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The unique bifurcation of electron flow at the ubihydroquinone-oxidation center of the cytochrome bc1 complex is the energy-conserving reaction of the protonmotive Q-cycle and is prerequisite to vectorial proton translocation. The widely accepted Q-cycle reaction scheme describes the overall electron and proton pathways, but does not address the detailed chemistry of this central step. Based on a model of the ubihydroquinone-oxidation pocket containing two ubiquinone molecules in a stacked configuration, a detailed model for the reactions during steady-state catalysis is proposed. In this proton-gated charge-transfer mechanism the reaction is controlled by the deprotonation of the substrate ubihydroquinone.

Mitochondria from the strobilurin A producing basidiomycetes Strobilurus tenacellus and Mycena galopoda exhibit natural resistance to (E)-beta-methoxyacrylate inhibitors of the ubiquinol oxidation center(center Qp) of the cytochrome bc1 complex. Isolated cytochrome bc1 complex from S. tenacellus was found to be highly similar to that of Saccharomyces cerevisiae with respect to subunit composition, as well as spectral characteristics and midpoint potentials of the heme centers. To understand the molecular basis of natural resistance, we determined the exon/intron organization and deduced the sequences of cytochromes b from S. tenacellus, M. galopoda and a third basidiomycete, Mycena viridimarginata, which produces no strobilurin A. Comparative sequence analysis of two regions of cytochrome b known to contribute to the formation of center Qp suggested that the generally lower sensitivity of all three basidiomycetes was due to the replacement of a small amino acid residue in position 127 by isoleucine. For M. galopoda replacement of Gly143 by alanine and Gly153 by serine, for S. tenacellus replacement of a small residue in position 254 by glutamine and Asn261 by aspartate was found to be the likely causes for resistance to (E)-beta-methoxyacrylates. The latter exchange is also found in Schizosaccharomyces pombe, which we found also to be naturally resistant to (E)-beta-methoxyacrylates.

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Publisher Summary This chapter focuses on the ubiquinol-cytochrome-c reductase from human and bovine mitochondria. Complex III is a membrane-bound multiprotein complex that forms the middle segment of the respiratory chain of the inner mitochondrial membrane. Complex III catalyzes the transfer of electrons from membrane-bound ubiquinol to ferricytochrome-c, which is located on the cytosolic side of the inner mitochondrial membrane. This redox reaction is linked to a transfer of protons across the membrane by the proton motive Q cycle. Complex III of mammalian mitochondria has been isolated by several different procedures. Each of these protocols, although finally yielding the complexes of the same protein subunit composition, has its special features that determine the choice of the optimal procedure for a special purpose. This chapter concludes with the discussion on the structure–function relationship of quinone reaction centers.

Ein freundlicher Herr in den besten Jahren, fruh ergraut und dabei noch ganz dynamisch wirkend, spaziert durch eine herrliche Landschaft, vielleicht einen Naturpark. Er spricht direkt in die Kamera: “Ich bin Arthur Brauss. Auch ich habe keine Erklarungen fur diese unglaublichen Geschichten und Berichte, die ich Ihnen gleich ausfuhrlich vorstellen werde. Sie beruhen angeblich auf wahren Begebenheiten und sind bezeugt von Leuten, die sie miterlebt haben. Sie als Lugner und Betruger abzutun, ist mir zu billig. Ich kann mir auch nicht vorstellen, warum jemand so ein Ereignis erfinden sollte. Trotzdem mochte ich es Ihnen uberlassen, herauszufinden, was denn das wahre Wunder an diesen Geschichten ist. Wie schon gesagt: manche sind wirklich unglaublich.”

