N-methyl oxidation in liver mitochondria of triiodothyronine-treated and thyroidectomized rats

The levels of sarcosine dehydrogenase and acid-nonextractable flavin in the inner matrix of mitochondria of rat liver are decreased in animals treated with triiodothyronine and are elevated in the mitochondria obtained from thyroidectomized animals. Administration of triiodothyronine does not affect the electron-transfer flavoprotein associated with the sarcosine dehydrogenase or the relative amounts of soluble and membrane-bound proteins of the mitochondria. In phosphate-washed mitochondria from either the controls or the triiodothyronine-treated rats, the O₂ uptake equals the total of the [¹⁴C]formaldehyde and [β-¹⁴C]serine isolated as reaction products of the sarcosine-[¹⁴C]methyl group. In contrast to its restraint of sarcosine or choline oxidation in preparations capable of oxidative phosphorylation, ADP does not inhibit the oxidation of these substrates in mitochondria of rats given triiodothyronine.     

Fluorescence of bacteriochlorophyll as related to the photochemistry of chromatophores of photosynthetic bacteria

Time courses and the emission spectra of fluorescence and light-induced absorption changes of P890 in chromatophores of the photosynthetic bacteria Chromatium D, Rhodopseudomonas spheroides and Rhodospirillum rubrum were investigated.

The time course of fluorescence in chromatophores was separated into two phases, i.e. an initial rapid rise (ƒ_i) and a subsequent slow increase towards a steady level of emission (ƒ_v). The ƒ_i and the ƒ_v components showed different emission spectra having different peak position. The ƒ_v component was emitted from the longest wavelength-absorbing form of bulk bacteriochlorophyll (B890), the ƒ_i component from both B890 and B850.

The magnitude of the ƒ_v component depended on experimental conditions controlling the states of the cyclic electron transport in chromatophores, including changes in levels of redox potential of the medium, additions of electron donors and inhibitors. The magnitude of the ƒ_i component was not affected by these experimental conditions. It was, therefore, concluded that only the ƒ_v component is related to the cyclic electron transport, and that the magnitude of ƒ_v is controlled by the oxidation-reduction state of the primary electron acceptor for the photochemical reaction center in chromatophores.     

The characterisation of inducible dehydrogenases specific for the oxidation of d-alanine,allohydroxy-d-proline,choline and sarcosine as peripheral membrane proteins in Pseudomonas aeruginosa

The interaction with the cytoplasmic membrane of the inducible, membrane-bound, cytochrome-linked dehydrogenases specific for the oxidation of d-alanine, allohydroxy-d-proline, choline and sarcosine in Pseudomonas aeruginosa was investigated. The susceptibility of d-alanine dehydrogenase to solubilisation by cation depletion or by washing with high ionic strength buffers indicated that it was a peripheral membrane protein. The effect of various divalent cations in reducing the amount of enzyme released by cation depletion suggests a requirement for Mg²⁺ in the binding of d-alanine dehydrogenase to the cytoplasmic membrane. The peripheral nature of all four dehydrogenases was confirmed by examination of the molecular properties and phospholipid content of preparations of the enzymes solubilised with 1 M phosphate buffer (pH 7.0). Additional confirmatory evidence was provided by Arrhenius plots of membrane-bound activity of d-alanine and allohydroxy-d-proline dehydrogenases which were monophasic and independent of the discontinuities attributable to membrane lipid phase separations which characterise such plots of the activity of integral membrane-bound enzymes. The shape of the Arrhenius plots obtained for the activities of known integral respiratory proteins of P. aeruginosa suggests that these enzymes may remain in a fluid environment throughout the course of the phase separation.     

Circular dichroism of cytochrome oxidase, cytochrome b566, and cytochrome c in beef heart mitochondrial membrane fragments

1. Circular dichroism spectra of the cytochromes in membrane fragments derived from sonicated beef heart mitochondria have been obtained in the wavelength region 400–480 nm in which the major absorbance maxima of the heme prosthetic groups are found.

2. 2. Cytochrome oxidase in the mitochondrial membrane fragments has a band of positive ellipticity at 426 nm in the oxidized form and a pronounced band of positive ellipticity at 445 nm in the reduced form. The reduced-minus-oxidized difference molar ellipticity at 445 nm, Δ[θ]₄₄₅ is 3.0·10⁵ degree·cm⁻²·dmole⁻¹ heme a for membrane-bound oxidase compared to 1.6·10⁵ degree·cm⁻²·dmole⁻¹ heme a for the purified oxidase. The membrane-bound oxidase in the reduced form also appears to have a band of negative ellipticity at 426 nm not found in the purified oxidase.

