The accessibility of the chloroplast cytochromes ƒ and b-559 to ferricyanide

(1) (a) A concentration range of ferricyanide ( 0.125–0.5 mM) can be found which in the dark causes oxidation of cytochrome ƒ with two distinct kinetic components of comparable amplitude. The slow oxidation has a half time of 1–2 min. (b) The oxidation of cytochrome ƒ by ferricyanide is rapid and monophasic after the chloroplasts are frozen and thawed. (c) The oxidation of cytochrome b-559 by ferricyanide in the dark is mostly monophasic with a time course similar to that of the fast component in the cytochrome ƒ oxidation. (d) Ascorbate reduction of cytochromes ƒ and b-559 appears monophasic. Reduction of cytochrome b-559 by ascorbate is somewhat faster, and that by hydroquinone somewhat slower, than the corresponding reduction of cytochrome ƒ.

(2) (a) The kinetics of dark ferricyanide oxidation of cytochrome ƒ after actinic preillumination in the presence of an electron acceptor are approximately monophasic with a half time of about 30 s and do not show the presence of the slowly oxidized component observed after prolonged dark incubation. (b) The effect of actinic preillumination in altering the time course of ferricyanide oxidation appears to persist for several minutes in the dark. (c) Preillumination causes an increase in the extent of cytochrome b-559 oxidation by low concentrations of ferricyanide. The increase is inhibited if 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea is present during the preillumination. (d) The presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea during preillumination does not inhibit the amplitude or rate of ferricyanide oxidation of cytochrome ƒ, although the presence of the inhibitor KCN does cause such inhibition.

(3) It is proposed that a significant fraction of the cytochrome ƒ population resides at a position in the membrane relatively inaccessible to the aqueous interface compared to high potential cytochrome b-559. Actinic illumination would cause a structural or conformational change in the cytochrome ƒ and/or the membrane resulting in an increase in accessibility to this fraction of the cytochrome ƒ population.     

Kinetics of the reduction and oxidation of cytochromes b6 and ƒ in isolated chloroplasts

Absorbance changes induced by flash illumination of a chloroplast suspension were monitored kinetically at selected wavelengths between 510 and 575 nm. Digital subtraction of the P518 component from the total transient signal permitted isolation of the responses due to cytochromes ƒ and b₆. The half rise time for cytochrome b₆ reduction (1.3±0.1 ms) was much greater than a previously reported value (< 10 μs); the half time for cytochrome b₆ oxidation was 35±4 ms. Cytochrome ƒ was oxidized with a half time of about 0.22 ms and the subsequent reduction occurred in two phases with half times of about 7.3 and 83 ms. These kinetic data show that cytochrome b₆ cannot be a primary electron acceptor in Photosystem 1. The rate of oxidation of cytochrome b₆ is consonant with this cytochrome being the source of electrons for the slower phase of cytochrome ƒ reduction.     

Characterization of a mutant of Schizosaccharomyces pombe lacking cytochrome b-566

1. A chromosomal respiration-deficient mutant of the petite-negative yeast Schizosaccharomyces pombe was isolated. Its mitochondria show respiration rates of about 7% of the wild-type respiration with NADH and succinate as substrate, and 45% with ascorbate in the presence of tetramethyl-p-phenylenediamine. Oxidation of NADH and succinate is insensitive to antimycin and cyanide and that of ascorbate is much less sensitive to cyanide than the wild type.

2. The amounts of cytochromes c₁ and aa₃ are similar in the mutant and wild type. Cytochrome b-566 could not be detected in low-temperature spectra after reduction with various substrates or dithionite. A b-558 is, however, present.

3. The b-cytochromes in the mutant are not reduced by NADH or succinate during the steady state even after addition of ubiquinone-1. QH₂-3: cytochrome c reductase activity is very low and succinate oxidation is highly stimulated by phenazine methosulphate.

4. Antimycin does not bind to either oxidized or reduced mitochondrial particles of the mutant.

5. In contrast to the b-cytochromes of the wild type, b-558 in the mutant reacts with CO.

6. Cytochromes aa₃, c and c₁ are partly reduced in aerated submitochondrial particles isolated from the mutant and the EPR signal of Cu (II), measured at 35°K, is detectable only after the addition of ferricyanide. In the mutant, a signal with a trough at g = 2.01 is found, in addition to the signal at g = 1.98 found in the wild type.

7. The ATPase activity of particles isolated from the mutant is much lower than in the wild type but is still inhibited by oligomycin.     

Photooxidation of cytochrome b-559 in leaves and chloroplasts at room temperature

1. Light-induced absorbance changes of cytochrome b-559 and cytochrome f in the -band region were examined in leaves and in isolated chloroplasts.

