High-potential Form Research Articles

Characterization of Three Photosystem II Mutants in Zea mays L. Lacking a 32,000 Dalton Lamellar Polypeptide

Two fully blocked and one partially blocked photosystem II nuclear mutants have been selected in Zea mays. The fully blocked mutants lack photosystem II activity, variable fluorescence, the light-inducible C-550 signal, the high potential form of cytochrome b-559, and most or all of the low potential form of the cytochrome. The block in these mutants may primarily affect the reducing side of photosystem II, inasmuch as chloroplasts isolated from both mutants exhibit an elevated F695 fluorescence emission peak. The partially blocked mutant exhibits partial photosystem II activity and a reduction, but not the total loss of the variable fluorescence yield, the C-550 signal, and the high potential form of cytochrome b-559. Lamellae isolated from the fully blocked mutants are greatly deficient for a major lamellar polypeptide with an apparent molecular weight of 32,000 daltons, whereas lamellae from the partially blocked mutant show the partial loss of this same polypeptide, suggesting that the 32,000 dalton polypeptide is necessary for the proper function of photosystem II.

Photosynthetic electron transport in a cell-free preparation from the thermophilic blue-green alga Phormidium laminosum.

1. A cell-free preparation of membrane fragments was prepared from the thermophilic blue-green alga Phormidium laminosum by lysozyme treatment of the cells followed by osmotic shock to lyse the spheroplasts. The membrane fragments showed high rates of photosynthetic electron transport and O2 evolution (180-250 mumol of O2/h per mg of chlorophyll a with 2,6-dimethyl-1,4-benzoquinone as electron acceptor). O2-evolution activity was stable provided that cations (e.g. 10mM-Mg2+ or 100mM-Na+) or glycerol (25%, v/v) were present in the suspending medium. 2. The components of the electron-transport chain in P. laminosum were similar to those of other blue-green algae: the cells contained Pigment P700, plastocyanin, soluble high-potential cytochrome c-553, soluble low-potential cytochrome c-54 and membrane-bound cytochromes f, b-563 and b-559 (both low- and high-potential forms). The amounts and midpoint potentials of the membrane-bound cytochromes were similar to those in higher-plant chloroplasts. 3. Although O2 evolution in P. laminosum spheroplasts was resistant to high temperatures, thermal stability was not retained in the cell-free preparation. However, in contrast with higher plants, O2 evolution in P. laminosum membrane fragments was remarkably resistant to the non-ionic detergent Triton X-100.

On the interaction of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) with bound electron carriers in spinach chloroplasts

2,5-Dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), when added to chloroplasts as the sole electron donor, is an effective reducing agent. Low concentrations of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone reduce cytochrome f, plastocyanin, and P700 in the dark but do not reduce the high-potential form of cytochrome b 559. 2,5-Dibromo-3-methyl-6-isopropylbenzoquinone appears to interact at or near the site of function of the “Rieske” iron-sulfur center, as evidenced by a shift in the g value of the electron paramagnetic resonance signal of the reduced center.

Cyanide reactivity of cytochrome c derivatives

The kinetic rates and equilibrium association constants for cyanide binding have been measured for a series of cytochrome c derivatives as a probe of heme accessibility. The series included horse and yeast cytochromes iodinated at Tyr 67 and 74, horse cytochrome formylated at Trp 59 in both a low and high redox potential form, the Met 80 sulfoxide derivative of horse cytochrome and the N-acylisourea heme propionate derivative of tuna cytochrome. Native cytochromes c are well known to bind cyanide slowly in a reaction simply first order both in cytochrome and cyanide up to at least 100 mM in cyanide. The derivatives demonstrate markedly different kinetics which indicate the following conclusions. (1) In spite of chemical modification at different loci, all the derivatives have highly similar reactivity, suggesting common ligation structures and mechanisms for reaction. (2) Compared to native cytochromes, reaction rates are 10–20 fold greater. This is in accord with a more accessible heme crevice, but not a completely opened crevice. For the completely opened case, rate increases are expected to be between three and five orders of magnitude. (3) Reaction rates are either independent of cyanide concentration (zero order) or show only slight variation. A mechanism which accounts for the data over four orders of magnitude in concentration postulates a protein conformation step, opening of the heme crevice, as the rate determining step. This conformation change has a limiting rate of 6 · 10 −2 s −1.

