Myxothiazol, a new inhibitor of the cytochrome b-c1 segment of the respiratory chain

Myxothiazol inhibited oxygen consumption of beef heart mitochondria in the presence and absence of 2,4-dinitrophenol, as well as NADH oxidation by submitochondrial particles. The doses required for 50% inhibition were 0.58 mol myxothiazol/mol cytochrome b for oxygen consumption of beef heart mitochondria, and 0.45 mol/mol cytochrome b for NADH oxidation by submitochondrial particles. Difference spectra with beef heart mitochondria and with cell suspensions of Saccharomyces cerevisiae revealed that myxothiazol blocked the electron transport within the cytochrome b-c₁ segment of the respiratory chain. Myxothiazol induced a spectral change in cytochrome b which was different from and independent of the shift induced by antimycin. Myxothiazol did not give the extra reduction of cytochrome b typical for antimycin. Studies on the effect of mixtures of myxothiazol and antimycin on the inhibition of NADH oxidation indicated that the binding sites of the two inhibitors are not identical.     

A kinetic completion of the cyclic photosynthetic electron pathway of Rhodopseudomonas sphaeroides: Cytochrome b-cytochrome c2 oxidation-reduction

In Rhodopseudomonas sphaeroides, following a single-turnover flash of light, cytochrome c₂ is oxidized by reaction center bacteriochlorophyll, and a cytochrome b is reduced by the primary electron acceptor, probably via ubiquinone. In this report we show that, in the uncoupled state, the rate of re-oxidation of the cytochrome b is identical to the rate of reduction of the cytochrome c₂, a kinetic completion of the cyclic photosynthetic electron transport system.     

The respiratory chain of plant mitochondria. XV. Equilibration of cytochromes c549, b553, b557 and ubiquinone in mung bean mitochondria: Placement of cytochrome b557 and estimation of the midpoint potential of ubiquinone

1. 1. Cycles of oxidation followed by reduction at pH 7.2 have been induced in uncoupled anaerobic mung bean mitochondria treated with succinate and malonate by addition of oxygen-saturated medium. Under the conditions used, cytochromes b₅₅₇, b₅₅₃, c₅₄₉ (corresponding to c₁ in mammalian mitochondria) and ubiquinone are completely oxidized in the aerobic state, but become completely reduced in anaerobiosis.

2. 2. The time course of the transition from fully oxidized to fully reduced in anaerobiosis was measured for cytochromes c₅₄₉, b₅₅₇, and b₅₅₃. The intramitochondrial redox potential (IMP_h) was calculated as a function of time for each of the three cytochromes from the time course of the oxidized-to-reduced transition and the known midpoint potentials of the cytochromes at pH 7.2. The three curves so obtained are superimposable, showing that the three cytochromes are in redox equilibrium under these conditions during the oxidized-to-reduced transition.

3. 3. This result shows that the slow reduction of cytochrome b₅₅₇ under these conditions, heretofore considered anomalous, is merely a consequence of its more negative midpoint potential of +42 mV at pH 7.2, compared to +75 mV for cytochrome b₅₅₃ and +235 mV for cytochrome c₅₄₉. Cytochrome b₅₅₇ is placed on the low potential side of coupling site II and transfers electrons to cytochrome c₅₄₉ via the coupling site.

4. 4. The time course of the transition from fully oxidized to fully reduced was also measured for ubiquinone. Using the change in intramitochondrial potential IMP_h with time obtained from the three cytochromes, the change in redox state of ubiquinone with IMP_h was calculated. When replotted as IMP_h versus the logarithm of the ratio (fraction oxidized)/(fraction reduced), two redox components with n = 2 were found. The major component is ubiquinone with a midpoint potential E_m7.2 = + 70 mV. The minor component has a midpoint potential E_m7.2 = − 12 mV; its nature is unknown.

Photooxidation of cytochrome b559 and the electron donors in chloroplast Photosystem II

The effect of light on the reaction center of Photosystem II was studied by differential absorption spectroscopy in spinach chloroplasts.

At − 196 °C, continuous illumination results in a parallel reduction of C-550 and oxidation of cytochrome b₅₅₉ high potential. With flash excitation, C-550 is reduced, but only a small fraction of cytochrome b₅₅₉ is oxidized. The specific effect of flash illumination is suppressed if the chloroplasts are preilluminated by one flash at 0 °C.

