Large-scale purification of active cytochrome b6/f complex from spinach chloroplasts

A preparation is described through which large quantities of pure, active cytochrome b6/f complex can be isolated from spinach chloroplasts. The resulting complex is at least 90% pure with respect to the maximum content of redox centers, consists of four polypeptides according to polyacrylamide gel electrophoresis, and lacks both ferredoxin: NADP+ oxidoreductase and the high molecular weight form of cytochrome f seen in some other preparations. The complex contains 2 mol b6 and 2 atoms of nonheme iron per mole of cytochrome f, and possesses a high plastoquinol-plastocyanin oxidoreductase activity (Cyt f turnover no. 20-35 s-1). The present preparation should be helpful in the effort to crystallize the cytochrome b6/f complex.     

Evidence for a cytochrome f-Rieske protein subcomplex in the cytochrome b6f system from spinach chloroplasts

The cytochrome b6f complex of spinach chloroplasts was prepared with minor modification according to the method of E. Hurt and G. Hauska (1981) Eur. J. Biochem. 117, 591-599) replacing, however, the final ultracentrifugation step by hydroxyapatite chromatography as suggested by M. F. Doyle and C.-A Yu (1985) Biochem. Biophys. Res. Commun. 131, 700-706). The purified complex was partially dissociated by treatment with 4 M urea or 0.1% sodium dodecyl sulfate (SDS) in the absence of reducing agents. A binary subcomplex consisting of cytochrome f and the Rieske iron-sulfur protein was observed under these conditions by three different methods: (a) hydroxyapatite chromatography; (b) extraction with an isopropanol/water/trifluoroacetic acid mixture; and (c) gel filtration in the presence of low SDS concentrations. The subcomplex dissociated into its components by treatment with mercaptoethanol. These results suggest a close interaction of the cytochrome f with the Rieske protein involving SH groups which under reducing conditions leads to complete dissociation of the subcomplex.     

Choline oxidation by intact spinach chloroplasts

Plants synthesize betaine by a two-step oxidation of choline (choline → betaine aldehyde → betaine). Protoplast-derived chloroplasts of spinach (Spinacia oleracea L.) carry out both reactions, more rapidly in light than in darkness (AD Hanson et al. 1985 Proc Natl Acad Sci USA 82: 3678-3682). We investigated the light-stimulated oxidation of choline, using spinach chloroplasts isolated directly from leaves. The rates of choline oxidation obtained (dark and light rates: 10-50 and 100-300 nanomoles per hour per milligram chlorophyll, respectively) were approximately 20-fold higher than for protoplast-derived chloroplasts. Betaine aldehyde was the main product. Choline oxidation in darkness and light was suppressed by hypoxia. Neither uncouplers nor the Calvin cycle inhibitor glyceraldehyde greatly affected choline oxidation in the light, and maximal choline oxidation was attained far below light saturation of CO₂ fixation. The light stimulation of choline oxidation was abolished by the PSII inhibitors DCMU and dibromothymoquinone, and was partially restored by adding reduced diaminodurene, an electron donor to PSI. Both methyl viologen and phenazine methosulfate prevented choline oxidation. Adding dihydroxyacetone phosphate, which can generate NADPH in organello, doubled the dark rate of choline oxidation. These results indicate that choline oxidation in chloroplasts requires oxygen, and reducing power generated from PSI. Enzymic reactions consistent with these requirements are discussed.     

Betaine aldehyde oxidation by spinach chloroplasts

Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H₂O₂-mediated reaction cannot be ruled out.     

Strong binding of cytochrome C on the envelope of spinach chloroplasts

Yeast cationic ferricytochrome c was able to bind to the spinach (Spinacia oleracea) chloroplast envelope with a low affinity (Kd = 1.1 mum). The total amount of low affinity binding sites was of the order of 50 nmol cytochrome c mg(-1) protein. We gave the evidence that binding of ferricytochrome c to the envelope was electrostatic and that the envelope membranes were strongly negatively charged. Addition of yeast ferricytochrome c to a preparation of intact washed chloroplasts (class I) induced a strong agglutination of chloroplasts.     