Redox potentials of the cytochromes in the respiratory chain of aerobically grown Escherichia coli

The midpoint oxidation-reduction potentials of the cytochromes of the respiratory chain of Escherichia coli were determined. Cytochromes d and a 1 had midpoint oxidation-reduction potentials of +260 and +147 mV, respectively. Cytochrome b was resolved into two major components, cytochrome b 558 and cytochrome b 556, by difference spectroscopy at 77 °K and by potentiometric titrations. These cytochromes had midpoint oxidation-reduction potentials of +165 and +35 mV, respectively, in membranes from exponential-phase cells. The amount of the high and low potential forms of cytochrome b varied with the growth phase.

Simultaneous Appearance of C‐550 and Cytochrome b559HP (High Potential Form) in Barley Leaves Greened under Intermittent Light

AbstractCytochrome b559HP has been detected by spectrochemical assays in plastids of barley leaves greened under intermittent light (flashed leaves). The amount of cytochrome b559HP in these plastids was nearly 10‐fold lower than in normal chloroplasts when the results were expressed on plastid number. The amount of cytochrome b559HP phototransformable at ‐170°C was similar, on a C‐550 basis, in the plastids of flashed and normal green leaves. The appearance of the 2 components was simultaneous during the greening process under intermittent light and it is suggested that it was parallel to the increase of appressed regions in thylakoids.The illumination of flashed leaves under continuous light for 5 minutes allowed the appearance of a normal Photosystem‐II activity, but had no effect either on the cytochrome b559HP content of the plastids or on the photoreactivity of this component at low temperature.

Further investigation of the light-induced cytochrome b-559 reaction in membrane fragments of the blue-green alga Anabaena variabilis

Oxidation-reduction reactions of the low redox potential cytochrome b-559 were studied for membrane fragments of the blue-green alga Anabaena variabilis. Cytochrome photooxidation was observable at room temperature when the membrane fragments had been preincubated at room temperature in the dark. A CCCP addition (10−4M) strongly enhanced the reaction. Oxidation consisted of a DCMU-sensitive and an insensitive reaction. The former depended on actinic illumination of short wavelength. The latter showed a dependency on longer wavelength light. The former was assumed to be induced by the action of photosystem II and the latter by that of photosystem I. The photosystem II oxidation was small before preincubation, and was enhanced by added DPIP or Ferro. This was interpreted as photosystem II action inducing oxidation as well as reduction; reduction being inactivated during dark incubation or being suppressed by added redox reagents which compete for electron acceptance from photosystem II so that oxidation was apparently enhanced. The oxidationreduction reactions of this cytochrome with low redox potential were assumed to be almost identical with those of the high redox potential form, at least in the membrane fragments of Anabaena variabilis.

La photooxydation du cytochrome b-559, en presence de [formula omitted] et de 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone, ou de p-benzoquinone, chez trois mutants non-photosynthétiques de Chlamydomonas reinhardti

Cytochrome b-559 photooxidation in the presence of carbonyl cyanide p-trifluorometh-oxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone or p-benzoquinone in three non-photosynthetic mutants of Chlamydomonas reinhardti Studies of absorbance changes related to the cytochrome b-559 photooxidation induced by FCCP, with and without addition of 3- p-chlorophenyl-1, 1-dimethylurea (CMU), DBMIB or p-benzoquinone, in whole cells and in chloroplast fragments of Chlamydomonas reinhardti, were carried out. In addition to the wild type, three strains of non-photosynthetic mutants were used: Fl 5, which lacks P 700; Fl 9 and Fl 15, which are deficient in bound cytochrome c-553 and in cytochrome b-563. In the presence of FCCP, whole cells and chloroplast fragments of the four strains showed a System II-dependent photooxidation of cytochrome b-559. This photooxidation was inhibited by CMU but it occurred again in presence of FCCP, CMU and DBMIB. In chloroplast fragments, cytochrome b-559 photooxidation was also inhibited by an excess of FCCP; it was recovered, likewise, by addition of DBMIB. In whole cells, the highest measured redox changes were: 1 μmol oxidized cytochrome b-559 per 1 mmol chlorophyll, corresponding approximately to about one seventh (wild type, Fl 5) or one fifth ( Fl 9, Fl 15) of the total amount of this cytochrome. Another kind of cytochrome b-559 photooxidation, CMU-insensitive, also occurred in the mutants Fl 9 and Fl 15 and in the wild type, but not in the mutant Fl 5. This latter kind of photooxidation was observed with chloroplast fragments in the presence of FCCP and CMU and also with whole cells in the presence of FCCP, CMU and p-benzoquinone. These reactions can be attributed to the Photosystem I; they do not require the intervention of the cytochrome c-553. A high-potential form of cytochrome b-559, hydroquinone-reducible, was involved in these two kinds of photooxidation. In addition, a lower potential form, reducible only by ascorbate, appeared to be able to interfere also. An interpretation is attempted, taking into consideration the various effects of FCCP and DBMIB, at different concentrations, on photosynthetic electron transport.

