Inorganic pyrophosphatase and photosynthesis by isolated chloroplasts. II. The controlling influence of orthophosphate

1. 1. It has been proposed that Mg²⁺, inorganic pyrophosphate and a protein fraction which exhibits fructose-1,6-diphosphatase activity may interact to regulate photosynthesis by isolated chloroplasts.

2. 2. Evidence is presented which confirms the interaction and regulation but shows that these effects are indirectly attributable to pyrophosphatase activity rather than fructose-1,6-diphosphatase.

3. 3. When provided with Mg²⁺ and PP_i the pyrophosphatase simply alters the proportions of orthophosphate and PP_i in the reaction mixture. As the P_i concentration is increased, it first stimulates and then inhibits, the degree of inhibition being enhanced by additional Mg²⁺. PP_i ameliorates the inhibition, possibly by chelation of Mg²⁺.

4. 4. It is concluded that the proposed regulation is ultimately governed by the P_i concentration and the known relationship between P_i uptake and triose phosphate export across the chloroplast envelope.

Activation of CO2 fixation in isolated spinach chloroplasts

1. 1. The effect of the Mg²⁺ concentration on the CO₂ fixation activity in situ in isolated and intact spinach chloroplasts upon suspension in hypotonic medium was examined. CO₂ fixation in the dark was activated 25–100 fold by 20 mM Mg²⁺ in the presence of added ATP plus either ribulose 5-phosphate or ribose 5-phosphate. 20 mM Mg²⁺-stimulated fixation only 2–3 fold in the presence of the substrate of fixation, ribulose 1,5-diphosphate. The highest Mg²⁺-stimulated rate of fixation in the dark observed with chloroplasts was 480 μmoles CO₂ fixed per mg chlorophyll per h.

2. 2. The concentration of bicarbonate at half of the maximal velocity (apparent K_m) during the Mg²⁺-stimulated fixation of CO₂ was 0.4 mM in the presence of ATP plus ribose 5-phosphate and 0.6 mM with ribulose 1,5-diphosphate.

3. 3. Dithioerythritol or light enhanced Mg²⁺-stimulated CO₂ fixation 1–3 fold in the presence of ATP plus ribose 5-phosphate but not ribulose 1,5-diphosphate.

4. 4. These results indicate that Mg²⁺ fluxes in the stroma of the chloroplast could control the activity of the phosphoribulokinase with a lesser effect on the ribulosediphosphate carboxylase. An increase in Mg²⁺ of 6–10 mM in the stroma region of the chloroplast would be enough to activate CO₂ fixation during photosynthesis.

Reaction of myosin with salicylaldehyde II. Effect of salicylalation on the ATPase activity of myosin

The effect of salicylalation on the biological properties of myosin was studied.

1. 1. The ATPase activity of myosin is affected by salicylalation if the treatment is carried out at higher pH than 6.5. The Mg²⁺-activated ATPase shows a maximal curve with 250–380% maximal activation when 25–70 moles of salicylaldehyde are bound per mole of myosin. The EDTA-activated ATPase decreases with increasing salicylalation. Ca²⁺-activated ATPase shows a small increase with increasing salicylalation.

2. 2. Less salicylaldehyde is bound if the treatment is carried out in the presence of ATP, while that of PP_i does not affect the degree of salicylalation. The enzymic properties of myosins salicylalated in the presence of ATP or PP_i are not different from those of the samples treated in their absence.

3. 3. Salicylalation decreases ATP sensitivity of ATPase and superprecipitation of actomyosins reconstituted from salicylalated myosins only if more than 50 moles of salicylaldehyde are bound per mole myosin.

Uptake and light activated esterification of P by isolated chloroplasts

1. 1. Esterification of ³²P₁ by illuminated chloroplasts prepared on a sucrose gradient was examined to establish the optimal incubation conditions.

2. 2. The evidence is consistent with phosphorylation being closely coupled to the sum of noncyclic and pseudocyclic electron flow and with the rate of electron flow responding to the availability of electron acceptors.

3. 3. Apparent K_m values for ADP and Mg²⁺ were found to be 40 and 250 μM, respectively. The K_m value for Mg²⁺ was increased by the presence of Ca²⁺. Two apparent values were observed for P₁ at 0.2 and 1.1 mM. Chloroplast damage resulted in increased apparent K_m (P₁) values.

4. 4. Acceleration of the esterification resulting from the addition of ADP and P₁ to the medium indicated that these compounds were able to penetrate to the active site of esterification.