At − 50 °C, continuous illumination results in the reduction of C-550 but little oxidation of cytochrome b₅₅₉. However, complete oxidation is obtained if the chloroplasts have been preilluminated by one flash at 0 °C. The effect of preillumination is not observed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

A model is discussed for the reaction center, with two electron donors, cytochrome b₅₅₉ and Z, acting in competition. Their respective efficiency is dependent on temperature and on their states of oxidation. The specific effect of flash excitation is attributed to a two-photon reaction, possibly based on energy-trapping properties of the oxidized trap chlorophyll.     

A simple procedure for the solubilization of NADH-cytochrome b5 reductase

NADH-cytochrome b₅ reductase has been solubilized by extraction of rabbit liver microsomes with 1 potassium phosphate buffer (pH 7.4), and has been purified to comparable purity with the Triton X-100-solubilized enzyme. Gel electrophoresis indicated an apparent molecular weight of 33,000 for both phosphate buffer-extracted and Triton X-100-solubilized enzymes. Phosphate buffer extraction provides a simple mild procedure for the extraction of NADH-cytochrome b₅ reductase that avoids detergents or proteolytic agents.     

Purification of cytochrome b6 A tightly bound protein in chloroplast membranes

Purification of cytochrome b₆ was pursued to further develop rational technology for purification, proof of purity, and study of properties of membrane proteins. Cytochrome b₆ was purified—the first time from any source—from spinach chloroplast membranes; yield of pure cytochrome b₆ was 30% of that found in ethanol-extracted particles. The three-step procedure (pH 8) employed: (I) extraction in Triton X-100-4 M (optionally 2 M) urea, (II) chromatography in a Bio-Gel A-1.5m Column (Triton X-100-4 M urea). Without this step, subsequent electrophoresis failed. (III) Preparative disc gel electrophoresis.

Properties of cytochrome b₆: Cytochrome b₆ migrated in undenatured form as a single band in disc electrophoresis (pH 8, 7 or 8.9). None of the limited, accepted properties of the cytochrome in particles was altered by the purification procedure: Reduced b₆ has absorption maxima (22 °C) at 434, 536, and 563 nm; at −199 °C the a absorption region shows two peaks of equal intensity at 561 and 557 nm. Cytochrome b₆ is reduced by dithionite (not by ascorbate) and is autoxidizable. The prosthetic group of b₆ is protohaemin and is fully extractable by acid-acetone. No non-haem iron is present. The millimolar extinction coefficient of reduced b₆ (563–600nm) per mole of haem is 21. The protein equivalent weight is 40000 g per mole of haem. Cytochrome b₄ is an intrinsically aggregatable molecule. The reduced cytochrome does not react with CO except when Triton X-100 is present.     

Cytochrome b5 reductase–cytochrome b5 as an active P450 redox enzyme system in Phanerochaete chrysosporium: Atypical properties and in vivo evidence of electron transfer capability to CYP63A2

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b₅ reductase–cyt b₅. In fungi, limited information is available for the cyt b₅ reductase–cyt b₅ system. Here we characterized the kinetic mechanism of (cyt b₅r)–cyt b₅ redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b₅r followed a “ping-pong” mechanism and could directly reduce cytochrome c. However, unlike other cyt b₅ reductases, Pc-cyt b₅r lacked the typical ferricyanide reduction activity, a standard for cyt b₅ reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b₅r–cyt b₅ complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b₅r–cyt b₅ complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b₅r–cyt b₅) in terms of supporting a P450 monooxygenase activity.     

Involvement of plastoquinone bound within the isolated cytochrome b6-f complex from chloroplasts in oxidant-induced reduction of cytochrome b6

(1) Oxidant-induced reduction of cytochrome b₆ is completely dependent on a reduced component within the isolated cytochrome b₆-f complex. This component can be reduced by dithionite or by NADH/N-methylphenazonium methosulfate. It is a 2H⁺/2e⁻ carrier with a midpoint potential of 100 mV at pH 7.0, which is very similar to the midpoint potential of the plastoquinone pool in chloroplasts. (2) Oxidant-induced reduction of cytochrome b₆ is stimulated by plastoquinol-1 as well as by plastoquinol-9. The midpoint potential of the transient reduction of cytochrome b₆, however, was not shifted by added plastoquinol. (3) Quinone analysis of the purified cytochrome b₆-f complex revealed about one plastoquinone per cytochrome f. The endogenous quinone is heterogeneous, a form more polar than plastoquinone-A, probably plastoquinone-C, dominating, This is different from the thylakoid membrane where plastoquinone-A is the main quinone. (4) The endogenous quinone can be extracted from the lyophilized cytochrome b₆-f complex by acetone, but not by hydrocarbon solvents. Oxidant-induced reduction of cytochrome b₆ was observed in the lyophilized and hexane-extracted complex, but was lost in the acetone-extracted complex. Reconstitution was achieved either with plastoquinol-1 or plastoquinol-9, suggesting that a plastoquinol molecule is involved in oxidant-induced reduction of cytochrome b₆.     

