (cyts b561) constitute a family of eukaryotic membrane proteins, catalysing ascorbate (Asc)-mediated trans-membrane electron transport, and hence likely involved in Asc regeneration. A class of proteins (DoH-CB) has been identified in plants and animals, containing the cyt b561 electron-transport domain (CB), combined with the catecholamine-binding regulatory domain of dopamine-beta-hydroxylase (DoH). A mammalian DoH-CB protein was previously reported to function as a cell-derived growth factor receptor (SDR2). We have performed an in silico analysis on DoH-CB proteins from Arabidopsis thaliana and demonstrate that structural features of both CB and DoH domains are well conserved. The combination of both domains may have evolved from a functional interaction between a cyt b561 and a DoH-containing protein, illustrating the so-called "Rosetta Stone" evolutionary principle, and this hypothesis is supported by sequence comparisons. DoH-CB proteins form a newly identified group of proteins, likely to play a key role in catecholamine action in plants. It is suggested that these proteins may function as trans-membrane electron shuttles, possibly regulated by catecholamines. The role and action of catecholamines in plants is poorly documented, but it is clear that they are involved in many aspects of growth and development. Whether the DoH-CB proteins functionally interact with Asc, as is the case for cyts b561, remains to be determined.     

The cytochrome b6f complex. Novel aspects

The Cyt b ₆ f complex from plant chloroplasts, the green alga Chlamydomonas reinhardtii , and the thermophilic cyanobacterium, Mastigocladus laminosus , can be isolated in a highly active state, in which it is dimeric and contains one bound chlorophyll a molecule per monomeric unit. The latter feature is a distinguishing trait compared to the b ₆ f complex of bacterial photosynthesis and the respiratory chain. In contrast to the trans-membrane domains of the b ₆ f complex, and of most other integral membrane proteins, which are characterized by an a -helical structure, the p -side peripheral domains, consisting of Cyt f and the Rieske protein, have a predominantly β-strand secondary structure motif. One consequence of this motif is an extension of these polypeptides from the membrane surface. For example, the length of Cyt f is 75 Å. The heme Fe is 45 Å from the α-carbon of Arg250 at the membrane bilayer interface and, even though Cyt f may be tilted relative to the membrane plane, the heme electron transfer reactions are carried out far from the membrane surface. The presence of an internal 5 water chain, which has the properties of a proton wire, with one water H-bonded to the histidine-25 heme ligand, also suggests that the pathway of long distance H⁺ translocation traverses the extended p -side protein domain of the b ₆ f complex. A mechanism of H⁺ transfer in the chain that is coupled to the redox state of the heme, in which a proton is transferred into the chain to compensate the extra electron in the ferro-heme, is proposed.     

The adaptation of the electron transfer chain of Rhodopseudomonas capsulata to different light intensities

(1) Cells of Rhodopseudomonas capsulata (wild-type) were grown photoheterotrophically in a turbidostat under very high and very low light intensity. Membranes were isolated from cells adapted to the respective light conditions and fractionated by sucrose density centrifugation. The molar ratios of ubiquinone and cytochromes c₂, c₁, b-561 and b-566 per reaction center were 3-fold to 5-fold higher in high-light than in low-light membranes. (2) Most of the Cyt(c₁ + c₂) and Cyt b-561 detected in dark redox titrations undergoes light-induced redox changes, both in high- and in low-light membranes. (3) The fractions of the total photooxidizable reaction center and Cyt(c₁ + c₂) oxidized under continuous light in the absence of antimycin are higher in membranes from low-light- than from high-light-grown cells. (4) From these data and results of kinetic studies it is proposed that cyclic electron flow under saturating light intensities is faster in high-light-grown cells.     

细胞色素c蛋白序列分析与结构比较

首先以马心细胞色素c(Horse Cytc)蛋白的氨基酸序列为查询序列,利用生物信息学方法进行相似性搜索,获得了一系列细胞色素c(Cytc)蛋白的氨基酸序列,然后对Cytc蛋白进行了多重对齐分析、进化分析和三维结构比较分析。分析结果表明:Cytc中某些特定部位的氨基酸残基高度保守;相近物种来源的Cytc具有较近的亲缘关系,而来源于同一物种不同部位的Cytc却具有较远的亲缘关系;来源于不同物种的Cytc,即使具有较远的亲缘关系,却具有极其相似的三维空间结构。这些研究结果将为基于Cytc进行蛋白分子设计与构建提供指导意义。     

Cytochrome b561 spectral changes associated with electron transfer in chromaffin-vesicle ghosts