The cytochrome bc1 complex is an oligomeric electron transfer enzyme located in the inner membrane of mitochondria and the plasma membrane of bacteria. The cytochrome bc1 complex participates in respiration in eukaryotic cells and also participates in respiration, cyclic photosynthetic electron transfer, denitrification, and nitrogen fixation in a phylogenetically diverse collection of bacteria. In all of these organisms, the cytochrome bc1 complex transfers electrons from ubiquinol to cytochrome c and links this electron transfer to translocation of protons across the membrane in which it resides, thus converting the available free energy of the oxidation-reduction reaction into an electrochemical proton gradient. The mechanism by which the cytochrome bc1 complex achieves this energy transduction is the protonmotive Q cycle. The Q cycle mechanism has been documented by extensive experimentation, and recent investigations have focused on structural features of the three redox subunits of the bc1 complex essential to the protonmotive and electrogenic activities of this membranous enzyme.

The iron-sulfur protein of the cytochrome bc1 complex oxidizes ubiquinol at center P in the protonmotive Q cycle mechanism, transferring one electron to cytochrome c1 and generating a low-potential ubisemiquinone anion which reduces the low-potential cytochrome b-566 heme group. In order to catalyze this divergent transfer of two reducing equivalents from ubiquinol, the iron-sulfur protein must be structurally integrated into the cytochrome bc1 complex in a manner which facilitates electron transfer from the iron-sulfur cluster to cytochrome c1 and generates a strongly reducing ubisemiquinone anion radical which is proximal to the b-566 heme group. This radical must also be sequestered from spurious reactivities with oxygen and other high-potential oxidants. Experimental approaches are described which are aimed at understanding how the iron-sulfur protein is inserted into center P, and how the iron-sulfur cluster is inserted into the apoprotein.

The Q cycle mechanism of the bc1 complex requires two quinone reaction centers, the hydroquinone oxidation (QP) and the quinone reduction (QN) center. These sites can be distinguished by the specific binding of inhibitors to either of them. A substantial body of information about the hydroquinone oxidation site has been provided by the analysis of the binding of QP site inhibitors to the bc1 complex in different redox states and to preparations depleted of lipid or protein components as well as by functional studies with mutant bc1 complexes selected for resistance toward the inhibitors. The reaction site is formed by at least five protein segments of cytochrome b and parts of the iron-sulfur protein. At least two different binding sites for QP site inhibitors could be detected, one for the methoxyacrylate-type inhibitors binding predominantly to cytochrome b, the other for the chromone-type inhibitors and hydroxyquinones binding predominantly to the iron-sulfur protein. The interactions with the protein segments, between different protein segments, and between protein and ligands (substrate, inhibitors) are discussed in detail and a working model of the QP pocket is proposed.

The physico-chemical properties and uncoupling activity of eight derivatives of N-phenyl-2-pyridinamines related to the fungicide fluazinam were analyzed using rat liver mitochondria. The uncoupling activity of these compounds relies on the deprotonable secondary amino group. One of the derivatives tested (B-3) was slightly more efficient than fluazinam. By phase-distribution analysis we could show that the N-phenyl-2-pyridinamines are chemicals with moderate hydrophobicity. Deprotonation of the compound reduces the water/octanol partition coefficient by about one order of magnitude. The pKA value of the deprotonable group is affected equally by electron withdrawing substituents of the phenyl- and the pyridinyl-ring, and could be predicted simply from the sum of the Hammett coefficients. The uncoupling efficiency was not dependent on the hydrophobicity of the compound, but appeared to be governed by the pKA of the deprotonable group. This structure/uncoupling characteristic is different from that of the generally more hydrophobic uncouplers of the salicylanilide-type. The pKA resulting in the most efficient uncoupling was found to lie in the range of the pH of the reaction medium. A model based on a solution complexation mechanism, which describes this behaviour, is presented. We conclude that the N-phenyl-2-pyridinamines uncoupled the mitochondria by a simple protonophoric cycle involving protonation/deprotonation in the bulk phase, and that the kinetics of uncoupling were primarily governed by the total concentration of the limiting uncoupler species.

