Energy-dependent accumulation of iron by isolated rat liver mitochondria. IV. Relationship to the energy state of the mitochondria

1. The energy-dependent accumulation of iron by isolated rat liver mitochondria, respiring on endogenous substrates, is strongly dependent on the efficiency of energy coupling in the respiratory chain as measured by respiratory control with ADP and the endogenous energy dissipation. The accumulation reached a saturation level at respiratory control with ADP values (with succinate as the substrate) of approx. 4.0.2. In the presence of exogenous substrate, the energy-dependent accumulation of iron was markedly reduced, primarily due to binding of iron as carboxylate complexes having less favourable dissociation constants than the iron(III)-sucrose complex(es).3. The effect of added ATP was at least 2-fold, i.e. that of providing energy and that of chelating iron. When the mitochondria respired on endogenous substrate, the energy-dependent accumulation of iron increased at low concentrations of ATP, whereas higher concentrations (> 50 μM) gradually inhibited the uptake.4. Energization of the mitochondria by the generation of an artificial K⁺ gradient across the inner membrane with valinomycin in a K⁺-free medium increased the energy-dependent accumulation of iron.     

Energy-dependent accumulation of iron by isolated rat liver mitochondria. III. Submitochondrial localization of the iron accumulated

Isolated rat liver mitochondria accumulate iron partly by an energy-dependent and partly by an energy-independent mechanism (Romslo, I. and Flatmark, T. (1973) Biochim. Biophys. Acta 305, 29–40). When the iron-loaded mitochondria were disrupted mechanically and the mitochondrial subfractions isolated by density gradient centrifugation, the iron accumulated by the energy-dependent mechanism was recovered mainly in the soluble matrix and intermembrane space (approx. 50% of the total activity) and the inner membrane (approx. 30%). A negligible contribution to the total iron content of the soluble fraction by intermembrane space was revealed by the preparation of ‘mitoplasts’. On the other hand, most of the energy-independent iron accumulation was confined to the outer and inner membranes (approx. 35% of the total activity in each).     

Studies on the mobilization of iron from ferritin by isolated rat liver mitochondria

Rat liver mitochondria and rat liver mitoplasts mobilize iron from ferritin by a mechanism which depends on a respiratory substrate (preferentially succinate), a small molecular weight electron mediator (FMN, phenazine methosulphate or methylene blue) and (near) anaerobic conditions.The release process under optimized conditions (approx. 50 μmol/l FMN, 1 mmol/l succinate, 0.35 mmol/l Fe(III) (as ferritin iron), 37°C and pH 7.40) amounts to 0.9–1.2 nmol iron/mg protein per min.The results suggest that ferritin might function as an intermediate in the cytosolic transport of iron to the mitochondria.     

Properties of photoreductions by photosystem II in isolated chloroplasts. III. The effect of uncouplers on phenylenediamine shuttles across the membrane in the presence of dibromothymoquinone

In the presence of the plastoquinone antagonist dibromothymoquinone the photoreduction of ferricyanide by isolated chloroplast membranes is attributed to Photosystem II. The reaction is stimulated by the addition of phenylenediamine or C-substituted phenylenediamines (which may form a diimine on oxidation) but not of N-substituted phenylenediamines (which form a stable radical on oxidation). Phenylenediamines also restore NADP reduction (and O₂ evolution) in 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)-treated chloroplasts. In this bypassing of the inhibition site, N-substituted phenylenediamines are very effective, whereas p-phenylenediamine and C-substituted phenylenediamines are inefficient. Uncouplers exhibit a surprising effect on these systems. Even under coupling conditions uncouplers inhibit electron flow to ferricyanide mediated by phenylenediamine in the pH range 7.3–8.0, whereas the restoration of the NADP system is stimulated.

For the interpretation of the results the side of the membrane involved is considered. It is proposed that in ferricyanide reduction by Photosystem II, a phenylenediimine/diamine shuttle operates which moves reducing equivalents from the inside to the outside across the membrane. This shuttle requires a pH gradient across the membrane because of different optimal ratios of diimine/diamine inside and outside. This pH difference is abolished by the uncoupler, accounting for the observed inhibition.

The restoration of electron flow from water to NADP in DBMIB-treated chloroplasts is assumed to be a bypass of the inhibition site inside the membrane via a phenylenediamine. Because the imine/amine ratio brought about by the pH gradient is not favorable for the inside oxidation an uncoupler stimulates NADP reduction even under coupling conditions.

Also in photoreductions by Photosystem I, for example NADP reduction at the expense of P-phenylenediamine/ascorbate, a shuttle of reducing equivalents across the membrane occurs but this time from outside to inside.     

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.     

Effects of 5,5'-diphenyl-2-thiohydantoin on respiration and oxidative phosphorylation of rat liver mitochondria.

