E.C. Slater

Uncoupling activity of long-chian fatty acids

1. 1. At all pH values between 6.0 and 9.5 unsaturated long-chain fatty acids are more effective than the saturated acids in stimulating the latent ATPase activity of fresh rat-liver mitochondria, provided Mg2+ is added. Oleic, linoleic, linolenic, arachidonic and elaidic acids are about equally effective. 2. 2. Whereas the saturated fatty acids scarcely stimulate above pH 8 the unsaturated acids are maximally active at PH 9. 3. 3. Mg2+ was required for maximal stimulation by oleic acid. In the absence of added Mg2+ oleic acid completely inhibited the ATPase stimulated by 2,4-dinitro-phenol. 4. 4. Oleic acid concentration that gave complete loss of ADP respiratory control or phosphate respiratory control lowered the P:O ratio with glutamate as substrate below I. With palmitic acid loss of respiratory control was only partial. 5. 5. Uncoupling by oleic could be reserved by adding serum albumin.

The value of ΔG° for the hydrolysis of ATP☆

Abstract 1. 1. The equilibrium constant of the reaction catalysed by glutamine synthetase ( l -glutamate: ammonia ligase (ADP), EC. 6.3.1.2), K obs = [ Gln ] · [ ADP ] · [ P i ] [ Glu ] · [ ammonia ] · [ ATP ] , was measured at 37 °C between pH 6.6 and 7.6 and [Mg2+] between 1 and 34 mM. 2. 2. The stability constant of the magnesium-glutamate complex was found to be 7.66 ± 0.26 (S.E.) M−1 at I 0.2 and 37 °C. 3. 3. By combining these results with published data for the equilibrium of the glutaminase reaction, the pK and binding constants of the magnesium complexes with Pi, ADP and ATP, activity coefficients and enthalpy data, the −ΔG° of the reaction ATPH3− + H2O ag ADPH2− + H2PO4−, at I o and 25 °C was calculated to be 7.53 ± 0.09 (S.E.) kcal/mole (31.5 ± 0.4 kJ/mole). 4. 4. This value is 2.33 kcal/mole less than that calculated by R. C. Phillips, P. George and R. J. Rutman (J. Biol. Chem., 244 (1969) 3330). The difference is caused mainly by (i) the lower value used by us for the equilibrium constant of the glutamine synthetase reaction, (ii) a lower value used for the stability constant of the magnesium-glutamate complex, (iii) allowing for the effect of lowering the temperature from 37 to 25 °C on the RT term. 5. 5. Calculations from data in the literature on the equilibria of the reactions catalysed by hexokinase and glucose-6-phosphatase yield a value similar to that reported above. 6. 6. Data are tabulated and plotted graphically for the value of −ΔG°obs at 25 and 37 °C, between pH 6 and 9 and for Mg2+ concentrations up to 50 mM. At pH 7.5, I 0.2 and 25 °C, ΔG°obs is −8.48 kcal/mole (−35 kJ/mole), 2.26 kcal/mole less negative than that calculated by Phillips et al.

The phosphorylation potential generated by respiring mitochondria

1. 1. The phosphorylation potential, ΔGP = ΔG0′ + 1.36 log ([ATP][ADP][Pi]), where ΔGO′ is the standard free energy of hydrolysis of ATP at a given pH, and [ATP], [ADP] and [Pi] refer to concentrations in the suspending medium, has been determined in rat-liver mitochondria under various conditions. 2. 2. The ATPADP 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 ATPADP 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 Pi 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-mans 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.

The reaction of antimycin with a cytochrome b preparation active in reconstitution of the respiratory chain

Abstract 1. Succinate-cytochrome c reductase activity was reconstituted by incubating a mixture of succinate dehydrogenase, cytochrome c1, ubiquinone-10, phospholipid and a preparation of cytochrome b, made by the method of Yamashita and Racker . 2. Preparations of cytochrome b active in reconstitution contained 5–28% native cytochrome b, as adjudged by reducibility with succinate in the reconstituted preparation and by lack of reaction with CO. Preparations of cytochrome b containing no native cytochrome b according to this criterion were inactive in reconstitution. 3. With a fixed amount of cytochrome b, the activity of the reconstituted preparation increased with increasing amounts of cytochrome c1 until a ratio of about 2b (total): 1c1 (allowing for the cytochrome c1 present in the cytochrome b preparation) was reached. 4. The amount of antimycin necessary for maximal inhibition of the reconstituted enzyme is a function of the amount of the cytochrome b and is independent of the amount of cytochrome c1. It is equal to about one half the amount of native cytochrome b. 5. Preparations of intact or reconstituted succinate-cytochrome c reductase or of cytochrome b completely quench the fluorescence of added antimycin, until an amount of antimycin equal to onehalf the amount of native cytochrome b present was added. Antimycin added in excess of this amount fluoresces with normal intensity. The quenching is only partial in the presence of Na2S2O4. Denatured cytochrome b does not quench the fluorescence. 6. Since preparations of cytochrome b active in reconstitution contained cytochrome c1 in an amount exceeding one half the amount of native cytochrome b present in the preparation, there is no evidence that native cytochrome b has been resolved from cytochrome c1. The stimulatory action of cytochrome c1 may be due to the restoration of a damaged membrane conformation. 7. Based on the assumption that the bc1 segment of the respiratory chain contains 2b:1c1:1 antimycin-binding sites, the specific quenching of antimycin fluorescence by binding to cytochrome b enables an accurate determination of the absorbance coefficients of cytochromes b and c1. These are 25.6 and 20.1 mM−1×cm−1 for the wavelength pairs 563–577 nm and 553–539 nm, respectively, in the difference spectrum reduced minus oxidized.