The succinate dehydrogenase isolated from Bacillus subtilis was found to catalyze the oxidation of succinate with hydrophilic quinones. Either naphthoquinones or benzoquinones served as acceptors. The enzyme activity increased with the redox potential of the quinone. The highest turnover number was commensurate with that of the bacterial succinate respiration in vivo. The succinate dehydrogenase was similarly active in fumarate reduction with quinols. The highest activity was obtained with the most electronegative quinol. The fumarate reductase isolated from Wolinella succinogenes catalyzed succinate oxidation with quinones and fumarate reduction with the corresponding quinols at activities similar to those of the B. subtilis enzyme. Succinate oxidation by the lipophilic quinones, ubiquinone or vitamin K-1, was monitored as cytochrome c reduction using proteoliposomes containing succinate dehydrogenase together with the cytochrome bc1 complex. The activity with ubiquinone or vitamin K-1 was commensurate with the succinate respiratory activity of bacteria or of the bacterial membrane fraction. The results suggest that menaquinone is involved in the succinate respiration of B. subtilis, although its redox potential is unfavorable.

Self-organization behavior and in particular pattern forming spatiotemporal dynamics play an important role in far from equilibrium chemical and biochemical systems. Specific external forcing and control of self-organizing processes might be of great benefit in various applications ranging from technical systems to modern biomedical research. We demonstrate that in a cellular chemotaxis system modeled by one-dimensional reaction-diffusion equations particular forms of spatiotemporal dynamics can be induced and stabilized by controlling spatially distributed influx patterns of a chemical species as a function of time. In our model study we show that a propagating wave with certain shape and velocity and static symmetrical and asymmetrical patterns can be forced and manipulated by numerically computing open-loop optimal influx controls.

The chloroplast cytochrome bf complex is unaffected by micromolar concentrations of almost all the reported antibiotic inhibitors of the bcl complexes [l], with the notable exception of stigmatellin which has been shown to have an analogous action in both systems [2,3]. As for the Qi site, only the rather unspecific inhibitor NQNO (and HQNO) has been shown to affect the Qi site of the bf complex [4,5]. This effector produces changes of the transient kinetics of the enzyme consistent with a block of electron transfer through the Qi site, and yet does not significantly alter the steady state turnover number of the enzyme [4] or its protonmotive action [6]. This is in contrast to the action of NQNO on the mammalian enzyme where a full block in the Qi site, together with a cessation of electron and proton transfer activity, is evident. Studies with NQNO in thylakoids are considerably hampered by its strong inhibitory action on photosystem 11 [7].

Stigmatellin and its derivatives represent a third class of Qo site inhibitors besides the hydroxyquinone derivatives and the E-beta-methoxyacrylate (MOA) inhibitors [von Jagow and Link (1986) Methods Enzymol. 126, 253-271]. The stigmatellins consist of a chromone ring system connected to an substituted alkenyl side chain. Alterations in the side chain, i.e. saturation of the C = C double bonds, shift of a methoxy group or loss of the methyl groups, specifically affect the binding characteristics. Besides changing the red shift spectrum of reduced cytochrome b566 and the EPR spectrum of the Rieske iron-sulfur cluster, the side chain alterations diminish the binding affinity and the extent of the midpoint potential shift of the iron-sulfur protein. Thus, the side chain of the molecule makes an essential contribution to the binding energy and is not necessary solely for partitioning the molecule into the hydrophobic phase, as assumed so far.

Die kompressible Stromung in einem Diffusor eines Radialverdichters wird durch ein numerisches Verfahren berechnet. Auser der Kompressibilitat werden auch noch die Effekte der Reibung berucksichtigt. Die Ergebnisse sind in Form von Isobarenfeldern bzw. in Diagrammen fur die Impulsverlustdicke und fur den Schubspannungskoeffizienten dargestellt. Zum Abschlus wird ein Vergleich mit Meswerten durchgefuhrt.

A new purification procedure for cytochrome-c oxidase from bovine heart mitochondria is described. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxyapatite and gel chromatography. The preparation was highly pure and active. The subunit composition and steady-state kinetics were found to be the same as thoes reported for other preparations. In contrast to most of the previously published protocols the method presented here resulted in a preparation which had a rapid intramolecular electron transfer from cytochrome a to cytochrome a3, i.e. it was found to have retained its pulsed state. This correlated with monoexponential cyanide-binding kinetics. The formation of resting kinetics biphasic cyanide-binding kinetics was shown to be induced by a short incubation at pH 5.0.