3. 3. When reduced with succinate in the presence of cyanide and oxygen, cytochrome oxidase in the membrane fragments has a positive band at 442 nm very similar to that observed with the purified oxidase.

4. 4. Cytochrome c, which has a positive band at 426 nm in the purified form when reduced, appears to have a negative band at this wavelength in the mito-chondrial membrane fragments which contributes to the pronounced negative band at 426 nm observed in the membrane fragments reduced with succinate in anaerobiosis. There is no evidence for a contribution to the CD spectra of the membrane fragments from cytochrome c₁ or from cytochrome b₅₆₁ in either the oxidized or the reduced form.

5. 5. Cytochrome b₅₆₆ in the mitochondrial membrane fragments has no detectable CD spectrum in the oxidized form, but has a small positive band at 427 nm and a small negative band at 436 nm in the reduced form. The same CD spectrum is observed with cytochrome b₅₆₆ reduced with succinate in the presence of antimycin A or 2-heptyl-4-hydroxyquinoline-N-oxide. The same increase in positive ellipticity is observed at 427 nm in the mitochondrial membrane fragments, treated with oligomycin to restore energy coupling, when cytochrome b₅₆₆ is reduced with succinate in the energized membrane, as is observed in the inhibitor-treated membrane fragments. The absence of a pronounced conformational change in cytochrome b₅₆₆ on energization, as revealed by its CD spectrum, favors the concept that its reduction by succinate in the energized state is due to reversed electron transport rather than an intrinsic shift in the cytochrome's midpoint redox potential.

Components of cytochrome c oxidase detectable by EPR spectroscopy

A procedure for the preparation from frozen beef heart mitochondria of cytochrome c oxidase (EC 1.9.3.1) of high heme ( 14 μmoles/mg protein) and low extraneous copper ( 1.1 atoms Cu/mole heme) and low lipid ( 0.05 g phospholipid/g protein) content is described. EPR signals observed with the enzyme between 6 and 100 °K at various states of oxidation and at different conditions of pH and presence of solutes are described in detail. The quantities of paramagnetic species represented by these signals are estimated. Under no conditions does the sum of the EPR detectable species represent more than approx. 50% of the potentially paramagnetic components of the enzyme. Comparisons are made to the corresponding signals as observed in whole tissue, mitochondria and submitochondrial particles from a number of species. The assignment of the observed signals to known components of cytochrome c oxidase is discussed briefly.     

Studies on electron transport associated with Photosystem I. I. Functional site of plastocyanin: Inhibitory effects of HgCl2 on electron transport and plastocyanin in chloroplasts

1. Incubation of chloroplasts with HgCl₂ at a molar ratio of HgCl₂ to chlorophyll of about unity, induced a complete inhibition of the methyl viologen Hill reaction, as well as methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol (DCIP) as electron donor. Photooxidation of cytochrome ? was similarly sensitive towards HgCl₂, whereas photooxidation of P700 was resistant to the poison. Photoreduction of cytochrome ? and light-induced increase in fluorescence yield were enhanced by the HgCl₂ treatment of chloroplasts.     

The respiratory system of the marine bacterium Beneckea natriegens. II. Terminal branching of respiration to oxygen and resistance to inhibition by cyanide