Tightly bound nucleotides of the energy-transducing ATPase of chloroplasts and their role in photophosphorylation.

1. Like other energy-transducing membranes, chloroplast membranes bear a coupling ATPase with especially tight binding sites for adenine nucleotides. Membranes washed several times still contain 2.5 nmol ATP and 1.3 nmol ADP bound per mg chlorophyll, which is equivalent to 1.9 ATP and 1.0 ADP per coupling ATPase. 2. In de-energized membranes, these nucleotides exchange to only a limited extent with added nucleotides. In membranes illuminated in the presence of pyocyanine, however, complete exchange of the bound nucleotides occurs rapidly, irrespective of whether ATP or ADP is present in the medium. 3. Pi can exchange into these nucleotided at both the beta and gamma positions when the membranes are energized in the presence of Mg-2+. Equilibrium with the beta and gamma groups of th ebound nucleotides is, however, not complete. 4. The inhibitors and uncouplers Dio-9, S13 and EDTA have different effects on the exchange of nucleotides, the exchange of inorganic phosphate and photophosphorylation. 5. The bound ATP level on the membrane is stable to a wide variety of conditions. The ADP level, however, drops to near zero under conditions of maximal activation of the emmbrane ATPase.

Monovalent nickel in hydrogenase from Chromatium vinosum: Light sensitivity and evidence for direct interaction with hydrogen

Redox titrations with hydrogenase from Chromatium vinosum show that its nickel ion can exist in 3, possibly 4, different redox states: the 3 +, 2 +, 1 + and possibly a zero valent state. The 1 + state is unstable: oxidation to Ni(II) occurs unless H2 gas is present. The Ni(I) coordination, but not that of Ni(III), is highly light sensitive. A photoreaction occurs on illumination. It is irreversible below 77 K, but reversible at 200 K. The rate of this photodissociation reaction in 2H2O is nearly 6‐times slower than in H2O, indicating the breakage of a nickel‐hydrogen bond. This forms the first evidence for an H atom in the direct coordination sphere of Ni in hydrogenase and for the involvement of this metal in the reaction with hydrogen.

The allosteric binding of antimycin to cytochrome b in the mitochondrial membrane

Abstract 1. 1. Based on the assumptions that the quenching of the fluorescence of antimycin on binding to the bc 1 segment of the respiratory chain (Complex III) is caused by energy transfer from antimycin to the cytochrome b haem, and the intrinsic fluorescence of antimycin bound to the complex is the same as that in ethanolic solution the distance between the fluorescent group of antimycin and the b haem was found to be 1.9 nm in oxidized Complex III and 2.4 nm in the reduced Complex. If the intrinsic enhancement of the fluorescence on binding to the Complex is the same as that on binding to serum albumin (about 9-fold), these distances become 1.35 nm and 1.7 nm, respectively. 2. 2. The binding of antimycin to oxidized (sub-)mitochondrial particles from beef heart is non-co-operative, with a binding constant of about 3 · 10 10 M −1 . 3. 3. In succinate-reduced mitochondria and sub-mitochondrial-particles from beef heart the binding of antimycin increases with increasing amounts of antimycin. At zero antimycin concentration the binding constant is about 3 · 10 9 M −1 . With high concentrations of antimycin the binding constant equals that for the oxidized particles. It is assumed that in succinate-reduced particles the antimycin-binding site is predominantly in a conformation (T state) with a lower binding constant than the conformation present in oxidized particles (R state) and that antimycin promotes the formation of the R state in succinate-reduced particles. 4. 4. Particles reduced with dithionite and beef-heart mitochondria reduced with succinate in the presence of ATP bind antimycin non-co-operatively with the same binding constant as for oxidized particles. Rat-liver mitochondria behave qualitatively the same as beef-heart mitochondria, but with a smaller affinity for antimycin. 5. 5. In cytochrome c -depleted particles reduced with succinate, the antimycin-effect curves are linear, and the antimycin-binding curves are non-co-operative with a binding constant equal to that of the T state. 6. 6. In pentane-extracted particles reduced with succinate, the binding is non-co-operative, with a binding constant a little less than in oxidized particles. 7. 7. Isolated Complex III in the oxidized state binds antimycin much more strongly than oxidized particles. The binding to Complex III reduced by succinate or dithionite is about the same as to oxidized particles. It is suggested that the site of splitting with detergent and salt is the same as the antimycin-binding site. 8. 8. The results with particles and mitochondria are explained on the basis of the allosteric model of Monod. Both ubiquinone and cytcchrome c are allosteric effectors of the bc 1 complex, while antimycin is an allosteric inhibitor. In mitochondria ATP also causes a shift of the T 2D R equilibrium towards the R state. 9. 9. Using the antimycin-induced inhibition of succinate oxidation or increased reduction of cytochrome b by succinate as a measure for the amount of R state present at a given antimycin concentration, the values of n and log L in the Monod allosteric model were calculated to be 7.5 and 5.85, respectively. The latter corresponds to an energy difference between T and R states of about 8 kcal/mole.

