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.     

Characterization of Nuclear Mutants of Maize Which Lack the Cytochrome f/b-563 Complex

Two high fluorescent, nuclear recessive mutants of maize (Zea mays L.), designated hcf-2 and hcf-6, are described which are missing the chloroplast cytochrome f/b-563 complex. Thylakoids from the mutants show a block in whole chain electron transport activity (H₂O to methyl viologen), while retaining activities associated with photosystem II (H₂O to phenylenediamine) and photosystem I (diaminodurene to methyl viologen). Chemically induced, optical difference spectra indicate a loss of cytochromes f and b-563. Cytochrome b-559 is present in both high and low potential forms. EPR analyses of thylakoid membranes of hcf-6 reveals the lack of a signal (g = 1.90) associated with the Rieske Fe-S center. Additionally, hcf-6 is lacking EPR signals at g = 6 (attributable to the high spin ferric heme of cytochrome b-563) and g = 2.5 (unidentified). The mutant retains signals at g = 2.9 (cytochrome b-559) and at g = 4.3 and 9 (both signals probably arising from a storage form of ferric iron).

Thylakoid polypeptides are examined using polyacrylamide gel electrophoresis. hcf-2 and hcf-6 have identical profiles, showing losses of polypeptides with apparent molecular masses of 33 (cytochrome f), 23 (cytochrome b-563), and 17.5 kilodaltons. The protein associated with the Rieske Fe-S center could not be determined from the gel profiles. Additionally, both mutants show an increase in a band with a molecular mass of 31 kilodaltons.

     

Simultaneous purification of a cytochrome P-450 and cytochrome b5 from the house fly,Musca domestica L.

Cytochrome P-450, cytochrome b₅ and cytochrome P-450 reductase were purified from house fly abdomens using high performance liquid chromatography (HPLC). Using a new technique, cytochrome P-450 was separated from the bulk of other proteins after polyethylene glycol fractionation and hydrophobic interaction chromatography (HIC) using a phenyl-5PW column. This technique resulted in 91% recovery of the cytochrome P-450s in a single concentrated fraction that also contained the remaining cytochrome b₅ and cytochrome P-450 reductase activity. Further purification by anion exchange on a DEAE-5SW column resolved the cytochrome P-450s, cytochrome b₅ and cytochrome P-450 reductase into individual fractions. The ion exchange step yielded one fraction that contained a high specific content of P-450 (14.4 nmol/mg protein). This cytochrome P-450 fraction ran as a single band at 54.3 kDa in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and had a carboxy ferrocytochrome absorbance maximum at 447 nm.Further purification of the anion exchange cytochrome b₅ fraction, by C8 reverse phase HPLC, resulted in a cytochrome b₅ fraction with a specific content of 51.8 nmol/mg protein and an apparent molecular mass of 19.7 kDa by SDS-PAGE. The anion exchange HPLC fraction containing the cytochrome P-450 reductase activity was further purified by NADP-agarose affinity chromatography. This step yielded cytochrome P-450 reductase with an apparent molecular mass of 72 kDa.     

Relationship between O2 evolution capacity and cytochrome b-559 high-potential form in Photosystem II particles

As previously shown for inside-out vesicles by Larsson et al. (Larsson, C., Jansson, C., Ljungberg, U.L., Åkerlund, H.E. and Anderson, B. (1984) in Advances in Photosynthesis Research, Vol. I, pp. 363–366 (Sybesma C., ed.), Martinus Nijhoff/Dr. W. Junk Publishers, Dordrecht, The Netherlands), we observed that NaCl 1 M washing of Photosystem II particles prepared by Triton X-100 treatment of spinach thylakoids induces both an inactivation of oxygen evolution and transformation of cytochrome b-559 from its high-potential to its low-potential form. A partial reactivation of water oxidation by 24 kDa polypeptide refixation is accompanied by a partial restoration of the cytochrome b-559 high-potential (HP) form. In contrast, reconstitution of water splitting by Ca²⁺ addition is not associated to a reestablishment of the cytochrome (HP) form. We conclude that cytochrome b-559 HP plays no role in water oxidation.     