The involvement of cytochrome b561, an integral membrane protein, in electron transfer across chromaffin-vesicle membranes is confirmed by changes in its redox state observed as changes in the absorption spectrum occurring during electron transfer. In ascorbate-loaded chromaffin-vesicle ghosts, cytochrome b561 is nearly completely reduced and exhibits an absorption maximum at 561 nm. When ferricyanide is added to a suspension of these ghosts, the cytochrome becomes oxidized as indicated by the disappearance of the 561 nm absorption. If a small amount of ferricyanide is added, it becomes completely reduced by electron transfer from intravesicular ascorbate. When this happens, cytochrome b561 returns to its reduced state. If an excess of ferricyanide is added, the intravesicular ascorbate becomes exhausted and the cytochrome b561 remains oxidized. The spectrum of these absorbance changes correlates with the difference spectrum (reduced-oxidized) of cytochrome b561. Cytochrome b561 becomes transiently oxidized when ascorbate oxidase is added to a suspension of ascorbate-loaded ghosts. Since dehydroascorbate does not oxidize cytochrome b561, it is likely that oxidation is caused by semidehydroascorbate generated by ascorbate oxidase acting on free ascorbate. This suggests that cytochrome b561 can reduce semidehydroascorbate and supports the hypothesis that the function of cytochrome b561 in vivo is to transfer electrons into chromaffin vesicles to reduce internal semidehydroascorbate to ascorbate.     

以谷胱甘肽为电子供体的细胞膜氧化还原系统

内载谷胱甘肽(GSH)的大豆(Glycine max L.)下胚轴正向型质膜囊泡具有以GSH为电子供体的跨膜电子传递活性,能还原膜外电子受体FeCN和细胞色素(Cyt)C,其还原速率分别为(21.6±0.6)nmolFeCN·min~(-1)·mg~(-1)蛋白和(6.6±1.0)nmol Cyt C·min~(-1)·mg~(-1)蛋白。这种跨膜电子传递能引起膜上Cyt P-450吸收光谱标志带(Soret带)的变化,表明Cyt P-450参与了这一氧化还原过程。在跨质膜电子传递的同时伴随着H~ 运输和膜电位的改变。     

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.     

Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.     

Demonstration of a collisional interaction of ubiquinol with the ubiquinol-cytochrome c2 oxidoreductase complex in chromatophores from Rhodobacter sphaeroides

Ubiquinone-10 can be extracted from lyophilized chromatophores of Rhodobacter sphaeroides (previously called Rhodopseudomonas sphaeroides) without significant losses in other components of the electron-transfer chain or irreversible damages in the membrane structure. The pool of ubiquinone can be restored with exogenous UQ-10 to sizes larger than the ones in unextracted membranes. The decrease in the pool size has marked effects on the kinetics of reduction of cytochrome b-561 induced by a single flash of light and measured in the presence of antimycin. The initial rate of reduction, which in unextracted preparations increases on reduction of the suspension over the Eh range between 170 and 100 mV (pH 7), is also stimulated in partially UQ-depleted membranes, although at more negative Eh's. When the UQ pool is completely extracted the rate of cytochrome (Cyt) b-561 reduction is low and unaffected by the redox potential. In membranes enriched in UQ-10 above the physiological level the titration curve of the rate of Cyt b-561 reduction is displaced to Eh values more positive than in controls. This effect is saturated when the size of the UQ pool is about 2-3 times larger than the native one. The reduction of Cyt b-561 always occurs a short time after the flash is fired; also the duration of this lag is dependent on Eh and on the size of the UQ pool. A decrease or an increase in the pool size causes a displacement of the titration curve of the lag to more negative or to more positive Eh's, respectively. Similarly, the lag becomes Eh independent and markedly longer than in controls when the pool is completely extracted. These results demonstrate that the rate of turnover of the ubiquinol oxidizing site in the b-c1 complex depends on the actual concentration of ubiquinol present in the membrane and that ubiquinol from the pool is oxidized at this site with a collisional mechanism. Kinetic analysis of the data indicates that this reaction obeys a Michaelis-Menten type equation, with a Km of 3-5 ubiquinol molecules per reaction center.     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b₅₆₁ proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b₅₆₁-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b₅₆₁ paralogs from Arabidopsis thaliana (Acytb₅₆₁-A, Acytb₅₆₁-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb₅₆₁-A resembles the best characterised member of the CYBASC family, the cytochrome b₅₆₁ from adrenomedullary chromaffin vesicles, and that Acytb₅₆₁-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (E_M) values were found to be fully consistent with ascorbate oxidation activities and Fe^3 +-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b₅₆₁ from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E_M values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E_M values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe^3 +-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E_M values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b₅₆₁ paralogs exist as homodimers.     

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 Deltavarepsilon in the alpha-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 alpha-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.