Photochemical activity and components of membrane preparations from blue-green algae. II. Low-temperature photooxidation of cytochrome b559

In view of some conflicting reports we investigated the properties of cytochrome b 559 present in membrane fragments of Nostoc muscorum (Strain 7119) cells capable of high rates of photooxidation of water. We found that (i) the membrane-bound cytochrome b 559 exists in more than one form, the high-potential form being the predominant one, (ii) the high-potential form of cytochrome b 559 was photooxidized at cryogenic temperatures (−196°C), and (iii) the low-temperature photooxidation was accomplished by short-wavelength light characteristic of Photosystem II but not by long-wavelength ligh characteristic of Photosystem I. Since similar properties of cytochrome b 559 have also been found in chloroplasts, they appear to be general characteristics of the O 2-evolving apparatus in photosynthesis.

Development of Photochemical Activity and the Appearance of the High Potential Form of Cytochrome b-559 in Greening Barley Seedlings

The development of photochemical activity during the greening of dark-grown barley seedlings (Hordeum vulgare L. cv. Svalöfs Bonus) was studied in relation to the formation of the high potential form of cytochrome b-559 (cytochrome b-559(HP)). Photosynthetic oxygen evolution from leaves was detected at 30 minutes of illumination. The rate of oxygen evolution per gram fresh weight of leaf was as high at 2 to 2.5 hours of greening as at 24 hours or in fully greened leaves. On a chlorophyll basis, the photosynthetic rate at 90 minutes of greening was 80-fold greater than the rate at 45 hours. It is concluded that the majority of photosynthetic units are functional at an early stage of greening, and that chlorophyll synthesis during greening serves to increase the size of the units.Plastids showed substantial photochemical oxygen evolution after a seedling greening time of 1 hour. However, a comparison of the relative activity of leaves and plastids at 2 hours and 24 hours of greening suggests that there was some inactivation of greening plastids during isolation. Appreciable photosystem I activity was observed as early as 15 minutes of greening.The synthesis of cytochrome b-559(HP) during greening does not correlate with the onset of oxygen evolution. Cytochrome b-559(HP) was absent from etioplasts and in most preparations of 2-hour plastids. The average amount of cytochrome b-559(HP) at 2 hours of greening was well below the level needed to provide 1 molecule of the carrier for each functional photosynthetic chain. The results suggest that cytochrome b-559(HP) is not essential for oxygen evolution. Cytochrome f, cytochrome b(6), and the low potential form of cytochrome b-559 were present in the etioplast. There was little increase in the levels of these cytochromes during 24 hours of greening.

Light-induced redox changes of cytochrome b-559

Dark incubation of spinach or pea chloroplasts with 10 μ m 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 μ m 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.