Evidence is presented that single electron reduction is sufficient for rapid electron transfer (k greater than 20 s-1 at pH 8.0 in 0.43 M potassium EDTA) between haem a/CuA and the binuclear centre in 'fast' oxidase, whereas in 'slow' oxidase intramolecular electron transfer is slow even when both CuA and haem a are reduced (k congruent to 0.01 s-1). However, while a single electron can equilibrate rapidly between CuA, haem a and CuB in 'fast' oxidase, it seems that equilibration with haem a3 is relatively slow (k congruent to 2 s-1). Electron transfer between cytochrome c and CuA/haem a is similar for both types of enzyme (k congruent to 2.4 x 10(5) M-1.s-1).

The large membrane protein complexes of mitochondrial oxidative phosphorylation are composed of central subunits that are essential for their bioenergetic core function and accessory subunits that may assist in regulation, assembly or stabilization. Although sequence conservation is low, a significant proportion of the accessory subunits is characterized by a common single transmembrane (STMD) topology. The STMD signature is also found in subunits of other membrane protein complexes. We hypothesize that the general function of STMD subunits is to organize the hydrophobic subunits of large membrane protein complexes in specialized environments like the inner mitochondrial membrane.

In addition to the 14 central subunits, respiratory chain complex I from the aerobic yeast Yarrowia lipolytica contains at least 24 accessory subunits, most of which are poorly characterized. Here we investigated the role of the accessory 39-kDa subunit which belongs to the heterogeneous short-chain dehydrogenase/reductase (SDR) enzyme family and contains non-covalently bound NADPH. Deleting the chromosomal copy of the gene that codes for the 39-kDa subunit drastically impaired complex I assembly in Y. lipolytica. We introduced several site-directed mutations into the nucleotide binding motif that severely reduced NADPH binding. This effect was most pronounced when the arginine at the end of the second β-strand of the NADPH binding Rossman fold was replaced by leucine or aspartate. Mutations affecting nucleotide binding had only minor or moderate effects on specific catalytic activity in mitochondrial membranes but clearly destabilized complex I. One mutant exhibited a temperature sensitive phenotype and significant amounts of three different subcomplexes were observed even at more permissive temperature. We concluded that the 39-kDa subunit of Y. lipolytica plays a critical role in complex I assembly and stability and that the bound NADPH serves to stabilize the subunit and complex I as a whole rather than serving a catalytic function.

While diagnosis and genetic analysis of mitochondrial disorders has made remarkable progress, we still do not understand how given molecular defects are correlated to specific patterns of symptoms and their severity. Towards resolving this dilemma for the largest and therefore most affected respiratory chain enzyme, we have established the yeast Yarrowia lipolytica as a eucaryotic model system to analyse respiratory chain complex I. For in vivo analysis, eYFP protein was attached to the 30-kDa subunit to visualize complex I and mitochondria. Deletions strains for nuclear coded subunits allow the reconstruction of patient alleles by site-directed mutagenesis and plasmid complementation. In most of the pathogenic mutations analysed so far, decreased catalytic activities, elevated KM values, and/or elevated I50 values for quinone-analogous inhibitors were observed, providing plausible clues on the pathogenic process at the molecular level. Leigh mutations in the 49-kDa and PSST homologous subunits are found in regions that are at the boundaries of the ubiquinone-reducing catalytic core. This supports the proposed structural model and at the same time identifies novel domains critical for catalysis. Thus, Y. lipolytica is a useful lower eucaryotic model that will help to understand how pathogenic mutations in complex I interfere with enzyme function.