5. 5. Ribose 5-phosphate (Rib-5-P) was shown to inhibit P₁ esterification without affecting the apparent K_m for ADP or P₁. The evidence suggests that Rib-5-P interferes with the uptake of P₁, and possibly ADP.

The mechanism of the oxidation of ascorbate and Mn by chloroplasts: The role of the radical superoxide

1. 1. The mechanism of the photooxidation of ascorbate and of Mn²⁺ by isolated chloroplasts was reinvestigated.

2. 2. Our results suggest that ascorbate or Mn²⁺ oxidation is the result of the Photosystem I-mediated production of the radical superoxide, and that neither ascorbate nor Mn²⁺ compete with water as electron donors to Photosystem II nor affect the rate of electron transport through the two photosystems: The radical superoxide is formed as a result of the autooxidation of the reduced forms of low potential electron acceptors, such as methylviologen, diquat, napthaquinone, or ferredoxin.

3. 3. In the absence of ascorbate or Mn²⁺ the superoxide formed dismutases either spontaneously or enzymatically producing O₂ and H₂O₂. In the presence of ascorbate or Mn²⁺, however, the superoxide is reduced to H₂O₂ with no formation of O₂. Consequently, in the absence of reducing compounds, in the reaction H₂O to low potential acceptor one O₂ (net) is taken up per four electrons transported where as in the presence of ascorbate, Mn²⁺ or other suitable reductants up to three molecules O₂ can be taken up per four electrons transported.

4. 4. This interpretation is supported by the following observations: (a) in a chloroplast-free model system containing NADPH and ferredoxin-NADP reductase, methylviologen can be reduced to a free radical which is autooxidizable in the presence of O₂; the addition of ascorbate or Mn²⁺ to this system results in a two fold stimulation of O₂ uptake, with no stimulation of NADPH oxidation. The stimulation of O₂ uptake is inhibited by the enzyme superoxide dismutase; (b) the stimulation of light-dependent O₂ uptake in the system H₂O → methylviologen in chloroplasts is likewise inhibited by the enzyme superoxide dismutase.

5. 5. In Class II chloroplasts in the system H₂O → NADP upon the addition of ascorbate or Mn²⁺ an apparent inhibition of O₂ evolution is observed. This is explained by the interaction of these reductants with the superoxide formed by the autooxidation of ferredoxin, a reaction which proceeds simultaneously with the photoreduction of NADP. Such an effect usually does not occur in Class I chloroplasts in which the enzyme superoxide dismutase is presumably more active than in Class II chloroplasts.

6. 6. It is proposed that since in the Photosystem I-mediated reaction from reduced 2,4-dichlorophenolindophenol to such low potential electron acceptor as methylviologen, superoxide is formed and results in the oxidation of the ascorbate present in the system, the ratio ATP/2e in this system (when the rate of electron flow is based on the rate of O₂ uptake) should be revised in the upward direction.

Chloroplast inorganic pyrophosphatase

An alkaline, Mg²⁺-dependent inorganic pyrophosphatase has been isolated from previously isolated spinach chloroplast. The activity of the enzyme was increased 100-fold, with a 42% yield, upon purification from the total soluble chloroplast enzymes. The pH optimum for the enzyme shifts from 9.0 at 5 mM Mg²⁺ to 7.0 at 40 mM Mg²⁺. The substrate for the reaction appears to be magnesium pyrophosphate, and anionic pyrophosphate is an effective inhibitor. There seems to be also an activating effect of Mg²⁺ on the enzyme at pH 7. No other cation substitutes for Mg²⁺ in activating the hydrolysis of pyrophosphate. Among anions tested, only F⁻ caused severe inhibition. The enzyme is inactive towards fructose 1,6-diphosphate, thiamine pyrophosphate, ATP, and ADP. The possibility that this enzyme is subject to metabolic regulation is discussed in relation to an indicated role of pyrophosphate in the regulation of photosynthetic carbon reduction.     

Studies on the ferrochelatase activity of isolated rat liver mitochondria with special reference to the effect of oxidizable substrates and oxygen concentration

The mitochondrial ferrochelatase activity has been studied in coupled rat liver mitochondria using deuteroporphyrin IX (incorporated into liposomes of lecithin) and Fe(III) or Co(II) as the substrates.

1. 1. It was found that respiring mitochondria catalyze the insertion of Fe(II) and Co(II) into deuteroporphyrin. When Fe(III) was used as the metal donor, the reaction revealed an absolute requirement for a supply of reducing equivalents supported by the respiratory chain.

2. 2. A close correlation was found between the disappearance of porphyrin and the formation of heme which allows an accurate estimate of the extinction coefficient for the porphyrin to heme conversion. The value Δ (mM⁻¹ · cm⁻¹) = 3.5 for the wavelength pair 498 509 nm, is considerably lower than previously reported.