Photochemical properties of a Photosystem II subchloroplast fragment

1. The Photosystem II fraction (D-10) obtained by incubation of spinach chloroplasts with digitonin was further purified by incubation with Triton X-100. The resulting Photosystem II subchloroplast fragment (DT-10) contained 1 mole of cytochrome b-559 per 170 moles of chlorophyll. It lacked cytochrome f and cytochrome b 6 and its content of P700 was low. 2. The DT-10 fragment showed only traces of photochemical activity with water as electron donor, but it was active in a Photosystem II reaction with 2,6-dichlorophenolindophenol as electron acceptor and diphenyl carbazide as donor. Photoreduction of NADP + with diphenyl carbazide as donor was negligible. There was some photoreduction of NADP + with ascorbate plus 2,6 dichlorophenolindophenol as donor but this activity could be accounted for by contamination with Photosystem I. These results are consistent with the Z-scheme of photosynthesis with Photosystems I and II operating in series for the reduction of NADP + from water. DT-10 subchloroplast fragments showed a light-induced rise in fluorescence yield at 20 °C in the presence of diphenyl carbazide. A light-induced fluorescence increase also was observed at 77 °K. 3. During the preparation of the DT-10 fragment, the high potential form of cytochrome b-559 was largely converted to a form of lower potential and C-550 was converted to the reduced state. A photoreduction of C-550 was observed at liquidnitrogen temperature, provided the C-550 was oxidised with ferricyanide prior to cooling. Some photooxidation of cytochrome b-559 was obtained at 77 °K if the preparation was reduced prior to cooling, but the degree of photooxidation was variable with different preparations. C-550 does not appear to be identical with the primary fluorescence quencher, Q. 4. Photosystem I subchloroplast fragments (D-144) released by the action of digitonin were compared with Photosystem I fragments (DT-144) released from D-10 fragments by Triton X-100. There were no significant differences between D-144 and DT-144 fragments either in chlorophyll a b ratio or in P700 content.

On the photooxidation of a high potential form of cytochrome b-559

On the photooxidation of a high potential form of cytochrome b-559

Extraction and Reconstitution of Photosystem II

Hill activity (oxygen evolution with ferricyanide as the electron acceptor), light-induced absorbance changes at liquid nitrogen temperature associated with the primary activity of photosystem II, and fluorescence yield changes at both low temperature and room temperature were measured with lyophilized spinach chloroplasts before and after extraction with hexane and reconstitution with beta-carotene and plastoquinone A. Extraction eliminated the Hill activity, and both beta-carotene and plastoquinone A were required for maximal restoration of activity to the reconstituted chloroplasts.Extraction also eliminated the light-induced absorbance changes at -196 C due to the photoreduction of C-550 and photooxidation of cytochrome b(559), and reconstitution with beta-carotene and plastoquinone A restored the low temperature photoreactions. However, only beta-carotene was essential for the restoration of the photoreactions. Cytochrome b(559) was modified, as a result of the extraction, to a lower redox potential, autooxidizable form and remained as such after reconstitution with beta-carotene. The beta-carotene-restored chloroplasts showed the photoreduction of C-550 but not the photooxidation of cytochrome b(559) because the cytochrome was already oxidized. When beta-carotene-reconstituted chloroplasts were suspended in buffer containing ascorbate prior to freezing, the cytochrome b(559) was reduced and could be photooxidized by irradiation at low temperature. After reconstitution with beta-carotene plus plastoquinone A the cytochrome b(559) was partially restored to its original high potential form and was in the reduced state so that both the photoreduction of C-550 and the photooxidation of cytochrome b(559) occurred on irradiation of the beta-carotene plus plastoquinone A-reconstituted chloroplasts. Reconstitution with plastoquinone A alone had essentially no effect on restoring the photoreactions.The fluorescence yield of dark-adapted lyophilized chloroplasts at -196 C showed an irreversible increase of about 2.5-fold during irradiation. After extraction the fluorescence yield of the chloroplasts was high (at the maximal light-induced level of the lyophilized control chloroplasts) and showed very little change in the light. Reconstitution with beta-carotene alone restored some fluorescence quenching which was relieved by irradiation at low temperature. Reconstitution with plastoquinone A alone restored a high degree of quenching, but this quenching was not relieved by light at low temperature. Fluorescence emission spectra at -196 C showed that the fluorescence of variable yield in the lyophilized and beta-carotene-reconstituted chloroplasts involved only the 680 and 695 nm emission bands but not the larger 730 nm emission band, whereas the irreversible quenching in plastoquinone A-reconstituted chloroplasts involved all wavelengths of emission. Extraction of the chloroplasts also eliminated the sharp 695 nm emission band at low temperature, and reconstitution with beta-carotene partially restored it.The fluorescence yield changes at room temperature differed from the low temperature measurements in that the strong fluorescence quenching restored to the plastoquinone A-reconstituted chloroplasts was relieved by light and reappeared in the dark. Thus plastoquinone A appeared to be much more effective than beta-carotene in restoring the fluorescence of variable yield in room temperature measurements. However, it is argued from the results at low temperature that the quenching in plastoquinone A-reconstituted chloroplasts, which is probably due to the oxidized form of the quinone, is nonspecific and a different quenching mechanism from that which obtains in normal chloroplasts.The results suggest that extraction with hexane removes plastoquinone A, which interrupts electron transport, and beta-carotene, which disrupts the primary photochemical activity of photosystem II. Reconstitution of the extracted chloroplasts with beta-carotene alone restores C-550 and the primary photochemical activity of photosystem II, and when the photosystem II reaction centers are restored the additional requirement of plastoquinone A for the Hill reaction can be demonstrated.