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica . Using 5–10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

The overall electron- and proton-pathways within the cytochrome-bc1 complex are described by a widely accepted mechanism known as the protonmotive Q-cycle. Within this reaction scheme, the unique bifurcation of electron flow into a high potential and a low potential pathway occurring at the ubihydroquinone-oxidation center is the energy conserving reaction. It is this reaction, which results in vectorial proton translocation, as it allows the ‘recycling’ of every second electron across the membrane onto the ubiquinone-reduction center. However, the Q-cycle reaction scheme does not address the detailed chemistry of this central step. Based on a structural model of the ubihydroquinone-oxidation pocket and the assumption that the reaction involves two ubiquinone molecules in a stacked configuration, here I propose a detailed chemical model for the reactions occurring during steady-state catalysis. In this proton-gated charge-transfer mechanism the reaction is controlled by the deprotonation of the substrate ubihydroquinone and not, as proposed earlier, by the formation of a highly unstable semiquinone species.

We determined the sites of artificial electron transfer onto 2-nitrosofluorene (NOF), a metabolite of carcinogenic 2-acetylaminofluorene in mitochondria and isolated cytochrome bc1 complex. NOF-induced O2 consumption in mitochondria was sensitive to antimycin A, but insensitive to myxothiazol. In the isolated cytochrome bc1 complex, NOF induced rapid MOA-stilbene-insensitive reoxidation of cytochrome b, whereas in the presence of antimycin A, reoxidation was very slow. The corresponding hydroxylamine, N-hydroxy-2-aminofluorene (NOHAF), reduced cytochrome b specifically through center N of the cytochrome bc1 complex. We conclude that NOF and NOHAF bind to center N of the cytochrome bc1 complex and acts as electron acceptor and donor, respectively. The NOHAF/NOF interconversion is considered to be involved in the cytotoxicity of 2-acetylaminofluorene in vivo.

Despite its central function in oxidative phosphorylation, the molecular mechanism of proton pumping respiratory complex I is still elusive. In recent years, considerable progress has been made towards understanding structure/function relationships in this very large and complicated membrane protein complex. Last year X-ray crystallographic analysis of bacterial and mitochondrial complex I provided important insights into its molecular architecture. Based on this evidence, here a hypothetical molecular mechanism for redox-driven proton pumping of complex I is proposed. According to this mechanism, two pump modules are driven by two conformational strokes that are generated by stabilization of the anionic forms of semiquinone and ubiquinol that are formed in the peripheral arm of complex I during turnover. This results in the experimentally determined pumping stoichiometry of 4 H+/2e−. In the two-state model, electron transfer from iron–sulfur cluster N2 is allowed only in the ‘E-state,’ while protonation of the substrate is only possible in the stabilizing ‘P-state.’ In the membrane arm, transition from the E- to the P-state drives the two pump modules via long range conformational energy transfer through the recently discovered helical transmission element connecting them. The proposed two-state stabilization-change mechanism is fully reversible and thus inherently explains the operation of complex I in forward and reverse mode. This article is part of a Special Issue entitled Allosteric cooperativity in respiratory proteins.

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of 3.8H→+/2e¯ indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of 3.6H→+/2e¯ . This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme.

Mitochondrial NADH:ubiquinone oxidoreductase is the largest and most complicated proton pump of the respiratory chain. Here we report the preparation and characterization of a subcomplex of complex I selectively lacking the flavoprotein part of the N-module. Removing the 51-kDa and the 24-kDa subunit resulted in loss of catalytic activity. The redox centers of the subcomplex could be reduced neither by NADH nor NADPH demonstrating that physiological electron input into complex I occurred exclusively via the N-module and that the NADPH binding site in the 39-kDa subunit and further potential nucleotide binding sites are isolated from the electron transfer pathway within the enzyme. Taking advantage of the selective removal of two of the eight iron–sulfur clusters of complex I and providing additional evidence by redox titration and site-directed mutagenesis, we could for the first time unambiguously assign cluster N1 of fungal complex I to mammalian cluster N1b.