Cytochrome c1 and b-type cytochrome with two heme centers in the photosynthetic membranes from Rhodopseudomonas palustris

The photosynthetic membranes from Rhodopseudomonas palustris contained one species of membrane-bound c-type cytochrome, presumably cytochrome c₁, and a b-type cytochrome with two heme centers. The molecular weight and midpoint potential of cytochrome c₁ were 30000 and 275 mV, respectively. The peak of the reduced-minus-oxidized difference spectrum of cytochrome c₁ was at 552 nm. Molecular weight of the b-type cytochrome was 32000 and the cytochrome had two midpoint potentials of 60 mV and −55 mV. The peaks of the reduced-minus-oxidized difference spectra of the high and low midpoint potential heme centers were at 560 and 562 nm, respectively. These results suggested that there was a cytochrome b-c₁ complex in Rps. palustris.     

Regulation of the photosynthetic electron transport chain

The regulation of electron transport between photosystems II and I was investigated in the plant Silene dioica L. by means of measurement of the kinetics of reduction of P₇₀₀ following a light-to-dark transition. It was found that, in this species, the rate constant for P₇₀₀ reduction is sensitive to light intensity and to the availability of CO₂. The results indicated that at 25 °C the rate of electron transport is down-regulated by approximately 40–50% relative to the maximum rate achievable in saturating CO₂ and that this down-regulation can be explained by regulation of the electron transport chain itself. Measurements of the temperature sensitivity of this rate constant indicated that there is a switch in the rate-limiting step that controls electron transport at around 20 °C: at higher temperatures, CO₂ availability is limiting; at lower temperatures some other process regulates electron transport, possibly a diffusion step within the electron transport chain itself. Regulation of electron transport also occurred in response to drought stress and sucrose feeding. Measurements of non-photochemical quenching of chlorophyll fluorescence did not support the idea that electron transport is regulated by the pH gradient across the thylakoid membrane, and the possibility is discussed that the redox potential of a stromal component may regulate electron transport. Received: 4 March 1999 / Accepted: 25 May 1999     

Single and multiple turnover reactions in the ubiquinone-cytochrome b-c2 oxidoreductase of Rhodopseudomonas sphaeroides. The physical chemistry of the major electron donor to cytochrome c2, and its coupled reactions

We have examined the thermodynamic properties of the physiological electron donor to ferricytochrome c₂ in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides. This donor (Z), which is capable of reducing the ferri-cytochrome with a halftime of 1–2 ms under optimal conditions, has an oxidation-reduction midpoint potential of close to 150 mV at pH 7.0, and apparently requires two electrons and two protons for its equilibrium reduction.

The state of reduction of Z, which may be a quinone · protein complex near the inner (cytochrome c₂) side of the membrane, appears to govern the rate at which the cyclic photosynthetic electron transport system can operate. If Z is oxidized prior to the flash-oxidation of cytochrome c₂, the re-reduction of the cytochrome takes hundreds of milliseconds and no third phase of the carotenoid bandshift occurs. In contrast if Z is reduced before flash activation, the cytochrome is rereduced within milliseconds and the third phase of the carotenoid bandshift occurs. The prior reduction of Z also has a dramatic effect on the uncoupler sensitivity of the rate of electron flow; if it is oxidized prior to activation, uncoupler can stimulate the cytochrome re-reduction after several turnovers by less than tenfold, but if it is reduced prior to activation, the stimulation after several turnovers can be as dramatic as a thousandfold. The results suggest that Z plays a central role in controlling electron and proton movements in the ubiquinone cytochrome b-c₂ oxido-reductase.     

Photoreactions of cytochrome b6 in systems using resolved chloroplast electron-transfer complexes

Photoreactions of cytochrome b₆ have been studied using resolved chloroplast electron-transfer complexes. In the presence of Photosystem (PS) II and the cytochrome b₆-f complex, photoreduction of the cytochrome can be observed. No soluble components are required for this reaction. Cytochrome b₆ photoreduction was found to be inhibited by quinone analogs, which inhibit at the Rieske iron-sulfur center of the cytochrome complex, by the addition of ascorbate and by depletion of the Rieske center and bound plastoquinone from the cytochrome complex. Photoreduction of cytochrome b₆ can also be demonstrated in the presence of the cytochrome complex and PS I. This photoreduction requires plastocyanin and a low-potential electron donor, such as durohydroquinone. Cytochrome b₆ photoreduction in the presence of PS I is inhibited by quinone analogs which interact with the Rieske iron-sulfur center. These results are discussed in terms of a Q-cycle mechanism in which plastosemiquinone serves as the reductant for cytochrome b₆ via an oxidant-induced reductive pathway.     