Inhibition of cytochrome b563-oxidation by triorganotins in spinach chloroplasts

The triorganotin compounds triphenyltin chloride and tributyltin chloride have been known as inhibitors of the transmembrane proton channel forming F₀-domain of ATPases at micromolar concentrations. We show that these compounds at higher concentrations (10–100 µM) also inhibit uncoupled electron transport in chloroplasts within the low potential chain of the cytochrome bf complex. They cause high levels of transiently reduced cytochrome b563 as they decelerate the reoxidation process in flash illuminated chloroplasts. At the same time they slow down the flash induced slow electrogenic step generated at the cytochrome bf complex. The inhibitory effect of triphenyltin chloride on cytochrome b563 turnover in chloroplasts is comparable to that of the Q_n-inhibitor MOA-stilbene, with even less side effects on the high potential chain. Studies on the isolated bf complex suggest different binding sites for triorganotins and the quinone analogue type Q_n-inhibitors. The results are interpreted within the framework of the modified Q-cycle model by a putative organotin sensitive proton translocating site which enables proton transfer from the outer aqueous face of the membrane to the hydrophobic quinone reduction site within the complex. Hence, cytochrome b563 oxidation and plastoquinone reduction may be inhibited as a consequence of proton transfer being suppressed by triorganotins. In analogy, the previously described inhibitory effect of Val/K⁺ at the n-side of the cytochrome bf complex [Klughammer and Schreiber (1993) FEBS 336: 491–495] may be rationalised by binding of the cyclic depsipeptide at the entrance of the proton path to the Q_n-site.     

Hydrogen peroxide-dependent oxidation of flavonols by intact spinach chloroplasts

Externally added quercetin (100 micromolar) was oxidized by intact spinach chloroplasts at a rate of 30 micromoles per mg chlorophyll per hour in the presence of 100 micromolar H₂O₂. The oxidation rate was increased by about 20% in a hypotonic reaction mixture. The thylakoid fraction also oxidized the flavonol in the presence of H₂O₂, and the rate was about 25% of that by intact chloroplasts. The oxidation of quercetin was inhibited by KCN and NaN₃. Ascorbate, which permeates slowly across chloroplast envelope, only slightly suppressed the initial rate of quercetin oxidation by intact chloroplasts, while the oxidation by ruptured chloroplasts was suppressed by ascorbate by about 60%. Quercetin glycosides, quercitrin and rutin, were also oxidized by chloroplasts in the presence of H₂O₂. These results suggest that flavonols are oxidized by peroxidase-like activity in chloroplasts and that externally added flavonols can permeate into the stroma through the envelope of intact chloroplasts.     

Post-illumination kinetics of cytochrome f reduction in chloroplast thylakoid in the presence of dibromothymoquinone

Addition of dibromothymoquinone (DBMIB) to isolated chloroplast thylakoids reduces cytochrome f in the dark. Reduced cytochrome f is oxidised when the thylakoids are illuminated, and is re-reduced in the subsequent darkness. The rate of re-reduction in the dark is faster after red (650 nm) illumination than after far red (713 nm) illumination. In the presence of DCMU or upon heat treatment or at high (greater than 10 microM) concentration of DBMIB the rate of dark reduction after red illumination becomes slower and equal to that after far red illumination, suggesting that photosystem II electron transfer at least upto plastoquinone facilitates DBMIB-mediated reduction of cytochrome f in the thylakoids.     

Effects of herbicides on binary oscillations of ultraviolet flash-induced absorption changes in chloroplasts

The competition between various herbicides and plastoquinone, at the Q_B site of Photosystem II, has been studied by measuring absorption changes between 300 and 360 nm in spinach and chenopod chloroplasts, in response to a train of saturating short xenon flashes. A complex pattern was observable without addition of chemicals interfering with electron flow. The effect of potassium ferricyanide, of hydroxylamine and of valinomycine-K⁺ permitted to simplify the pattern and, in particular, to observe binary oscillations with flash number, attributable to the functioning of the two-electron gate Q_B. Herbicides belonging to different classes block electron transfer when added at high concentration. At low concentration, however, inverted binary oscillations become observable. When the chloroplasts have been first oxidized with ferricyanide, this behaviour develops progressively in response to illumination. Varying the herbicide concentration, it appears that the concentration inducing maximum binary oscillations correlates with inhibition of linear electron transfer, within each class of herbicides. Phenolic herbicides induce the largest oscillations and ureas the smallest. The binary oscillations have the spectrum of the plastoquinone anion. The results clearly show that the studied herbicides compete efficiently with Q_B, but not with Q⁻_B, at the Q_B binding site. Atrazine-resistant chenopod chloroplasts still display normal binary oscillations in the absence of herbicide, or in the presence of atrazine alone. They are highly sensitive to DCMU and to i-dinoseb, but no inverted binary oscillations could be observed with these herbicides.     

