Light-induced redox changes of cytochrome b-559

Dark incubation of spinach or pea chloroplasts with 10 μm carbonylcyanide m-chlorophenylhydrazone (CCCP) had a negligible effect either on the redox state or the redox potential of the high potential form of cytochrome b-559 (cytochrome b-559_hp). A similar result was obtained with spinach chloroplasts on incubation with 3.3 μm carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), but pea chloroplasts showed a decrease of 10–20% in the amount of reduced cytochrome b-559.Light-induced redox changes of cytochrome b-559 were not observed in untreated spinach chloroplasts. In the presence of CCP or FCCP, cytochrome b-559 was photooxidized both in 655 nm actinic light and in far-red light. Addition of the plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) to CCCP- or FCCP-treated chloroplasts had only a small effect on the photooxidation of cytochrome b-559 in 655 light, but it completely inhibited the oxidation in far-red light.Electron flow from water to 2,3′,6-trichlorophenolindophenol was partly inhibited by CCCP or FCCP, but the degree of inhibition does not appear to be sufficient to account for the photooxidation of cytochrome b-559.The photooxidation of cytochrome b-559 by 655 nm light at liquid nitrogen temperature was not influenced by prior treatment of the chloroplasts at room temperature with CCCP, DBMIB, or CCCP + DBMIB.The results cannot be explained by the presence of two independent pools of cytochrome b-559 in CCCP-treated chloroplasts, one photooxidized by Photosystem II and the other photooxidized by Photosystem I and photoreduced by Photosystem II.     

Purification of cytochrome b-245 from human neutrophils.

The low potential cytochrome b (b-245) of the microbicidal oxidase of phagocytic cells has been purified from neutrophils from patients with chronic myeloid leukaemia. Cells were homogenized in the presence of proteinase inhibitors and centrifuged to remove the cytoplasm. The pellets containing membranes, granules and other organelles (15 mg/ml) were then washed with buffered sodium cholate (5 mg/ml). Residual pellets were subsequently solubilized with the non-ionic detergent Triton N 101 (10 mg/ml) which extracted about 60% of the cytochrome b. About 10% of the cytochrome b was of mitochondrial origin which was removed on a column of n-amino-octyl-Sepharose that did not adsorb cytochrome b-245. Cytochrome b-245 was chromatographed on a column of heparin-agarose and eluted with NaCl to give a peak specific content of 11-16 nmol of cytochrome b-245/mg of protein, representing a 140-200-fold purification with a recovery of 15%. This technique results in the purification of approx. 100-150 nmol of highly purified cytochrome b-245 from (3-5) X 10(11) cells within 4 days. The most purified material gave a broad band with an apparent Mr of between 68 000 and 78 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, but gel filtration indicated an aggregated form of the protein in Triton N101 . Purified protein (14 nmol of haem/mg of protein) did not contain FAD or FMN and had no NADPH-dependent O2--generating activity.     

The enigmatic cytochrome b-559 of oxygenic photosynthesis

The ubiquitous and obligatory association of cytochrome b -559 with the photosystem II reaction center of oxygenic photosynthesis is a conundrum since it seems not to have a function in the primary electron transport pathway of oxygen evolution. A model for the cytochrome structure that satisfies the cis -positive rule for membrane protein assembly consists of two short, non-identical hydrophobic membrane-spanning polypeptides (α and β), each containing a single histidine residue, as ligands for the bridging heme prosthetic group that is on the side of the membrane opposite to the water splitting apparatus. The ability of the heterodimer, but not the single α-subunit, to satisfy the cis -positive rule implies that the cytochrome inserts into the membrane as a heterodimer, with some evidence implicating it as the first membrane inserted unit of the assembling reaction center. The very positive redox potential of the cytochrome can be explained by a position for the heme in a hydrophobic niche near the stromal aqueous interface where it is also influenced by the large positive dipole potential of the parallel α-helices of the cytochrome. The requirement for the cytochrome in oxygenic photosynthesis may be a consequence of the presence of the strongly oxidizing reaction center needed for H₂O-splitting. This may lead to the need, under conditions of stress or plastid development, for an alternate source of electrons when the H₂O-splitting system is not operative as a source of reductant for the reaction center.     

Concerning the enigmatic cytochrome b-559 of oxygenic photosynthesis

Photosynthesis Research - Although there is an extensive literature on the properties and possible electron transfer pathways of cytochrome b-559, which is a prominent subunit of the multi-subunit...     