2. Absorbance changes of cytochrome b-559 were not detected in untreated leaves or in most preparations of isolated chloroplasts. After treatment of leaves or chloroplasts with carbonyl cyanide m-chlorophenylhydrazone, high rates of photooxidation of cytochrome b-559 were obtained, both in far-red (>700 nm) and red actinic light. Cytochrome f was photooxidized in far-red light, but in red light it remained mainly in the reduced state. The initial rates of photooxidation of cytochrome b-559 in leaves or chloroplasts treated with carbonyl cyanide m-chlorophenylhydrazone were considerably decreased by 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea.

3. A slow photoreduction of cytochrome b-559 was observed in aged mutant pea chloroplasts in red light.

4. The results do not support the view that cytochrome b-559 is a component of the electron transport chain between the light reactions. It is suggested that cytochrome b-559 is located on a side path from Photosystem II, but with a possible additional link to Photosystem I.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain

1. Formate inhibits cytochrome $\text{c}$ oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome $\text{aa}{}_3$. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome $\text{aa}{}_3\text{(a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}$) and in the half-reduced species ($\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}$). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the $\text{aa}{}_3$ spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of $\text{a}{}_3{}^{\mathrm{2+}}$ minus $\text{a}{}_3{}^{\mathrm{3+}}$, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome $\text{a}{}_3{}^{\mathrm{3+}}$) and azide (which induces peak shifts of cytochrome $\text{a}{}^{\mathrm{2+}}$ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome $\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$ is faster than its rate of dissociation from $\text{a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$, especially in the presence of cytochrome $\text{c}$. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome $\text{c}$ reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus $\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenyl-enediamine}$ oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome $\text{c}$ reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome $\text{aa}{}_3$ level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

Counterflow efflux of thiamin in Escherichia coli

A resting cell of Escherichia coli lacking thiamin kinase incorporated external thiamin with an energy-dependent counterflow efflux (C-efflux). This C-efflux could be separated from an energy-dependent exit by a selective inhibition of exit by $\text{2 · 10}{}^{\mathrm{?2}}\text{M}$ NaN₃. The extracellular thiamin could be replaced by thiamin diphosphate, resulting in the same rate of C-efflux, but the rate of C-efflux of intracellular thiamin diphosphate against the external thiamin was markedly low. This low rate of C-efflux of thiamin diphosphate could explain the higher accumulation of the compound than that of free thiamin in the thiamin-kinase-defective mutant as well as in its wild-type parent. Basic characteristics of free thiamin uptake and exit in E. coli W mutant were compared with those reported in K 12 mutant: a marked difference existed in the rate of exit. The low rate of exit in E. coli W 70-23-102 was inferred as the reason for the absence of an overshoot phenomenon of thiamin uptake in this strain.     

Reconstitution of plastoquinone in the D1/D2/cytochrome b-559 photosystem II reaction centre complex

Reconstitution of plastoquinone in the photosystem II D1/D2/cytochrome b-559 reaction centre complex, in the presence of the detergent Triton X-100, is reported. Illumination of the reconstituted system results in the reduction of cytochrome b-559, the process being partly herbicide-sensitive. In addition, the reconstitution of plastoquinone results in the ability of the isolated reaction centre to catalyse the photoreduction of 2,6-dichlorophenolindophenol in the presence of the exogenous electron donor diphenylcarbazide.     

Biotransformation of p-, m-, and o-hydroxybenzoic acids by Panax ginseng hairy root cultures

The regioselective glycosylation of three isomers of hydroxybenzoic acids was observed in Panax ginseng hairy root cultures. p-Hydroxybenzoic acid (1) and m-hydroxybenzoic acid (2) were converted into their corresponding glycosides (1a and 2a) and glycosyl esters (1b and 2b) while no metabolite of o-hydroxybenzoic acid (3) was detected. A new compound, m-hydroxybenzoic acid β-d-xylopyranosyl (1 → 6)-β-d-glycopyranosyl ester (2c) was identified as a biotransformation product of 2. Further time-course studies of the biotransformation reactions showed that the glycosides were major products in the latter stage. The addition of carbohydrates or antioxidants increased glycosyl esters formation.     

Reactions of b-cytochromes in the red alga Porphyridium aerugineum

1. 1. The kinetics of light-induced absorbance changes due to oxidation and reduction of cytochromes were measured in a suspension of intact cells of the unicellular red alga Porphyridium aerugineum. Absorbance changes in the region 540–570 nm upon alternating far-red light and darkness indicated the oxidation of cytochrome ƒ and reduction of cytochrome b₅₆₃ upon illumination. The relative efficiencies of far-red and orange light indicated that both reactions were driven by Photosystem I.