1. Cell-free extracts of the marine bacterium Beneckea natriegens, derived by sonication, were separated into particulate and supernatant fractions by centrifugation at 150 000 × g.2. NADH, succinate, d(?)- and l(+)-lactate oxidase and dehydrogenase activities were located in the particles, with 2- to 3-fold increases in specific activity over the cell free extract. The d(?)- and l(+)-lactate dehydrogenases were NAD⁺ and NADP⁺ independent. Ascorbate-N,N,N′,N′-tetramethylphenylenediamine (TMPD) oxidase was also present in the particulate fraction; it was 7–12 times more active than the physiological substrate oxidases.3. Ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide. Succinate, NADH, d(?)-lactate and l(+)-lactate oxidases were inhibited in a biphasic manner, with 10 μM cyanide causing only 10–50 % inhibition; further inhibition required more than 0.5 mM cyanide, and 10 mM cyanide caused over 90 % inhibition. Low sulphide (5 μM) and azide (2 mM) concentrations also totally inhibited ascorbate-TMPD oxidase, but only partially inhibited the other oxidases. High concentrations of sulphide but not azide caused a second phase inhibition of NADH, succinate, d(?)-lactate and l(+)-lactate oxidases.4. Low oxidase activities of the physiological substrates, obtained by using non-saturating substrate concentrations, were more inhibited by 10 μM cyanide and 2 mM azide than high oxidase rates, yet ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide over a wide range of rates of oxidation.5. These results indicate terminal branching of the respiratory system. Ascorbate-TMPD is oxidised by one pathway only, whilst NADH, succinate, d(?)-lactate and l(+)-lactate are oxidised via both pathways. Respiration of the latter substrates occurs preferentially by the pathway associated with ascorbate-TMPD oxidase and which is sensitive to low concentrations of cyanide, azide and sulphide.6. The apparent K_m for O₂ for each of the two pathways was detected using ascorbate-TMPD and NADH or succinate plus 10 μM cyanide respectively. The former pathway had an apparent K_m of 8–17 (average 10.6) μM and the latter 2.2–4.0 (average 3.0) μM O₂.     

Alkali-induced reduction of the b-cytochromes in purified Complex III from beef heart mitochondria

Approx. 40–50% of the cytochrome b in purified Complex III is reduced by ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine or phenazine methosulfate at neutral pH. The remaining cytochrome b, including cytochrome b-565, is reduced by increasing the pH. The apparent pK for this reduction is between pH 10 and 11, and is more than two pH units higher than a similar alkali-induced transition in Mg-ATP particles. Alkali-induced reduction of cytochrome b occurs concomitantly with the exposure of hydrophobic tyrosine and tryptophan residues to a more hydrophilic environment. The relationship of these findings to the presence of a substrate accessibility barrier in Complex III is discussed.     

Trypsin inhibition of electron transport

1. 1. A relaxation spectrophotometer was employed to measure the effects of trypsin treatment on electron transport in both cyclic and non-cyclic chloroplast reactions. The parameters measured were electron flow rate through P700 (flux) and the time constant for dark reduction of P700.

2. 2. In the reduction of methyl viologen by the ascorbate-2,6-dichlorophenol-indophenol (DCIP) donor couple, there was no effect of trypsin on P700 flux or on the time constant for dark reduction of P700. In the phenazine methosulfate (PMS) cyclic system, trypsin had either a slightly stimulatory or slightly inhibitory effect on the P700 flux, depending on the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU): either effect being marginal compared to trypsin effects on Photosystem II.With both ferricyanide and methyl viologen reduction from water, trypsin treament gave a first order decline in P700 flux: which matched the trypsin-induced decline in electron transport with the water to DCIP system, measured by dye reduction. This implies that Photosystem II is inhibited. The inhibition of Photosystem II was up to 90% with a 6–10-min trypsin treatment. This result is consistent with the concept of Photosystem I (P700) being in series with Photosystem II in the electron transfer sequence.

3. 3. Cyclic phosphorylation was severely inhibited (85%) by trypsin treatment which had a somewhat stimulatory effect on P700 flux, indicating uncoupling. Non-cyclic phosphorylation was uncoupled as well as electron flow being inhibited since the P/2e ratio decreased more rapidly as a function of trypsin incubation time than inhibition of electron flow. The two effects, uncoupling and non-cyclic electron flow inhibition, are separate actions of trypsin. It is probably that the uncoupling action of trypsin is due to attack on the coupling factor protein, known to be exposed on the outer surface of thylakoids.

4. 4. Trypsin treatment caused an increase in the rate constant, k_d, for the dark H⁺ efflux, resulting in a decreased steady state level of proton accumulation. The increased proton efflux and the inhibition of phosphorylation are consistent with an uncoupling effect on trypsin.

5. 5. Trypsin treatment did not reduce the manganese content of chloroplasts: as reported by others, Tris washing did remove about 30% of the chloroplast manganese.

6. 6. Electron micrographs of both negatively stained and thin-sectioned preparations showed that, under these conditions, trypsin does not cause a general breakdown of chloroplast lamellae. Inhibition by trypsin must therefore result from attacks on a few specific sites.

7. 7. Both System II inhibition and uncoupling occur rapidly when trypsin treatment is carried out in dilute buffer, a condition which leads to thylakoid unstacking, but both are prevented by the presence of 0.3 M sucrose and 0.1 M KCl, a condition that helps maintain stacked thylakoids. Evidently vulnerability to trypsin requires separation of thylakoids.

