Jan A. Berden

A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations

A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are “silent,” that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing “metabolic snapshots,” can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY—an abbreviation for functional analysis by co-responses in yeast.

Prohibitins act as a membrane‐bound chaperone for the stabilization of mitochondrial proteins

Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co‐overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse–chase experiments with mitochondria isolated from phb1/phb2‐null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins.

Differential investigation of the capacity of succinate oxidation in human skeletal muscle.

Procedures are described for the estimation of the succinate:ubiquinone oxidoreductase and succinate:phenazine methosulfate oxidoreductase activities in post-nuclear supernatants of human skeletal muscle homogenates using 2,6-dichlorophenol indophenol as the terminal electron acceptor. The influence of ionic strength and of sucrose upon these assays and upon the succinate:cytochrome c oxidoreductase activity has been investigated. Sucrose markedly interferes with the activation of the succinate dehydrogenase complex. Succinate:cytochrome c oxidoreductase activity and succinate:phenazine methosulfate oxidoreductase activity are inhibited by increasing concentrations of ions and of sucrose. Our results lead us to propose the existence of a single acceptor site for phenazine methosulfate at the succinate dehydrogenase complex, not involved in the physiological electron flux across ubiquinone. Estimation of the enzymatic activities mentioned above allows differential investigation of the functional integrity of a large part of the respiratory chain in patients suspected of suffering from a neuromuscular disorder.

Oxidoreduction of cytochrome b in the presence of antimycin

Abstract 1. The effect of oxidizing equivalents on the redox state of cytochrome b in the presence of antimycin has been studied in the presence and absence of various redox mediators. 2. The antimycin-induced extra reduction of cytochrome b is always dependent on the initial presence of an oxidant such as oxygen. After removal of the oxidant this effect remains or is partially (under some conditions even completely) abolished depending on the redox potential of the substrate used and the leak through the antimycin-inhibited site. 3. The increased reduction of cytochrome b induced by oxidant in the presence of antimycin involves all three spectroscopically resolvable b components (b-562, b-566 and b-558. 4. Redox mediators with an actual redox potential of less than 100–170 mV cause the oxidation of cytochrome b reduced under the influence of antimycin and oxidant. 5. Redox titrations of cytochrome b with the succinate/fumarate couple were performed aerobically in the presence of cyanide. In the presence of antimycin two b components are separated potentiometrically, one with an apparent midpoint potential above 80 mV (at pH 7.0), outside the range of the succinate/fumurate couple, and one with an apparent midpoint potential of 40 mV and an n value of 2. In the absence of antimycin cytochrome b titrates essentially as one species with a midpoint potential of 39 mV (at pH 7.0) and n = 1.14. 6. The increased reducibility of cytochrome b induced by antimycin plus oxidant is considered to be the result of two effects: inhibition of oxidation of ferrocytochrome b by ferricytochrome c1 (the effect of antimycin), and oxidation of the semiquinone form of a two-equivalent redox couple such as ubiquinone/ubiquinol by the added oxidant, leading to a decreased redox potential of the QH2/QH• couple and reduction of cytochrome b.

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.

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.

Binding of HQNO to beef-heart sub-mitochondrial particles

1. The fluorescence spectra of HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) in water at pH 7.5 show an emission maximum at 480 nm and an excitation maximum at 355 nm. 2. The fluorescence is enhanced by binding to bovine serum albumin, and is completely quenched by binding to sub-mitochondrial particles of beef heart. 3. Binding experiments reveal specific binding of HQNO to sub-mitochondrial particles with a dissociation constant of 64 nM and, depending on the protein concentration, a considerable amount of aspecific binding. 4. The concentration of specific binding sites for HQNO is identical with that of antimycin-binding sites. Furthermore, the presence of antimycin prevents the binding of HQNO and antimycin releases HQNO from its binding site. 5. The binding of HQNO is not sensitive to the redox state of the respiratory-chain components. 6. Inhibition of electron transfer by HQNO is caused by binding to the specific binding site. 7. The relation between inhibition of NADH or succinate oxidation and saturation of the binding site is hyperbolic. 8. The increase in the reduction level of cytochrome b on addition of HQNO in the presence of succinate and oxygen, either in the presence or absence of cyanide, does not parallel the inhibition of overall electron transfer. 9. All data can be quantitatively described and analysed using the model for electron transfer proposed by Wikström and Berden in 1972 (Wikström, M.K.F. and Berden, J.A. (1972) Biochim. Biophys. Acta 283, 403-420).

The effect of pH, ubiquinone depletion and myxothiazol on the reduction kinetics of the prosthetic groups of ubiquinol: Cytochrome c oxidoreductase

(1) The kinetics of the reduction by duroquinol of the prosthetic groups of QH2:cytochrome c oxidoreductase and of the formation of ubisemiquinone have been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) The formation of the antimycin-sensitive ubisemiquinone anion parallels the reduction of both high-potential and low-potential cytochrome b-562. (3) The rates of reduction of both the [2Fe-2S] clusters and cytochromes (c + c1) are pH dependent. There is, however, a pH-dependent discrepancy between their rate of reduction, which can be correlated with the difference in pH dependencies of their midpoint potentials. (4) Lowering the pH or the Q content results in a slower reduction of part of the [2Fe-2S] clusters. It is suggested that one cluster is reduced by a quinol/semiquinone couple and the other by a semiquinone/quinone couple. (5) Myxothiazol inhibits the reduction of the [2Fe-2S] clusters, cytochrome c1 and high-potential cytochrome b-562. (6) The results are consistent with a Q-cycle model describing the pathway of electrons through a dimeric QH2:cytochrome c oxidoreductase.

Properties of a semiquinone anion located in the QH2:Cytochrome c oxidoreductase segment of the mitochondrial respiratory chain

The way in which electrons are transferred from ubiquinol to cytochrome c is still under discussion. The results of potentiometric titrations and measurements of the pre-steady-state kinetics monitored optically, mainly giving information on the redox properties of the cytochromes, have led to proposals [ 1,2] for electron transfer through QH,:cytochrome c oxidoreductase, in which the formation of a semiquinone is a prerequisite for electron transfer. The presence of a semiquinone in preparations of the respiratory chain has, indeed, been identified by EPR studies [3-IO]. Ohnishi and Trumpower have detected two different populations of ubisemiquinone in isolated succinate:cytochrome c oxidoreductase [ 101, SQ, and SQ,, differing in relaxation time (see also 11 r11. The results in this paper indicate the existence of a very stable semiquinone anion located in QH, :cytochrome c oxidoreductase, presumably corresponding to SQ, in [lo]. Quantitation of the EPR signal of the semiquinone anion showed that the maximal concentration is I/2 that of the cytochrome cr. In order adequately to describe the effect of pH on the semiquinone anion concentration in the Nernst equation, a Limited capacity of the binding site for the semiquinone anion must be taken into account .