Involvement of plastoquinone and lipids in electron transport reactions mediated by the cytochrome b6-f complex isolated from spinach

The isolation of a cytochrome b₆-f complex from spinach, which is depleted of plastoquinone (and lipid), is reported. The depleted complex no longer functions as a plastoquinol-plastocyanin oxidoreductase but can be reconstituted with plastoquinone and exogenous lipids. The lipid classes digalactosyldiacylglycerol, phosphatidylglycerol and phosphatidylcholine were active in reconstitution while monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol were not. Neither plastoquinone nor lipid alone fully reconstitutes electron transport in the depleted complex. Saturation of plastoquinol-plastocyanin oxidoreductase activity in the depleted complex occurs at 1 plastoquinone per cytochrome f.     

Purification by hydrophobic chromatography of soluble cytochrome b5 of human erythrocytes

Soluble cytochrome b₅ of human erythrocytes was purified very effectively by hydrophobic chromatography using a butyl-Toyopearl 650 column. Cytochrome b₅ was adsorbed tightly on the column in the presence of 60% saturated ammonium sulfate, and was eluted at 40% saturation of ammonium sulfate in the elution buffer. The chromatography gave a good yield of cytochrome b₅ of the highest purity so far reported as estimated from the 414 nm to 280 nm absorbance ratio of the oxidized form of the cytochrome b₅. The value obtained wit the cytochrome b₅ purified in this study was 6.57, and is higher than the previously reported highest value of 6.4 (Hultquist, D.E., Dean, R.T. and Douglas, R.H. (1974) Biochem. Biophys. Res. Commun. 60, 28–34). Spectral properties including molecular absorption coefficients were determined using the cytochrome b₅ purified by this method.     

Modulation of the reductive metabolism of halothane by microsomal cytochrome b5 in rat liver

To study the modulation of the reductive metabolism of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) by microsomal cytochrome b₅, formation of 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), major reduced metabolites of halothane, was analyzed in vivo and in vitro. Rats were pretreated with both malotilate (diisopropyl-1,3-dithiol-2-ylidenemalonate) and sodium phenobarbital (malotilate-treated rats) or only with sodium phenobarbital (control rats). The microsomes of malotilate-treated rats had significantly more cytochrome b₅ than the controls, whereas the cytochrome P-450 content was not different between the two groups. At the end of 2-h exposure to 1% halothane in 14% oxygen, the ratio of CDE to CTE in arterial blood was significantly higher in malotilate-treated rats than in the controls. Under anaerobic conditions, the formation of CDE and the ratio of CDE to CTE were significantly greater in microsomal preparations of malotilate-treated rats than those of the controls. In a reconstituted system containing cytochrome P-450_PB purified from rabbit liver, addition of cytochrome b₅ to the system enhanced the formation of CDE and increased the ratio of CDE to CTE. These results suggested that cytochrome b₅ enhances the formation ratio of CDE to CTE by stimulating the supply of a second electron to cytochrome P-450, which might reduce radical reactions in the reductive metabolism of halothane.     

The influence of cytochrome b559 on the fluorescence yield of chloroplasts at low temperature

The maximum light-induced fluorescence yield, F_M, of spinach chloroplasts at − 196 °C was less when the chloroplasts were oxidized with ferricyanide prior to freezing; the minimum fluorescence yield, F₀, of the dark-adapted chloroplasts at − 196 °C was unaffected. The ratio of the fluorescence yields, F_M/F₀, measured at 695 nm at low temperature was 4.5–5.0 for normal chloroplasts and 2.0–2.5 in the presence of ferricyanide. The oxidative titration curve of F_M followed a 1 electron Nernst equation with a midpoint potential of 365 mV and followed closely to the oxidation of cytochrome b₅₅₉. The photoreduction of C−550 at low temperature was the same at all redox potentials over the range of 200–500 mV. It is suggested that a relatively strong oxidant associated with the water-splitting side of Photosystem II, possibly the primary electron donor, can chlorophyll fluorescence of Photosystem II as well as the primary electron acceptor.     

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.