2. 2. Experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), with anaerobic cells and in alternating far-red and orange light indicated that cytochrome b₅₆₃ reacts in a cyclic chain around Photosystem I, and that the reduced cytochrome does not react with oxygen or with another oxidized product of Photosystem II. The quantum requirement for the photoreduction was about 6 quanta/equiv at 700 nm. A low concentration of N-methylphenazonium methosulphate (PMS) enhanced the rate of reoxidation of cytochrome b₅₆₃ in the dark. In the presence of higher concentrations of PMS a photooxidation, driven by Photosystem I, instead of reduction was observed. These observations suggest that PMS enhances the rate of reactions between reduced cytochrome b₅₆₃ and oxidized products of Photosystem I.

3. 3. In the presence of carbonylcyanide m-chlorophenylhydrazone (CCCP) a light-induced decrease of absorption at 560 nm occurred. Spectral evidence suggested the photooxidation of cytochrome b₅₅₉ under these conditions. Inhibition by DCMU and a relatively efficient action of orange light suggested that this photooxidation is driven by Photosystem II.

Spectral properties of stabilized D1/D2/cytochrome b-559 photosystem II reaction center complex Effects of Triton X-100, the redox state of pheophytin, and β-carotene

Absorption, fluorescence, and CD spectral properties of the isolated D1/D2/cytochrome b-559 photosystem II reaction center complex were examined in stabilized reaction center material at 77 K. Spectral properties were dependent on the presence or absence of 0.05% Triton X-100 in the RC suspension medium, on the redox state of pheophytin, and on the state of inactivation of the complex. The specific spectral properties of the PS II RC complex in the red suggest that the primary donor is not a bacterial-type special pair and could be a monomer. Furthermore, the spectral properties in the PS II RC may be the result of excitonic interactions among all the porphyrin molecules in the complex. Interactions between β-carotene and porphyrins indicate a significant role for β-carotene in the PS II RC.     

Structural analysis of the reaction products of chitosan with o-, m- and p-phthalaldehydes

Chitosan, a $\text{(I}\text{a}\text{?}\text{4)-}\text{linked}$ 2-amino-2-deoxy-β-d-glucan, was allowed to react with o-, m- and p-phthalaldehydes in aqueous acetic acid-methanol at room temperature to give the corresponding Schiff's base derivative (d.s. ～ 1.0/hexosaminyl residue) in a gel form. The dry product was isolated in yields of 90–97%, and the fine structure was analysed. Both the crosslinking and N-formylbenzylidene structures were present in an almost equal molar ratio in the reaction product of chitosan with p-phthalaldehyde and at a molar ratio of 0.1:0.9 in the reaction product of chitosan with m-phthalaldehyde. However, both structures were absent in the reaction product of chitosan with o-phthalaldehyde, and an intramolecular cyclic structure was present.     

The relationship between Q, C-550 and cytochrome b 559 in photoreactions at −196° in chloroplasts

Absorbance changes and fluorescence yield changes induced by irradiating spinach chloroplasts with red light at −196° were measured as a function of the redox potential of the chloroplast suspension. Absorbance changes at 546 nm indicate the photoreduction of C-550 and changes at 556 nm indicate the photooxidation of cytochrome b 559. The changes of fluorescence yield indicate the photoreduction of Q, the fluorescence quencher of chlorophylla a in Photosystem II. The titration curves for all three changes were essentially the same and showed the same midpoint potential. In other experiments as well, it was found that when C-550 is in the reduced state the fluorescence yield of the chloroplasts is high and the low-temperature photooxidation of cytochrome b 559 is blocked. These data indicate that C-550 may be equivalent to Q and that cytochrome b 559 serves as the electron donor for the photoreduction of C-550 at low temperature.     

Quantitation of o-, m- and p-cresol and deuterated analogs in human urine by gas chromatography with electron capture detection

A gas chromatographic method for the analysis of cresol metabolites of toluene and [²H₈]toluene in urine was developed. Cresol glucuronides and sulfates in urine were hydrolyzed with β-glucuronidase and arylsulfatase. Following extraction with tert.-butyl methyl ether and solvent exchange into benzene, the cresols were derivatized with heptafluorobutyric anhydride to form the heptafluorobutyrate esters. The derivatives were analyzed by gas chromatography with electron capture detection. Chromatographic resolution was achieved between all cresol isomers and their ²H₇ analogs. Calibration ranged from 0.001 to 500 μg/ml. Recoveries were 55–97% and showed no trend with respect to analyte concentration. Within-day precision of analyses of benchmark urine samples had a coefficient of variation of less than 4%. The assay sensitivity was limited by chromatographic background but was sufficient for quantification of the unlabeled cresols in urine from men with only environmental exposure to toluene. Average levels in urine samples from 45 men were 0.023, 0.054 and 37 μg/ml for o-, m- and p-cresol, respectively.     

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.     