On the structure and function of cytochrome b-559

A sumary of biochemical, biophysical, and molecular biological data is presented which led to the identification of two different polypeptides ( and , MW=9.16 and 4.27 kDa) in the cytochrome b-559 protein. The presence of a single His residue on each polypeptide, and the conclusion from spectroscopy that the heme coordination must be bis-histidine led to an obligatory requirement for coordination of a single heme through a heme cross-linked dimer. This structure does not have a precedent among soluble or membrane bound cytochromes. The possible participation of the cytochrome in the pathway of photoactivation is discussed.     

Presence of cytochrome b-245 in NADPH oxidase preparations from human neutrophils

The composition of NADPH oxidase purified by Red Sepharose chromatography of extracts from human neutrophil membranes was investigated. In contrast to that was recently reported by others, the enzyme isolated according to this procedure contained a high concentration of cytochrome b-245 and little FAD. The results reinforce the belief that cytochrome b-245 is a major component of the NADPH oxidase and plays a fundamental role in the formation of O2-by neutrophils.     

Phagocytosis-induced release of arachidonic acid from human neutrophils

The phospholipids of human neutrophils were labeled with [³H] arachidonic acid and [¹⁴C] palmitic acid. Phagocytosis of opsonized zymosan resulted in rapid release of free arachidonic acid but not of palmitic acid. Arachidonic acid was not released when the cells were exposed to unopsonized zymosan, zymosan-activated serum, or phorbol myristate acetate. These observations suggest that phagocytosis of opsonized zymosan results in the activation of a phospholipase A₂.     

Lumen-side topography of the alpha-subunit of the chloroplast cytochrome b-559

Removal of the extrinsic 33 kDa polypeptide increased the accessibility to trypsin of a COOH-terminal tridecapeptide epitope of the alpha subunit of cytochrome b-559 (psbE gene product). The sensitivity of the cytochrome epitope to trypsin was not measurably affected by removal of the 16 and 23 kDa extrinsic polypeptides, nor increased by removal of the OEC manganese along with the 33 kDa protein. While protecting alpha-cytochrome b-559 against trypsin, the 33 kDa protein is also proteolyzed, suggesting the possibility of an additional protein component involved in the shielding of the cytochrome. Shielding of the COOH-terminal epitope of alpha-cytochrome b-559 by the OEC 33 kDa protein implies that these COOH-terminal chains of the cytochrome are part of a protein network in the lumen space near the photosystem II reaction center. This network may contain residues that are involved in the binding of essential OEC metal ions.     

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.     

Evidence for a hetero-oligomeric structure of the chloroplast cytochrome b-559

A second nonhomologous polypeptide in the thylakoid membrane cytochrome b-559 has been suggested by the finding of a smaller reading frame just slightly downstream from that corresponding to the 9 kDa cytochrome polypeptide that is dominant on a Coomassie-stained gel. This reading frame encoded a 39-residue polypeptide that was similar in having a central hydrophobic domain of 25–26 residues and a single His residue at the same position in the hydrophobic domain. The smallest polypeptide seen on SDS gels of the cytochrome was isolated by high-performance liquid chromatography (HPLC). The NH₂-terminal sequence matched that of the downstream gene. The stoichiometry of the 2 gene products separated by HPLC was approx. 1:1, based on the molecular masses of 9.16 and 4.27 kDa calculated from the nucleotide sequence. It is concluded that the cytochrome contains both the 9.16 kDa (α) and 4.27 kDa (β) polypeptides. These data, the single His residue on each polypeptide, and the previous finding of a bis-histidine coordination, imply that the unit heme binding structure of the cytochrome is a heme cross-linked dimer. If the cytochrome contains a single heme, the dimer structure would be (αβ). If there are 2 hemes/cytochrome, the more likely structure would be (αβ)₂, a tetramer consisting of 2 heme cross-linked hetero-dimers.     

叶绿体中的细胞色素b-559

细胞色素b-559是由叶绿体基因编码α、β亚基为单位构成的一种血红素蛋白,是光系统II反应中心的重要组分。以叶绿体为实验材料的研究表明,细胞色素b-559可通过氧化还原变化调节光系统II的光抑制敏感性,并对发生在供体侧和受体侧抑制的光系统II反应中心具有保护作用,但对整体植物在生理条件下的作用却未得到证实,这也正是今后需要研究的问题。     

叶绿体中的细胞色素b—559

细胞色素b-559是由叶绿体基因编码α,β亚基为单位构成的一种血红蛋白,是光系统II反应中心的重要组分。以叶绿体为实验材料的研究表明,细胞色素b-559可通过还原变化调节光系统Ⅱ的光抑制敏感性,并对发生在供体侧和受体侧抑制的光系统II反应中心具有保护作用,但对整体植物在生理条件下的作用却未得到证实,这也正是今后需要研究的问题。     

Differential inhibition by plastoquinone analogues of photoreduction of cytochrome b-559 in chloroplasts