Purification and Spectral Characteristics of Cytochrome b-559 from Oxygen-evolving Photosystem ￠ò Core Complexes of Spinach and Rice

Cytochrome b-559 in photosystem Ⅱ reaction center was purified from spinach (Spinacia oleracea L.) and rice (Oryza sativa L.) by a rapid and simple procedure. Their low temperature fluorescence emission and excitation spectra, ultraviolet fluorescence spectra and absolute absorption spectra were presented. The author's purification methods, which enhanced the yield of pure protein and shorted the time for isolation, have several advantages: 1. use of oxygen-evolving PSⅡ core complexes as the starting material in order to avoid disturbing from other cytochromes; 2. isocratic elution of cytochrome b-559 from a DEAE-Sephacel column for eliminating the impurity and yielding the protein in pure state; 3. a simple column procedure for removal of excess Triton X-100. Purified cytochromes b-559 from these species have similar optical spectra and mobility during gel electrophoresis under native conditions. From the results of novel electrophoresis (Tricine-SDS-PAGE), cytochrome b-559 from both spinach and rice reveal two polypeptide bands (apparent molecular weight 9 kD and 4 kD, respectively). By measuring of 77 K fluorescence spectra, it was shown that for the purified cytochrome b-559 there were two excitation peaks at 439 nm and 413 nm, and two emission peaks at 563 nm and 668 nm. This is the first indication that Cyt b-559 is able to emit fluorescence and also transfer excited electrons to chlorophyll. By the use of ultraviolet fluorescence spectra, it was demonstrated for the first time that the location of Trp residue could be in the hydrophobic transmembrane region of cytochrome b-559.     

Stoichiometry of components in the photosynthetic oxygen evolution system of Photosystem II particles prepared with Triton X-100 from spinach chloroplasts

The stoichiometry of the proteins of the photosynthetic oxygen evolution system and of the electron transport components in Photosystem II particles prepared with Triton X-100 from spinach chloroplasts were determined. Per about 220 chlorophyll molecules, there were one reaction center II, one molecule each of the 33, 24 and 18 kDa proteins, four Mn atoms, two cytochromes b-559 (one high-potential, the other low-potential), and 3.5 plastoquinone-9 molecules, but practically no cytochrome b-563, cytochrome f, phylloquinone, α-tocopherol or α-tocopherylquinone.     

Photoreactions of cytochrome b-559 and cyclic electron flow in Photosystem II of intact chloroplasts

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO₂ showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.     

Characterization of a resolved oxygen-evolving Photosystem II preparation from spinach thylakoids

An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg²⁺. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS II_α. (5) An E_m = ? 10 mV has been measured at pH 7.8 for the primary electron acceptor Q_α of PS II_α. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$. In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.     

Polypeptide composition of a Photosystem II core complex. Presence of a herbicide-binding protein

The polypeptide composition of a Photosystem II (PS II) core complex from higher plant chloroplasts has been characterized by subjecting the isolated complex to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides in the 40–50 kDa size class, attributed to the chlorophyll a-binding apoproteins of PS II, were resolved when the urea concentration in the SDS-polyacrylamide gel electrophoresis was greater than 1 M. The two chlorophyll a-binding proteins were dissimilar in their primary structure based upon their different hydrolysis products on SDS-polyacrylamide gel electrophoresis following papain treatment. The core complex contained three additional polypeptides. Two polypeptides in the 30–34 kDa size class were resolved when the urea concentration in the gel system was increased to greater than 4 M. One of the polypeptides in this size class was identified as the herbicide-binding protein from azido[¹⁴C]atrazine labeling studies. The herbicide-binding protein displayed an anomalous electrophoretic migration behavior in SDS-polyacrylamide gel electrophoresis in the presence or absence of urea; its apparent molecular weight decreased when the urea concentration increased. The fifth protein component of the core complex was attributed to cytochrome b-559 which was found to consist of the ascorbate- and dithionite-reducible forms in the samples prior to SDS solubilization.     

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 $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 25–35}\text{ms}$. The addition of uncouplers, which caused a 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.     