5,5'-Diphenyl-2-thiohydantoin (DPTH) administered in vitro, inhibited state 3 oxidation, stimulated state 4 oxidation and decreased ADP:O ratio when 3-hydroxybutyrate and succinate were used as substrates. Considerably lower DPTH concentrations were required for the inhibition of 3-hydroxybutyrate oxidation (50% inhibition occurred at approximately 0.17 mumoles DPTH/mg protein) than were needed for inhibition of succinate oxidation (50% inhibition occurred at about 0.62 mumoles DPTH/mg protein). DPTH showed no inhibitory effects when ascorbate plus tetramethylphenylenediamine (TMPD) served as the substrate. The inhibition of state 3 respiration was not reversed by 2,4-dinitrophenol (DNP), although there was a slight increase in the DNP rate:state 3 rate suggesting the presence of a weak DPTH inhibotory site located within the Site I energy transport chain. Uncoupling, in the presence of DPTH, was observed with all substrates. In experiments utilizing sonicated mitochondria, DPTH inhibited NADH-linked oxidation, but did not inhibit succinate or ascorbate plus TMPD oxidation. The effects of DPTH were reversed by dilution and by addition of albumin. DPTH concentrations which produced inhibition of state 3 respiration in vitro were reached, in vivo, in the livers of rats receiving a single oral dose of 40 mg/kg of DPTH.     

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.     

Photooxidase system of Rhodospirillum rubrum. I. Photooxidations catalyzed by chromatophores isolated from a mutant deficient in photooxidase activity

The aerobic photooxidations of reduced 2,6-dichlorophenolindophenol and of reaction-center bacteriochlorophyll (P-870) have been investigated in membrane vesicles (chromatophores) isolated from a non-phototrophic Rhodospirillum rubrum strain. In aerobic suspensions of wild-type chromatophores, continuous light elicits an increase of the levels of 2,6-dichlorophenolindophenol and of oxidized P-870, which reach steady-state values shortly after the onset of illumination. In contrast, light induces in mutant suspensions a transient increase of the levels of 2,6-dichlorophenolindophenol and of oxidized P-870, which fall to low steady-state values within a few seconds. These observations suggest that the mutation has altered a redox constituent located on the low-potential side of the photochemical reaction center, between a pool of acceptors and oxygen.Since endogenous cyclic photophosphorylation is catalyzed by mutant chromatophores at normal rates, it appears that the constituent altered by the mutation does not belong to the cyclic electron-transfer chain responsible for photophosphorylation. However, the system which mediates the aerobic photooxidations and the cyclic system are not completely independent: endogenous photophosphorylation is inhibited by oxygen in wild-type chromatophores but not in mutant chromatophores; in addition, the inhibitor of cyclic electron flow, 2-heptyl-4-hydroxyquinoline-N-oxide, enhances the aerobic photooxidation of reduced 2,6-dichlorophenolindophenol by chromatophores from both strains.These results support a tentative branched model for light-driven electron transfer. In that model, the constituent altered in the mutant strain is located in a side electron-transfer chain which connects the cyclic acceptors to oxygen.     

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.     

The electric generator in photosynthesis of green plants. I. Vectorial and protolytic properties of the electron transport chain

Light induces the generation of an electrochemical potential difference across the functional membrane of photosynthesis of green plants. Experimental results on the electrochemical phenomena have been largely interpreted in terms of a vectorial alternating electron hydrogen transport system as originally hypothesized by Mitchell.We asked whether or not the reaction coordinate of the electron transport crosses the membrane, and whether or not the protolytic reactions at either side of the membrane can be understood from the protolytic properties of the redox components involved. For this we studied the flash-light-induced protolytic reactions in the outer and the inner aqueous phase of the chloroplast inner disk membranes. Four sites of protolytic reactions were identified, two at either side of the membrane. One of these sites had to be attributed to the reduction of the terminal electron acceptor at the outer side of the membrane. Evidence is presented for the coupling of the other sites to the oxidation of water at the inner side of the membrane, to the reduction of plastoquinone at the outer side and its oxidation at the inner side, respectively. These results support Mitchell's hypothesis for the generation of an electrochemical potential difference by a vectorial electron transport system.     

The respiratory chain of Azotobacter vinelandii II. The effect of cyanide on cytochrome d

1. Cyanide causes a slow disappearance of the oxidized band (648 nm) of cytochrome d in particles of Azotobacter vinelandii and inhibits the appearance of the reduced band (631 nm). No effect of cyanide is found on the reduced band of cytochrome d.

2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide deviates from first-order kinetics at lower temperatures (22 °C) indicating that at least two conformations of the enzyme are involved. At higher temperatures (32 °C) the observed kinetics of the cyanide reaction are first order with a k_on = 0.7 M⁻¹·s⁻¹ and with an estimated k_off of approximately 5·10⁻⁵ s⁻¹.

3. The value of the k_off (7·10⁻⁴−14·10⁻⁴ s⁻¹ at 32 °C) determined from the rate of reduction of cyanocytochrome d by Na₂S₂O₄ or NADH is one order of magnitude larger than the k_off value found when the enzyme is in its oxidized state.

4. No effect of cyanide is found on the spectrum of cytochrome a₁.     

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active.These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H₂O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.