Laser flash-induced kinetic analysis of cytochrome f oxidation by wild-type and mutant plastocyanin from the cyanobacterium Nostoc sp. PCC 7119

Oxidation of the soluble, truncated form of cytochrome f by wild-type and mutant species of plastocyanin has been analyzed by laser flash absorption spectroscopy in the cyanobacterium Nostoc (formerly, Anabaena) sp. PCC 7119. At low ionic strengths, the apparent electron transfer rate constant of cytochrome f oxidation by wild-type plastocyanin is 1.34 x 10(4) s(-)(1), a value much larger than those determined for the same proteins from other organisms. Upon site-directed mutagenesis of specific residues at the plastocyanin interaction area, the rate constant decreases in all cases yet to varying extents. The only exception is the D54K variant, which exhibits a higher reactivity toward cytochrome f. In most cases, the reaction rate constant decreases monotonically with an increase in ionic strength. The observed changes in the reaction mechanism and rate constants are in agreement with the location of the mutated residues at the interface area, as well as with the peculiar orientation of the two partners within the Nostoc plastocyanin-cytochrome f transient complex, whose NMR structure has been determined recently. Furthermore, the experimental data herein reported match well the kinetic behavior exhibited by the same set of plastocyanin mutants when acting as donors of electrons to photosystem I [Molina-Heredia, F. P., et al. (2001) J. Biol. Chem. 276, 601-605], thus indicating that the copper protein uses the same surface areas-one hydrophobic and the other electrostatic-to interact with both cytochrome f and photosystem I.     

On the nature of the g = 6 EPR signal in isolated cytochrome b6f complex from spinach chloroplasts

Cytochrome b₆ in freshly prepared, active cytochrome t₆f complex from spinach chloroplasts shows a broad, low-spin EPR signal around g_z = 3.6. Maximally half of the hemes of cytochrome b₆ can be changed to high spin with a signal at g = 6 by inactivating treatments, or by isolating cytochrome b₆. In this state the heme reacts with NO. Reduction rates suggest that it is the low-potential heme which changes. The change is accompanied by the loss of the shift in the g_y signal of the Rieske FeS-center by quinone analogs.     

Light-induced changes in chlorophyll a fluorescence and cytochrome f in intact spinach chloroplasts: The site of light-dependent regulation of electron transport

Dark-adapted intact spinach chloroplasts exhibited two peaks,P and M₁, at the early phase of fluorescence induction and atransient reduction of cytochrome f shortly after its initialphotooxidation and in parallel to the appearance of P. Analysisof the peak P and the transient reduction of cytochrome f indicatedthat electron transport in intact spinach chloroplasts was regulatedby light: electron transport was inactivated at the reducingside of photosystem I in the dark-adapted chloroplasts but rapidlyreactivated by illumination. The fluorescence peak M₁ was correlatedto the proton gradient formed across the thylakoid membrane. Effects on P and transient reduction of cytochromef of NO₂^–,3-phosphoglycerate (PGA) and oxalacetate (OAA), which can penetrateinto intact chloroplasts and accept electrons at different sitesafter photosystem I, were studied to determine the site of thelight regulation. NC₂^–, which receives electrons fromreduced ferredoxin, markedly diminished both P and the transientreduction of cytochrome.f, whereas PGA and OAA, the reductionsof which are NADP-dependent, failed to affect the two transients.The ineffectiveness of PGA and OAA could not be attributed tothe dark inactivation of glyceraldehyde-3-phosphate and malicdehydrogenases, because dark-adapted chloroplasts still retainedsufficiently high levels of the enzyme activities. The resultsindicate that electron transport in intact spinach chloroplastsis regulated by light after ferredoxin but before NADP, i.e.,at the reducing terminal of the electron transport chain. (Received May 29, 1980; )     

Effects of copper on photosynthetic electron transport systems in spinach chloroplasts

The effects of copper on photosynthetic electron transfer systemsin isolated spinach chloroplasts were studied. Two differentinhibitions were observed. First, copper markedly inhibitedferredoxin-catalyzed reactions such as NADP⁺ photoreduction.The concentration required for 50% inhibition was about 2 µMof cupric sulfate. However, electron flow from reduced 2,6-dichloroindophenol(DCIP) to methyl viologen was not affected. The dissociationconstant between ferredoxin and ferredoxin-NADP⁺ reductase wasunchanged in the presence of 2.5 µM of cupric sulfate.In enzymic reaction systems, the ferredoxin-dependent electronflow from NADPH to cytochrome c was also strongly inhibitedin the presence of cupric sulfate, while DCIP reduction withNADPH as the electron donor was not affected. Second, DCIP photoreductionwas weakly blocked by copper and the lost activity could notbe recovered by adding 1,5-diphenylcarbazide (DPC). It can be concluded that copper directly interacted with ferredoxincausing inhibition of ferredoxin-dependent reactions. Further,copper caused weak inactivation between the oxidizing side ofthe reaction center of photosystem II and the electron donatingsite of DPC. (Received August 8, 1977; )