Localization of Cytochrome b-559 in the Chloroplast Thylakoid Membranes in Spinach

Cytochrome b-559 was purified from spinach leaves and antibodies were made against it in rabbit. Using affinity-purified, monospecific antibodies, we have found that cytochrome b-559, which is closely associated with the primary photochemical activity of photosystem II, is localized exclusively in the grana thylakoids.     

Cytochromes and prenylquinones in preparations of cytoplasmic and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans

The cytochrome and prenylquinone compositions were compared for cytoplasmic membranes and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans. Reduced-minus-oxidized difference absorption spectra at ?196°C indicated that the thylakoid membranes contained photosynthetic cytochromes such as cytochrome ?, cytochrome b-559 and cytochrome b₆, while cytochromes c-549 and c-552 were detected spectrophotometrically only after their release by sonic oscillation. The cytoplasmic membrane preparation contained one or two low-potential cytochrome(s) with α-band maxima at 553 and 559 nm at ?196°C, which differed from the cytochromes in the thylakoid membranes. A cytochrome specific to the cytoplasmic membranes was also found by heme-staining after lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Both types of membranes contained the three prenylquinones plastoquinone-9, phylloquinone and 5′-monohydroxyphylloquinone, but in different proportions.     

Photoreduction of cytochrome b559 in a Photosystem-II subchloroplast particle

A Photosystem-II reaction-center particle derived from spinach chloroplasts by Triton treatment contains only one kind of cytochrome, namely, cytochrome b₅₅₉, in the amount of slightly more than 2 per 100 total chlorophyll molecules. Cytochrome b₅₅₉ is present in the oxidized form, has a standard redox potential of 58 mV, and undergoes photoreduction.     

A New 4.8-kDa Polypeptide Intrinsic to the PS II Reaction Center, as Revealed by Modified SDS-PAGE with Improved Resolution of Low-Molecular-Weight Proteins

Low-molecular-weight polypeptides in various PS II preparationsfrom spinach and wheat were analyzed by modified SDS-PAGE, whichgave good resolution of low-molecular-weight proteins with minimizedinterference by lipids. PS II membrane fragments contained atleast nine low-molecular-weight polypeptides of between 3.9kDa and 11 kDa, and all of them were identified in thylakoidmembranes. Of these nine polypeptides, the 10-kDa phosphoprotein,the 5-kDa, 4.8-kDa, and 4.1-kDa polypeptides, and the two subunitsof cytochrome b₅₅₉ were commonly found in O₂-evolving core complexesof wheat and spinach. In contrast, PS II reaction center complexesthat consisted of D1, D2 and two cytochrome b₅₅₉ polypeptidesretained only the 4.8- kDa polypeptide. Analysis by Westernblotting revealed that the 4.8-kDa polypeptide is an intrinsiccomponent of the PS II reaction center. (Received May 30, 1988; Accepted August 19, 1988)     

Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach

Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl--D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.Abbreviations Bis-Tris bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane - Chl chlorophyll - CP29 Chl a/b protein of 29 kDa - Cyt b ₅₅₉ cytochrome b ₅₅₉ - DCBQ 2,5-dichloro-p-benzo-quinone - LHC II light-harvesting complex II, predominant Chl a/b protein - MES 2-[N-Morpholino]ethanesulfonic acid - Pheo pheophytin - PS H photosystem II - Q_A bound plastoquinone, serving as the secondary electron acceptor in PS II (after Pheo) - SDS sodiumdodecylsulfate     

Photooxidation of the cytochrome b-559 in the presence of various substituted 2-anilinothiophenes and of some other compounds,in Chlamydomonas reinhardtii