3. 3. The maximal rate of deuteroheme synthesis was found to be approx. 1 nM · min⁻¹ · mg⁻¹ of protein at 37 °C, pH 7.4 and optimal substrate concentrations, i.e. 75 μM Fe(III) and 50 μM deuteroporphyrin.

4. 4. Provided the mitochondria are supplemented with an oxidizable substrate, the presence of oxygen has no effect on the rate of deuteroheme synthesis.

Biochemical and biophysical studies on cytochrome aa3 VIII. Effect of cyanide on the catalytic activity

1. 1. Cyanide inhibits the catalytic activity of cytochrome aa₃ in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·10³ M⁻¹·s⁻¹ and a K_i that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3.

2. 2. When cyanide is added to the ascorbate-cytochrome c-cytochromeaa₃−O₂ system a biphasic reduction of cytochrome c occurs corresponding to an initial K_i of 0.8 μM and a final K_i of about 0.1 μM for the cytochrome aa₃−cyanide reaction.

3. 3. The inhibited species (a²+a₃³⁺HCN) is formed when a²⁺a₃³⁺ reacts with HCN, when a²⁺a₃²⁺HCN reacts with oxygen, or when a³⁺a₃³⁺HCN (cyano-cytochrome aa₃) is reduced. Cyanide dissociates from a²⁺a₃³⁺HCN at a rate of 2·10⁻³ s⁻¹ at 22 °C, pH 7.3.

4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a²⁺a₃³⁺ than to a³⁺a₃³⁺.

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

Some properties of, and the effect of temperature on the (Mg + Ca) ATPase from immature and adult rat brain

1. 1. (Mg²⁺ + Ca²⁺) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg²⁺ + Ca²⁺) ATPase seen during development was greatest in the synaptic membrane preparations.

2. 2. At 37°C both Na⁺ or K⁺ at concentrations higher than 30 mM inhibited the microsomal Mg²⁺ ATPase, but the (Mg²⁺ + Ca²⁺) ATPase was stimulated by both Na⁺ and K⁺. Synaptic membrane Mg²⁺ ATPase was inhibited by concentrations higher than 100 mM K⁺; Na⁺ however stimulated this enzyme at all concentrations. Much of this Na⁺ stimulated activity was ouabain sensitive. Synaptic membrane (Mg²⁺ + Ca²⁺) ATPase was stimulated by Na⁺ or K⁺, this stimulation follows approximate saturation kinetics with an apparent K_m of 18.8 mM Na⁺ or K⁺.

3. 3. Arrhenius plots of microsomal (Mg²⁺ + Ca²⁺) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole⁻¹ above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg²⁺ + Ca²⁺) ATPase from both immature and adult brain.

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver

1. 1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis.

2. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enzyme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane.

3. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-P_i exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin.

4. 4. The decrease in ATP synthesis and ATP-P_i exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase.

5. 5. The following electron transport activities in the megamitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc₁-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%.

6. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.

Oxidative phosphorylation coupled to oxygen uptake and nitrate reduction in Micrococcus denitrificans

A procedure is described for preparing particles from cells of Micrococcus denitrificans which were broken osmotically after treatment with lysozyme.

1. 1. The preparations catalysed ATP synthesis coupled to O₂ uptake or NO₃⁻ reduction. With NADH or succinate as the electron donors the P:O ratios were about 1.5 and 0.5, respectively; and the P:NO₃⁻ ratios were about 0.9 and 0.06, respectively.

2. 2. Addition of ADP or P_i to the reaction mixture increased the rates of NADH-dependent O₂ uptake and NO₃⁻ reduction. Addition of 1 mM 2,4-dinitrophenol, which inhibited phosphorylation by 50–60%, increased the basal rates of electron transport.

3. 3. Evidence derived from spectrophotometry and from the differential inhibition by antimycin A of O₂ and NO₃⁻ reduction leads to the conclusion that the nitrate reductase interacted with the respiratory chain in the region of the b-type cytochrome, and that the c-type cytochrome present was not involved in the reduction of NO₃⁻ to NO₂⁻.

Affinities of ATP for the dinitrophenol-induced ATPase

1. 1. The effect of Mg²⁺ on ATP-dependent processes catalysed by intact rat-liver mitochondria can be explained quantitatively by the formation of Mg-ATP complexes that cannot act as a substrate for the adenine nucleotide translocator.