The high-potential form of cytochrome b-559 (b-559 HP) is closely linked to the oxygenic photosystem (photosystem II) but its relation to other redox components of the photosynthetic apparatus, including plastoquinone, is still obscure. We investigated the photoreduction of cytochrome b-559 HP by isolated chloroplasts in the presence of 3 antagonists of plastoquinone, of which, DBMIB (dibromothymoquinone) and DNP-INT (dinitrophenyl ether of iodonitrothymol) are known to inhibit the oxidation of the plastoquinone pool (PQ) by the FeS-cytochrome ƒ/b₆ complex and one, UHDBT (5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) is known to inhibit the reduction of PQ by Q_B.Q_B is a protein-bound plastoquinone that serves as a two-electron gate for the reduction of PQ. We found that DBMIB and DNP-INT did not inhibit but low concentrations of UHDBT severely inhibited the photoreduction of cytochrome b-559 HP. These results suggest that the electron donor for the reduction of cytochrome b-559 HP was either Q_B or a portion of the PQ pool that was oxidized by a new pathway free of binding sites for DBMIB and DNP-INT.     

Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.     

Mechanism of arachidonic acid liberation in platelet-activating factor-stimulated human polymorphonuclear neutrophils

Upon stimulation of human polymorphonuclear neutrophils with platelet-activating factor (PAF), arachidonic acid (AA) is released from membrane phospholipids. The mechanism for AA liberation, a key step in the synthesis of biologically active eicosanoids, was investigated. PAF was found to elicit an increase in the cytoplasmic level of free Ca2+ as monitored by fluorescent indicator fura 2. When [3H] AA-labeled neutrophils were exposed to PAF, the enhanced release of AA was observed with a concomitant decrease of radioactivity in phosphatidylinositol and phosphatidylcholine fractions. The inhibitors of phospholipase A2, mepacrine and 2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, effectively suppressed the liberation of [3H]AA from phospholipids, indicating that liberation of AA is mainly catalyzed by the action of phospholipase A2. The extracellular Ca2+ is not required for AA release. However, intracellular Ca2+ antagonists, TMB-8 and high dose of quin 2/AM drastically reduced the liberation of AA induced by PAF, indicating that Ca2+ is an essential factor for phospholipase A2 activation. PAF raised the fluorescence of fura 2 at concentrations as low as 8 pM which reached a maximal level about 8 nM, whereas more than nM order concentrations of PAF was required for the detectable release of [3H]AA. Pretreatment of neutrophils with pertussis toxin resulted in complete abolition of AA liberation in response to PAF. However, the fura 2 response to PAF was not effectively inhibited by toxin treatment. In human neutrophil homogenate and membrane preparations, guanosine 5'-O-(thiotriphosphate) stimulated AA release and potentiated the action of PAF. Guanosine 5'-O-(thiodiphosphate) inhibited the effects of guanosine 5'-O-(thiotriphosphate). These results suggest several points: 1) PAF stimulates human polymorphonuclear neutrophils to liberate AA mainly by the action of phospholipase A2; 2) Ca2+ mobilization alone is not sufficient to stimulate AA release, although Ca2+ is the important factor for phospholipase A2 activation; and 3) a pertussis toxin-sensitive GTP-binding protein may be implicated in activation of phospholipase A2.     

Kinetics of the photoreduction of cytochrome b-559 by photosystem II in chloroplasts.

The kinetics of the photoreduction of cytochrome b-559 and plastoquinone were measured using well-coupled spinach chloroplasts. High potential (i.e, hydroquinone reducible) cytochrome b-559 was oxidized with low intensity far-red light in the presence of N-methyl phenazonium methosulfate or after preillumination with high intensity light. Using long flashes of red light, the half-reduction time of cytochrome b-559 was found to be 100 +/- 10 ms, compared to 6-10 ms for the photoreduction of the plastoquinone pool. Light saturation of the photoreduction of cytochrome b-559 occurred at a light intensity less than one-third of the intensity necessary for the saturation of ferricyanide reduction under identical illumination conditions. The photoreduction of cytochrome b-559 was accelerated in the presence of dibromothymoquinone with a t 1/2 = 25-35 ms. The addition of uncouplers, which caused stimulatory effect on ferricyanide reduction under the same experimental conditions resulted in a decrease in the rate of cytochrome b-559 reduction. The relatively slow photoreduction rate of cytochrome b-559 compared to the plastoquinone pool implies that electrons can be transferred efficiently from Photosystem II to plastoquinone without the involvement of cytochrome b-559 as an intermediate. These results indicate that it is unlikely that high potential cytochrome b-559 functions as an obligatory redox component in the main electron transport chain joining the two photosystems.