Five substituted 2-anilinothiophenes and two substituted carbonylcyanide-phenylhydrazones were comparatively studied with respect to their capacities for inducing photooxidation of the cytochrome b-559 in chloroplast fragments and in whole cells of Chlamydomonas reinhardtii (wild type and P-700-lacking mutant Fl 5). In addition, some other compounds: antimycin A, picric acid, tetraphenylboron and NH₄Cl were also tested.Cytochrome b-559 photooxidations were clearly observed in the presence of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), 2-(3,4,5-trichloro)anilino-3,5-dinitrothiophene (ANT 2s), 2-(4-chloro)anilino-3,5-dinitrothiophene and, with greater amplitudes, in the presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone and carbonylcyanide-m-chlorophenylhydrazone, both in whole cells and in chloroplast fragments. Picric acid, antimycin A and tetraphenylboron were also able to induce cytochrome b-559 photooxidation in chloroplast fragments, but not in whole cells. In the wild type, the highest photoinduced redox changes were 1.1 (carbonylcyanide-p-trifluoromethoxyphenylhydrazone, carbonylcyanide-m-chlorophenylhydrazone) and 0.6 (ANT 2p, ANT 2s) μmol of oxidized cytochrome b-559/1 mmol of chlorophyll, corresponding to 40% and 23% of the redox changes which could be induced chemically. All these cytochrome b-559 photooxidations, the greater part of which was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and occurred in the mutant Fl 5, appeared to be mainly Photosystem II-dependent reactions. But 3-(3,4-dichlorophenyl)-1,1-dimethylureainsensitive Photosystem I-dependent photooxidations of cytochrome b-559 occurred also in the wild type. On the other hand, 2-(4-dimethylamine)-anilino-3,5-dinitrothiophene, 2-N-methyl-(3-chloro-4-trifluoromethyl)anilino3,5-dinitrothiophene and NH₄Cl did not induce any cytochrome b-559 photooxidation.These results were discussed taking in consideration the nature of the molecular substitutions of the various tested substances and their respective acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis capacities which had been defined elsewhere by Renger (Renger, G. (1972) Biochim. Biophys. Acta 256, 428–439) for spinach chloroplasts. Like the acceleration of the deactivation reactions of the water-splitting enzyme system Y effect, the capacity for inducing the cytochrome b-559 photooxidation appeared dependent on the acidity of the NH group and on the number of halogenous substituents in the aromatic ring of the molecule. The greatest action towards cytochrome b-559 photooxidation was obtained with the most active acceleration of the deactivation reactions of the water-splitting enzyme system Y agents: carbonylcyanide-p-trifluoromethoxyphenylhydrazone, ANT 2p and ANT 2s.     

On the question of the identity of cytochrome b-560 in thylakoid stromal membranes

Stromal membranes enriched in PS I contain a low potential cytochrome with a reduced -band peak close to 560 nm. The identity of this cytochrome component has been ascribed either to a low potential form of the Photosystem II cytochrome b-559 or to a different cytochrome with a reduced -band of 560 nm. The half-bandwidth of the 560 nm component in stromal membranes is identical to that of purified cytochrome b-559. Western blots show that the stromal membranes contain an amount of PS II cytochrome b-559 -subunit that is more than sufficient to account for the cytochrome b-560 detected spectrophotometrically in these membranes. These immunochemical data and the similarity of (i) the spectral peaks, and (ii) the redox properties of low potential PS II cytochrome b-559 and the b-560 component, suggest that the simplest inference is that the cytochrome b-560 protein in stromal membranes is identical to the PS II cytochrome b-559.Abbreviations: A absorbance - cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - E_mx midpoint potential at pH x - hbw half-bandwidth - LP low potential - MD menadiol - MES 2-(N-morpholino)ethanesulfonic acid - MHQ methylhydroquinone - PS I-PS II photosystems I, II - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

The orientation of the magnetic axes of membrane-bound iron-sulfur clusters and a cytochrome b-559 in the green halophilic alga Dunaliella parva

Photosynthetic membrane fragments separated from whole cells of the green alga Dunaliella parva, were oriented by incorporation into multilayers on thin Mylar films. These partially dehydrated films were then examined by EPR spectroscopy for evidence of orientation of paramagnetic components. Five previously identified paramagnetic components, the reduced states of iron-sulfur clusters A and B, the intermediate acceptor X^?, the reduced Rieske iron-sulfur cluster, and oxidized cytochrome b-559, displayed EPR signals showing orientation. In addition, several previously unknown paramagnetic components were also observed to be oriented. Four components, previously characterized in spinach chloroplast preparations, the iron-sulfur clusters A and B, the intermediate acceptor X^?, and cytochrome b-559, were shown to be similar in the green alga, D. parva. The orientations of iron-sulfur clusters A and B, however, were determined unambiguously in this preparation; this was not possible in previous work with spinach. The heme plane orientation of cytochrome b-559 was found to be perpendicular to the membrane plane in agreement with the results in spinach preparations. A new photoinduced EPR signal with g values of 1.88, 1.97 and 2.12 was seen only in the oriented preparations and was indicative of a reduced iron-sulfur cluster with an orientation different from that of iron-sulfur cluster A or B. This suggests the existence of a previously unidentified acceptor in Photosystem I of green plants. These studies clearly show that the orientation of these components in bioenergetic membranes are conserved over a large span of evolutionary development and are, therefore, an important aspect of the mechanism of electron transfer.     