The emerging X-ray structures of the cytochrome bc1 complexes from bovine and chicken heart mitochondria support the protonmotive Q-cycle as the overall electron- and proton-pathway within the cytochrome bc1 complex. The energy conserving reaction within this reaction scheme is the unique bifurcation of electron flow into a high potential and a low potential pathway occurring at the ubihydroquinone-oxidation center (center P or Qo). This step is prerequisite for the ‘recycling’ of every second electron across the membrane onto the ubiquinone-reduction center, which results in vectorial proton translocation. It has been shown that during steady-state the step controlling this reaction is the first deprotonation of ubihydroquinone and not, as proposed earlier, the formation of a highly unstable semiquinone species. Ubiquinone has not yet been detected at the ubihydroquinone-oxidation center of the protein structures now available, but the pocket seems spacious enough to accommodate two ubiquinone molecules. This is in line with recent enzymological studies, which have shown that not only two ubiquinones, but also two inhibitor molecules can bind to center P. The most striking result from the structures is that the hydrophilic domain of the ‘Rieske’ protein can be found in two different positions which seem to allow electron transfer between the iron-sulfur cluster and either ubiquinone binding at center P or heme c1. This provides strong support for the ‘catalytic switch’ model proposed earlier based on detailed analysis of inhibitor binding to cytochrome bc1 complex in different redox states.

The amino acid sequence of subunit 9 of the bovine heart cytochrome bc1 complex is identical to the 78-amino acid presequence that is removed post-translationally from the Rieske iron-sulfur protein as it is imported and targeted to the mitochondrial cytochrome bc1 complex. Iron-sulfur protein precursor, generated by in vitro transcription and translation, is processed to mature size in a single step when incubated with rat liver mitochondria, and generates a peptide that comigrates on SDS-polyacrylamide gel electrophoresis with subunit 9. These results suggest that the Rieske protein is processed in a single proteolytic step after it is inserted into the cytochrome bc1 complex in mammals, and that the processed presequence remains as a subunit of the complex. This is apparently the first instance in which a cleaved targeting presequence has been shown to be retained in the cell, possibly exhibiting a second function in addition to its function in protein trafficking.

The binding of specific inhibitors to the ubiquinol oxidation pocket ("QP center") of cytochrome c reductase was analyzed before and after removal of bound phospholipid and the "Rieske" iron-sulfur protein using optical spectroscopy and fluorescence quench binding assays. The enzyme lacking iron-sulfur protein showed almost unchanged, tight binding of the E-beta-methoxyacrylate inhibitors oudemansin A and MOA-stilbene, whereas binding of the chromone inhibitor stigmatellin was almost completely abolished. The affinity of the weak inhibitor 3-undecyl-2-hydroxy-naphthoquinone was decreased. Oudemansin A binding to the defective pocket of the iron-sulfur protein-depleted enzyme was lowered by added phospholipid. It was deduced from these results that the QP center is a spacious pocket formed by domains of cytochrome b, bearing the E-beta-methoxcyacrylate binding site, and the iron-sulfur protein, bearing the stigmatellin binding site. Moreover, removal of the iron-sulfur protein leaves this pocket defective but essentially unchanged in its remaining binding capability. The affinity of three preparations of cytochrome c reductase, the complete, the delipidated, and the iron-sulfur depleted enzyme for E-beta-methoxyacrylate-stilbene, was analyzed for different redox states of the catalytic centers of cytochrome c reductase. The apparent Kd values for the different redox states were interpreted in terms of two conformational states. It is suggested that these changes reflect the two states of the "catalytic switch" proposed recently for the QP pocket of cytochrome c reductase (Brandt, U., and von Jagow, G. (1991) Eur. J. Biochem. 195, 163-170). According to the refined model presented in this work, changeover to the "b" state is triggered by reduction of the iron-sulfur cluster, and changeover back to the "FeS" state is triggered by electron transfer from the low potential onto the high potential heme b center. Our interpretation implies that the stability of the two states is affected by the redox states of the enzyme, but that additionally changing the redox states of the two centers is required for "switching" on a catalytic time scale.