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     

EPR study of 1Asp-3Cys ligated 4Fe-4S iron-sulfur cluster in NB-protein (BchN-BchB) of a dark-operative protochlorophyllide reductase complex

Dark-operative protochlorophyllide oxidoreductase, a nitrogenase-like enzyme, contains two [4Fe–4S] clusters, one in the L-protein ((BchL)₂) and the other in the NB-protein ((BchN–BchB)₂). The reduced NB-cluster in the NB-protein, which is ligated by 1Asp/3Cys residues, showed a broad S = 3/2 electron paramagnetic resonance signal that is rather rare in [4Fe–4S] clusters. A 4Cys-ligated NB-cluster in the mutated variant BchB–D36C protein, in which the Asp36 was replaced by a Cys, gave a rhombic normal S = 1/2 signal and lost the catalytic activity. The results suggest that Asp36 contributes to the low redox potential necessary to reduce protochlorophyllide.     

Noncyclic electron transport in chromatophores from photolithotrophically grown Rhodobacter sulfidophilus

Chromatophores isolated from the marine phototrophic bacterium Rhodobacter sulfidophilus were found to photoreduce NAD with sulfide as the electron donor. The apparent K _m for sulfide was 370 M and the optimal pH was 7.0. The rate of NAD photoreduction in chromatophore suspensions with sulfide as the electron donor (about 7–12 M/h·mol Bchl) was approximately onetenth the rate of sulfide oxidation in whole cell suspensions. NAD photoreduction was inhibited by rotenone, carbonyl cyanide-m-chlorophenylhydrazone, and antimycin A. Sulfide reduced ubiquinone in the dark when added to anaerobic chromatophore suspensions. These results suggest that electron transport from sulfide to NAD involves an initial dark reduction of ubiquinone followed by reverse electron transport from ubiquinol to NAD mediated by NADH dehydrogenase.Abbreviations Bchl bacteriochlorophyll - CCCP carbonyl cyanide-m-chlorophenylhydrazone - MOPS 3(N-morpholino)-propane sulfonate - Uq ubiquinone     

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650R668, P650R688, and P650R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643–644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658–711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.Abbreviations P650 protochlorophyll(ide) with absorption maximum at 650 nm - C697/684 chlorophyllide with fluorescence maximum at 695 nm and absorption maximum at 684 nm - R697 intermediate with absorption maximum at 697 nm     

木本植物阳生和阴生叶片叶绿体O2和NO2-光还原作用

在有PCR和PCO环活性抑制剂甘油醛和光合磷酸化解偶联剂NH4Cl存在下,比较了生长于3种光环境的乔木黧蒴和灌木九节幼苗阳生和阴生叶片叶绿体的O2和NO2－光还原速率,全自动光下两种植物阳生叶片的叶绿体O2的光还原速率最高,占总光合电子传递活性的66％－68％,NO2－光还原速率也有类似趋势,占总电子传递的11％－15％左右。36％和16％自然光下阴生叶片O2和NO2－光还原速率及O2光还原电子传递的比较显著降低,但NO2－光还原电子传递的比例不受影响,与NO2－光还原相关的叶片NiR和NR活性及NiR/NR活性比也因叶片接受光强度大小而异,随光强减弱,黧蒴的NiR活性降低,九节的NR活性增高,但黧蒴的NR活性和九节的NiR活性变化未达差异显著性。     

The biosynthesis of chlorophyll in greening barley (Hordeum vulgare). Is there a light-independent protochlorophyllide reductase?

Recently, some evidence for the occurence of a light-independent protochlorophyllide-reducing enzyme in greening barley plants has been presented. In the present work this problem was reinvestigated. -[¹⁴C] Aminolevulinic acid was fed to isolated barley shoots from plants which had been preilluminated for various lengths of time. Porphyrins which had been synthesized during the dark incubation were analyzed by high-performance liquid chromatography. There was no evidence for a light-independent synthesis of chlorophyll(ide). The ¹⁴C-labelled precursor was incorporated almost exclusively into protochlorophyllide. The reduction of labelled protochlorophyllide to chlorophyllide was strictly light-dependent. These results are not consistent with the existence of a light-independent protochlorophyllide-reductase in barley as proposed previously.Abbreviation HPLC high-performance liquid chromatography     

Oxygen reduction in a plastoquinone pool of isolated pea thylakoids

Oxygen uptake in isolated pea thylakoids in the presence of an inhibitor of plastoquinol oxidation by b ₆/f-complex dinitrophenylether of 2-iodo-4-nitrothymol (DNP-INT) was studied. The rate of oxygen uptake in the absence of DNP-INT had a distinct maximum at pH 5.0 followed by a decline to pH 6.5 and posterior slow rise, while in the presence of an inhibitor it increased at an increasing pH from 4.5 to 6.5 and then kept close to the rate in its absence up to pH 8.5. Gramicidin D substantially affected the oxygen uptake rate in the absence of DNP-INT, and only slightly in its presence. Such differences pointed to the presence of special oxygen reduction site(s) in photosynthetic electron transport chain `before' cytochrome complex. Oxygen uptake in membrane fragments of Photosystem II (BBY-particles) was low and did not depend on pH. This did not support the participation of Q_B in oxygen reduction in DNP-INT-treated thylakoids. Oxygen uptake in thylakoids in the presence of DNP-INT was inhibited by DCMU as well as by catalase in whole pH range. The catalase effect indicated that oxygen uptake was the result of dioxygen reduction by electrons derived from water, and that H₂O₂ was a final product of this reduction. Photoreduction of Cyt c in the presence of DNP-INT was partly inhibited by superoxide dismutase (SOD), and this pointed to superoxide formation. The latter was confirmed by a rise of the oxygen uptake rate in the presence of ascorbate and by suppression of this rise by SOD. Both tests showed that the detectable superoxide radicals averaged 20–25% of potentially formed superoxide radicals the quantity of which was calculated from the oxygen uptake rate. The obtained data implies that the oxygen reduction takes place in a plastoquinone pool and occurs mainly inside the membrane, where superoxide can be consumed in concomitant reactions. A scheme for oxygen reduction in a plastoquinone pool in thylakoid membranes is proposed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans

Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)₂ with the catalytic (BchY/BchZ)₂ protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF₄⁻. Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)₂. A second [4Fe-4S] cluster was identified on (BchY/BchZ)₂. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.