2. 2. The dinitrophenol-induced ATPase is characterized by two affinities of ATP: K_m(1) = 6.7 μM and K_m(2) = 63 μM, which contribute to the extent of 70% and 30%, respectively, to the total ATPase activity under the standard conditions employed.

3. 3. K_m(1) of ATP is competitively increased by atractyloside, and is insensitive to changes in cation concentration or to oligomycin or aurovertin.

4. 4. K_m(2) is as sensitive to atractyloside as the K_m(1) and is also insensitive to oligomycin. However, it is increased by decreasing the cation concentration, and disappears in the presence of aurovertin.

5. 5. It is proposed that two conformations of the adenine nucleotide translocator exist, characterized by their different affinities for ATP. The distribution of the enzyme over these two conformations appears to be a function of the energy state of the mitochondria (coupled or uncoupled).

The site of phosphorylation associated with Photosystem I

1. 1. Small particles prepared from spinach chloroplasts after treatment with digitonin, exhibited Photosystem I reactions, including phosphorylation, at rates as high as those in chloroplasts, whereas electron flow from water to NADP⁺ or ferricyanide through Photosystem II was completely lost. Mediators of cyclic electron flow, such as pyocyanine, or N-methylphenazonium methosulfate in red light, had to be reduced to support photophosphorylation.Diaminodurene at high concentrations catalyzed cyclic phosphorylation under anaerobic conditions without addition of a reductant. In fact, addition of ascorbate gave rise to a marked inhibition which was released by addition of a suitable electron acceptor such as methylviologen.

2. 2. Under aerobic conditions a low O₂ uptake, observed in the presence of diaminodurene, was stimulated several-fold upon addition of methylviologen and was stimulated again several-fold on further addition of ascorbate. The rate of phosphorylation, however, remained the same. The low P/2e ratio obtained under these conditions was not decreased at lower light intensities.

3. 3. These findings suggest a phosphorylation site associated with cyclic electron flow through Photosystem I without participation of the electron carriers of Photosystem II. A non-cyclic electron flow to O₂ can be induced in this system by addition of methylviologen which effectively competes with the electron acceptors of cyclic flow. This non-cyclic electron flow still involves the same phosphorylation site. A scheme for electron transport and for the location of phosphorylation sites in chloroplasts is proposed.

Effects of substrate and substrate analogues on the trinitrophenylation of myosin

The effects of ATP, ATP analogues, Mg²⁺ and actin on the trinitrophenylation of myosin and on the enzymic properties of trinitrophenylated samples were studied.

1. 1. Trinitrophenylation of myosin was inhibited by the presence of ATP and its analogues during the treatment in the order ADP > ATP > pyrophosphate > AMP.

2. 2. The alteration of the enzymic properties due to trinitrophenylation of myosin was prevented by the presence of ATP or ADP and somewhat less by that of pyrophosphate and AMP during trinitrophenylation, but only if Mg²⁺ was also present.

3. 3. Neither the degree of trinitrophenylation nor the enzymic properties of the trinitrophenylated myosin were influenced by the presence of actin during the treatment of myosin.

Acetylene reduction with physiological electron donors by extracts and particulate fractions from nitrogen-fixing Azotobacter chroococcum

1. 1. Preparations were obtained from Azotobacter chroococcum which reduced acetylene to ethylene using physiological electron donors instead of sodium dithionite. These preparations fell into two categories: those which required catalytic amounts of benzyl viologen for acetylene reduction and those that did not.

2. 2. Acetylene reduction without benzyl viologen or sodium dithionite was observed only with particles that sedimented at 40 000 × g after disrupting bacteria in the French press or with preparations obtained by disrupting bacteria protected by a mixture of defatted bovine serum albumin-Ficoll-MgCl₂ with liquid N₂; supernatant fractions required benzyl viologen for acetylene reduction.

3. 3. Added ATP inhibited acetylene reduction by large particles; ATP and MgCl₂ were necessary for maximum acetylene reduction with bovine serum albumin-protected preparations.

4. 4. NADH and carbon substrates acted as electron donors but H₂ did not; NAD⁺ was necessary for maximum acetylene reduction with carbon substrates.

5. 5. Anaerobic conditions were necessary for maximum acetylene reduction in all cases.

Influence of temperature changes on the ability of gangliosides to complex with Ca

1. 1.|The influence of temperature changes on Ca²⁺-binding to brain ganglioside mixtures of different polarity, to single gangliosides (G_M1, G_D1a, G_T1b) and to their deceramide was investigated potentiometrically by means of ion-selective electrodes.

2. 2.|Following cooling (3.5°C/min.) from 37 to 13°C the Ca²⁺-binding to gangliosides, except G_M1, was increased (7–30%).