The redox states of respiratory-chain components in ratliver mitochondria. III. ‘Cross-over points’ in site III

1. When ascorbate is used as a substrate for rat-liver mitochondria in the presence of rotenone, antimycin and N, N, N′, N′-tetramethyl- p-phenylenediamine or phenazine methosulphate, a cross-over point is seen between cytochrome a 3 and oxygen. 2. This is shifted to between cytochromes c and a or below cytochrome c on adding varying amounts of terminal respiratory inhibitors. 3. With low substrate concentrations, positive cross-over points were observed between cytochromes c and a and between cytochromes a 3 and oxygen, and a negative cross-over between cytochromes a and a 3. 4. The cross-over data are consistent with a mechanism for Site-III phosphorylation that involves a high-potential form of one carrier (maybe copper) and a low-potential form of a 3, analogous to the mechanism of Site-II phosphorylation previously proposed.

Three forms of cytochrome b 559 and their relation to the photosynthetic activity of chloroplasts.

Spinach chloroplasts were found to contain three forms of cytochrome b(559) that have the same alpha-peak at 559 nm, but are distinguished from one another by their oxidation-reduction potentials. The high-potential (H) form (E(m) about 330-350 mV) is reducible by hydroquinone, the middle-potential (M) form (E(m) about 50-80 mV) is reducible by ascorbate but not by hydroquinone, and the low-potential (L) form is reducible by dithionite but not by ascorbate. The H form was the predominant one in freshly prepared chloroplasts and was strongly correlated with Photosystem II activity. Chloroplast treatments such as aging, sonication, and mild heating, singly or in combination, brought about a shift of the H to the M form. More drastic treatments resulted in shifts of the H and M forms to the L form. The role of cytochrome b(559) as an electron carrier in System II of plant photosynthesis is discussed.

Changes in oxidation-reduction potential of cytochrome b observed in the presence of antimycin A

The cytochrome b of sonic particles of mitochondria or the isolated segment of the respiratory chain containing cytochromes b and c 1 (Complex III) was 80–95% reducible with Q 1H 2 (ubiquinol-5) in the presence of antimycin plus selected electron acceptors added externally (i.e., oxidants which reacted preferentially with respiratory components on the oxygen side of the point of inhibition by antimycin) such as oxygen or ferricyanide depending on whether sonic particles or isolated Complex III was used. In contrast, less than 40% of the cytochrome b was reduced by Q 1H 2 in the absence of either antimycin or the external electron acceptor. In the presence of antimycin ascorbate or mercaptoethanol, which behaved as mild reducing agents, completely inhibited the reduction of cytochrome b by Q 1H 2. Reduction of cytochrome b by varying mixtures of Q 1 and Q 1H 2 yielded data which could be fitted by potentiometric titration curves. Midpoint potentials of cytochrome b estimated from these curves obtained in the presence of antimycin and added electron acceptor gave values of E m7 = 0.15–0.19 V; whereas, E m7 values of ca. 0.05 V were obtained in the same system but without the added electron acceptor. In the absence of both antimycin and electron acceptor, titration curves corresponding to a cytochrome b with low values of E m at low potentials ( E h) but with anomalously steep slopes were obtained. The ΔE m ΔpH between pH values 6.0 and 7.0 for cytochrome b in the presence of antimycin and an electron acceptor was −0.05 V at 0 ° which corresponds to the dissociation of one proton from cytochrome b concomitant with oxidation of cytochrome b. The requirement of both antimycin and an added electron acceptor for measurement of the high potential form of cytochrome b is strongly suggestive that the state of oxidation of a component distinct from cytochrome b controls the midpoint potential of cytochrome b. Only when this component is in its oxidized form is cytochrome b in its high-potential state.