The pathway of electrons through QH2:cytochrome c oxidoreductase studied by pre-steady-state kinetics☆

The kinetic behaviour of the prosthetic groups and the semiquinones in in QH2:cytochrome c oxidoreductase has been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) In the absence of antimycin, cytochrome b-562 is reduced in two phases separated by a lag time. The initial very rapid reduction phase, that coincides with the formation of the antimycin-sensitive Qin, is ascribed to high-potential cytochrome b-562 and the slow phase to low-potential cytochrome b-562. the two cytochromes are present in a 1:1 molar ratio. The lag time between the two reduction phases decreases with increasing pH. Both the [2 Fe-2S] clusters and cytochrome c1 are reduced monophasically under these conditions, but at a rate lower than that of the initial rapid reduction of cytochrome b-562. (3) In the presence of antimycin and absence of oxidant, cytochrome b-562 is still reduced biphasically, but there is no lag between the two phases. No Qin is formed and both the Fe-S clusters and cytochrome c1 are reduced biphasically, one-half being reduced at the same rate as in the absence of antimycin and the other half 10-times slower. (4) In the presence of antimycin and oxidant, the recently described antimycin-insensitive species of semiquinone anion, Qout (De Vries, S., Albracht, S.P.J., Berden, J.A. and Slater, E.C. (1982) J. Biol. Chem. 256, 11996-11998) is formed at the same rate as that of the reduction of all species of cytochrome b. In this case cytochrome b is reduced in a single phase. (5) The reversible change of the line shape of the EPR spectrum of the [2Fe-2S] cluster 1 is caused by ubiquinone bound in the vicinity of this cluster. (6) The experimental results are consistent with the basic principles of the Q cycle. Because of the multiplicity, stoicheiometry and heterogeneous kinetics of the prosthetic groups, a Q cycle model describing the pathway of electrons through a dimeric QH2:cytochrome c oxidoreductase is proposed.

Tight binding of adenine nucleotides to beef-heart mitochondrial ATPase

Abstract Mitochondrial ATPase (F1) isolated from beef heart contains 5 moles of tightly bound adenine nucleotide per mole of enzyme—3 moles ATP and 2 moles ADP. These are retained after repeated precipitation of the enzyme with (NH4)2SO4 and are only partially removed by treating the enzyme with activated charcoal or filtering through Sephadex. Cold denaturation, however, causes complete release of the adenine nucleotides from the protein. The exchange of the bound nucleotides with added nucleotides is slow.

The nature and mechanism of action of uncoupling agents present in mitochrome preparations

Abstract 1. 1. The uncoupling activity of mitochrome preparations was not affected by addition of Na2S2O4, K3Fe(CN)6, CO or IO −4M KCN, all of which bring about changes in the absorption spectrum of mitochrome. 2. 2. The uncoupling activity was also not affected by precipitation of the protein by heating the solution or adding trichloroacetic acid. 3. 3. The substance(s) responsible for the uncoupling activity could be completely extracted by organic solvents; the mitochrome remained in the water layer. 4. 4. Purified preparations of cytochrome (a + a3) or cytochrome b changed into mitochrome on standing at 4° for a few days. Simultaneously, there was an increase in the amount of uncoupling material which could be extracted from these preparations by organic solvents. 5. 5. The uncoupling activity of an iso-octane extract of mitochrome resides in the unsaturated fatty acids, chiefly oleic and linoleic acids, which it contains.