3. 3.|Subsequent rewarming from 13 to 37°C resulted in up to 100% release of previously-bound Ca²⁺.

4. 4.|When comparing the maximal absolute binding difference of Ca²⁺ to gangliosides during temperature changes a decrease of these differences could be stated which corresponds to an increase in the polarity of the gangliosides.

5. 5.|From these experiments it is concluded that a higher polarity of neuronal gangliosides is responsible for a lower thermal sensitivity of Ca²⁺-binding to these compounds. This may be involved in the process of thermal adaptation of ectothermic vertebrates.

Transport of glutamate in rat-liver mitochondria

The transport of glutamate across the membrane of rat-liver mitochondria has been studied.

1. 1. Glutamate can be transported across the mitochondrial membrane in exchange for OH⁻ (or together with H⁺).

2. 2. Intramitochondrial glutamate is not extruded from the mitochondria by addition of aspartate when the mitochondria are preloaded with glutamate.

3. 3. N-Ethylmaleimide is a specific inhibitor of the movement of glutamate across the mitochondrial membrane.

Interaction of oxidized and reduced N-methylphenazonium methosulfate (PMS) with Photosystem II

In 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) poisoned chloroplasts, the restoration of the fluorescence induction is presumed to be due to a back reaction of the reduced primary acceptor (Q⁻) and the oxidized primary donor (Z⁺) of Photosystem II. Carbonylcyanide m-chlorophenylhydrazone (CCCP) is known to inhibit this back reaction. The influence of reduced N-methylphenazonium methosulfate (PMS) in the absence of CCCP and of oxidized PMS in the presence of CCCP on the back reaction was investigated and the following results were obtained:

1. (1) Reduced PMS at the concentration of 1 μM inhibits the back reaction as effectively as hydroxylamine, suggesting an electron donating function of reduced PMS for System II.

2. (2) The inhibition of the back reaction by CCCP is regenerated to a high degree by oxidized PMS which led to assume a cyclic System II electron flow catalysed by PMS.

3. (3) At concentrations of reduced PMS higher than 1 μM it is shown that both the fast initial emission and more significantly the variable emission are quenched.

The phosphorylation potential generated by respiring mitochondria

1. 1. The phosphorylation potential, ΔG_P = ΔG₀′ + 1.36 log ([ATP]/[ADP][P_i]), where ΔG_O′ is the standard free energy of hydrolysis of ATP at a given pH, and [ATP], [ADP] and [P_i] refer to concentrations in the suspending medium, has been determined in rat-liver mitochondria under various conditions.

2. 2. The ATP/ADP ratio is relatively constant, over a 10-fold range of phosphate concentration. Thus, the phosphate potential is higher at low phosphate concentration. State-4 rat-liver mitochondria in the presence of succinate, oxygen and low concentrations of phosphate in State 4 maintain a phosphorylation potential of 16.1 kcal (67.3 kJ) per mole ATP.

3. 3. High concentrations of ATP inhibit ADP uptake, and it is suggested that this is the reason for the independence of the ATP/ADP ratio on the phosphate concentration. A steady-state ratio is set up dependent upon two processes that are relatively slow compared with State-3 respiration, namely ADP transport and ATP hydrolysis.

4. 4. The phosphorylation potential calculated from the concentrations of total ADP, ATP and P_i within State-4 mitochondria is 4.5 kcal/mole less than that in the suspending medium.

5. 5. It was shown experimentally that the phosphorylation potential cannot be calculated from the ΔG of the redox couple, the respiratory-control ratio and the P:O ratio, as has been suggested in the literature.

6. 6. The measured phosphorylation potential is 83% of that calculated from the span succinate to oxygen, assuming thermodynamic equilibrium, and 95% of that calculated from the span NADH to oxygen.

7. 7. Based on the measurements of the phosphorylation potential and of the redox potentials and redox states of redox components in mitochondria, ubiquinone and cytochrome b are found at their expected position at the junction of the phosphorylations at Sites 1 and 2. The iron-sulphur centres 2 and 5 and the iron-sulphur centre of succinate dehydrogenase also probably lie at this junction. Cytochrome a₃ lies at its expected junction between phosphorylation Sites 2 and 3. A number of electron carriers (cytochromes c, c₁, and a, the iron-sulphur centre of Complex III and the EPR-detectable copper), however, lie in the ‘no-man's land’ within Site 2.

8. 8. A phosphorylation potential of 16.1 kcal/mole corresponds to a membrane potential of 350 mV in State 4, on the basis of the chemiosmotic hypothesis.