Concentration and function of membrane-bound cytochromes in cyanobacterial heterocysts

Membranes isolated from heterocysts and vegetative cells of Anabaena 7120 were assayed for content of cytochrome f, cytochrome b-563, cytochrome b-559_HP, cytochrome b-559_LP, and cytochrome aa₃ by use of reduced-minus-oxidized difference spectra. The level of cytochrome aa₃ in heterocyst membranes was 4 to 100 times higher than that in vegetative cells of Anabaena 7120 or other species of cyanobacteria. Heterocyst membranes lack cytochrome b-559_HP but contain cytochrome b-559_LP (E_m7.5 = +77 millivolts, n = 1) at approximately the same concentration as cytochrome f. The role of cytochrome b-559_LP in the hydrogenase-dependent electron transfer pathway was investigated with the inhibitor 2-(n-heptyl)-4-hydroxyquinoline N-oxide which blocks electron flow from hydrogenase to acceptors reacting with the plastoquinone pool. Addition of inhibitor elicited no change in the reduction level of cytochrome b-559_LP indicating that this cytochrome is not directly involved in this pathway.     

Gel electrophoresis of chloroplast membranes of mutants of Chlamydomonas reinhardii which have impaired Photosystem II function and lack photosynthetic cytochromes

Photosystem (PS) II-enriched particles or chloroplast fragments of the wild type and of three nonphotosynthetic mutants of Chlamydomonas reinhardii, which lack chloroplast cytochromes, were analyzed by lithium dodecyl sulfate polyacrylamide gel electrophoresis at 4°C to locate which chlorophyll complexes and which proteins are associated with cytochrome b-559. Two mutants, Fl 39 and Fl 50, have previously been shown to contain, respectively, 3.6- and 2.7-times less hydroquinone-reducible high-potential cytochrome b-559 than the wild type. They have impaired PS II functions. In the presence of ADRY agents: carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p) or 2-(3,4,5-trichloro)-anilino-3,5-dinitrothiophene (ANT 2s), Fl 50 carried out photo-oxidation of cytochrome b-559 with half the amplitude of that of the wild type. No photo-oxidation was observed with Fl 39. We show here that in both these mutants chlorophyll-protein complexes CP III, CP IV and CP V were missing. There were traces of the corresponding apoproteins (45 000, 42 000 and 33 000 daltons, respectively) in Fl 50 but none in Fl 39. In addition, a 19 000 dalton protein was missing in Fl 39 and present in a very small amount in Fl 50. In another mutant, Fl 9, previously characterized as lacking both cytochromes b-563 and c-553 with a normal cytochrome b-559 content, CP III-CP V and the 19 000 dalton protein were detected. CP I (110 000 daltons) and CP II (24 000 daltons) were present in all strains. These observations confirmed the close relationship between deficiencies in cytochrome b-559, lack of CP III and CP IV and anomalies in the photochemistry of PS II. They provided additional evidence that CP V and a 19 000 dalton protein are also involved in this PS II photochemistry. Staining of the gels with 3,3′,5,5′-tetramethylbenzidine and H₂O₂ allowed us to distinguish clearly four heme protein bands having peroxidase activity. Three of these bands (45 000, 42 000 and 19 000 daltons), which were shown in wild-type, Fl 39 and Fl 50 preparations but not in Fl 9, appeared related to cytochromes b-563 and c-553. The fourth heme protein (14 000 daltons) occurred in wild type and Fl 9 but was missing in Fl 39 and Fl 50; it appeared related to cytochrome b-559.