Catalytic and chemical competence of regulation of cdc25 phosphatase by oxidation/reduction

Cdc25 phosphatases belong to the family of protein tyrosine phosphatases (PTPs) that contain an active-site cysteine and form a phosphocysteine intermediate. Recently, oxidation/reduction of active-site cysteines of PTPs, including Cdc25, has been proposed to serve as a form of reversible regulation for this class of enzymes. Here we provide in vitro evidence that supports the chemical and kinetic competence for oxidation/reduction of the active-site cysteines of Cdc25B and Cdc25C as a mechanism of regulation. Using kinetic measurements and mass spectrometry, we have found that the active-site cysteines of the Cdc25's are highly susceptible to oxidation. The rate of thiolate conversion to the sulfenic acid by hydrogen peroxide for Cdc25B is 15-fold and 400-fold faster than that for the protein tyrosine phosphatase PTP1B and the cellular reductant glutathione, respectively. If not for the presence of an adjacent (back-door) cysteine in proximity to the active-site cysteine in the Cdc25's, the sulfenic acid would rapidly oxidize further to the irreversibly inactivated sulfinic acid, as determined by using kinetic partitioning and mass spectrometry with mutants of these back-door cysteines. Thus, the active-site cysteine is protected by rapid intramolecular disulfide formation with the back-door cysteines in the wild-type enzymes. These intramolecular disulfides can then be rapidly and effectively rereduced by thioredoxin/thioredoxin reductase but not glutathione. Thus, the chemistry and kinetics of the active-site cysteines of the Cdc25's support a physiological role for reversible redox-mediated regulation of the Cdc25's, important regulators of the eukaryotic cell cycle.     

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

“Reduced minus oxidized” difference extinction coefficients Δ? in the α-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1 ± 1.0 mM⁻¹ cm⁻¹ and 27.0 ± 1.0 mM⁻¹ cm⁻¹ were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from −250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E_m values at pH 6.5 of 244 ± 11 mV and −94 ± 21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the α-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q_B site and the microenvironment of the heme group of Cyt b559 are discussed.     

Control of respiration in proteoliposomes containing cytochrome aa3 II. Inhibition by carbon monoxide and azide

1. Carbon monoxide (CO) acts competitively towards oxygen when the latter is taken up in respiration by cytochrome aa₃-containing proteoliposomes, both in the presence of p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin (deenergized state) and in their absence (energized state). At high levels of CO, the double reciprocal plots ($\text{1}\text{v}$ vs. $\text{1}\text{[}\text{O}{}_2\text{]}$) in the energized and deenergized states are parallel, i.e. energization acts “anti-competitively” towards oxygen, and the “respiratory control ratio” decreases as the oxygen concentration decreases.2. Azide acts non-competitively towards cytochrome c when the latter is oxidized by cytochrome aa₃-containing proteoliposomes both in the energized and deenergized (plus p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin) conditions. At low azide concentrations the apparent K_i for azide is unaffected by energization, but at high azide levels the K_i increases in energized liposomes, i.e. the “respiratory control ratio” decreases as the azide concentration increases.3. It is concluded that the inhibitor experiments are consistent with but do not prove the concept that the oxidase molecules in a single vesicle are responding to a single “energization state” or set of electrochemical gradients. This and other models are discussed.     

Oxidation of both termini of p- and m-xylene by Escherichia coli transformed with xylene monooxygenase gene

Xylene monooxygenase (XMO) from Pseudomonas putida mt-2 catalyzes oxidation of methyl group of toluene and xylenes. While it has been postulated that this enzyme oxidizes one methyl group of xylene, we observed that both methyl groups in p- and m-xylene were oxidized to alcohol and aldehyde when the relevant genes (xylM and xylA) were co-expressed in Escherichia coli C600 and MC4100. When p-xylene was used as a substrate, p-hydroxymethylbenzaldehyde and p-xylyleneglycol were identified, in addition to p-methylbenzylalcohol and p-tolualdehyde. When m-xylene was used as a substrate, m-hydroxymethylbenzaldehyde and m-xylyleneglycol were identified, in addition to m-methylbenzylalcohol and m-tolualdehyde. Ratio of the products varied significantly according to the reaction condition and host strain, presumably reflecting the relative activity of XMO and host-derived dehydrogenase(s). Using various oxidized compounds as substrates, it was indicated that dialcohol (p- or m-xylyleneglycol) was formed via p- or m-hydroxymethylbenzaldehyde, respectively, rather than directly from corresponding monoalcohol (p- or m-methybenzylalcohol).     

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol have been determined by single-crystal X-ray diffraction studies. The space group of the α-cyclodextrin–p-chlorophenol complex is P2₁2₁2₁ with unit cell dimensions of a=15.299(3), b=24.795(5), c=13.447(5) Å, and that of the α-cyclodextrin–p-cresol complex is P2₁ with unit cell dimensions of a=7.927(7), b=13.568(7), c=24.54(1) Å, β=90.41(8)°. In spite of the similar structures of guest molecules, both complexes have different inclusion modes and packing structures.