8. 8. Since trypsin does not appear to disrupt thylakoids nor prevent their normal aggregation in high sucrose-salt medium and since the trypsin molecule is probably impermeable, it is probable that the site(s) of trypsin attack in System II are exposed on the outer thylakoid surface.

Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii

A membrane-bound cytochrome oxidase from Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using an ascorbate-TMPD oxidation assay. The oxidase was ‘solubilized’ from a sonic-type electron-transport particle (R₃ fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27–70% (NH₄)₂SO₄. The highly purified cytochrome oxidase has a V of 60–78 μgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN₃ and NH₂OH; NaNO₂ (but not NaNO₃) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c₄ and cytochrome o; cytochromes a₁ and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c₄?o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a₃ oxidase of mammalian mitochondria.     

Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP⁺). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, is defined as the actinic light intensity that, at a given concentration of electron donor, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ is compared with another parameter, equivalent to $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, that is obtained independently of the reaction-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow.The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP⁺ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.     

Solubilization and purification of cytochrome a1 from Nitrosomonas

Cytochrome a₁ was solubilized with Triton X-100 from a membrane-envelope preparation of Nitrosomonas and partially purified by repeated fractionation with (NH₄)₂SO₄. The purified fraction of cytochrome a₁ was enriched over the crude extract by a factor of 16 and 300 with respect to protein and c-type cytochrome, respectively. The cytochrome was characterized as cytochrome a₁ on the basis of (a) reduced absorption maxima at 444 nm and 595 nm, (b) acid acetone extractibility and ether solubility of the heme and (c) absorption maximum of 587 nm of the ferro-hemochrome in alkaline pyridine. The α absorption band shifted from 600 nm to 595 nm upon solubilization of the cytochrome with Triton X-100. Spectral shifts were observed in the presence of cyanide and azide and the cytochrome changed with aging to a form with a reduced absorption band at 422 nm. Cytochrome a₁ was reduced anaerobically in the presence of reduced mammalian cytochrome c and was rapidly reoxidized in the presence of O₂. CO caused a shift in the soret peak of the reduced form but did not prevent reoxidation of cytochrome a₁ in the presence of CO-O₂ (95:5, v/v).     

Light-induced slow changes in chlorophyll a fluorescence in isolated chloroplasts: Effects of magnesium and phenazine methosulfate

We have investigated the possible relationships between the cation-induced and phenazine methosulfate (PMS)-induced fluorescence changes and their relation to light induced conformational changes of the thylakoid membrane.1. In isolated chloroplasts, PMS markedly lowers the quantum yield of chlorophyll a fluorescence (φ_f) when added either in the presence or the absence of dichloro-phenyldimethylurea (DCMU). In contrast, Mg²⁺ causes an increase in φ_f. However, these effects are absent in isolated chloroplasts fixed with glutaraldehyde that retain (to a large extent) the ability to pump protons, suggesting that structural alteration of the membrane—not the pH changes—is required for the observed changes in φ_f. The PMS triggered decrease in φ_f is not accompanied by any changes in the emission (spectral) characteristics of the two pigment systems, whereas room temperature emission spectra with Mg²⁺ and Ca²⁺ show that there is a relative increase of System II to System I fluorescence.2. Washing isolated chloroplasts with 0.75 mM EDTA eliminates (to a large extent) the PMS-induced quenching and Mg²⁺-induced increase of φ_f, and these effects are not recovered by the further addition of dicyclohexyl carbodiimide. It is known that washing with EDTA removes the coupling factor, and thus, it seems that the coupling factor is (indirectly) involved in conformational change of thylakoid membranes leading to fluorescence yield changes.3. In purified pigment System II particles, neither PMS nor Mg²⁺ causes any change in φ_f. Our data, taken together with those of the others, suggest that a structural modification of the thylakoid membranes (not macroscopic volume changes of the chloroplasts) containing both Photosystems I and II is necessary for the PMS-induced quenching and Mg²⁺-induced increase of φ_f. These two effects can be explained with the assumption that the PMS effect is due to an increase in the rate of internal conversion (k_h), whereas the Mg²⁺ effect is due to a decrease in the rate of energy transfer (k_t), between the two photosystems.4. From the relative ratio of φ_f with DCMU and DCMU plus Mg²⁺, we have calculated k_t (the rate constant of energy transfer between Photosystems II and I to be 4.2·10⁸ s^?1, and φ_t (quantum yield of this transfer) to be 0.12.