The Q-cycle reviewed: How well does a monomeric mechanism of the bc1 complex account for the function of a dimeric complex?

Recent progress in understanding the Q-cycle mechanism of the bc₁ complex is reviewed. The data strongly support a mechanism in which the Q_o-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron–sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe–2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q_o-site, and the reduced iron–sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c₁ and liberate the H⁺. When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O₂ is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b_L to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b_L to enhance the rate constant. The acceptor reactions at the Q_i-site are still controversial, but likely involve a “two-electron gate” in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b₁₅₀ phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed.The mechanism discussed is applicable to a monomeric bc₁ complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b_L hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.     

Investigation of the mechanism of proton translocation by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria: does the enzyme operate by a Q-cycle mechanism?

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the membrane-bound electron transport chain in mitochondria. It conserves energy, from the reduction of ubiquinone by NADH, as a protonmotive force across the inner membrane, but the mechanism of energy transduction is not known. The structure of the hydrophilic arm of thermophilic complex I supports the idea that proton translocation is driven at (or close to) the point of quinone reduction, rather than at the point of NADH oxidation, with a chain of iron-sulfur clusters transferring electrons between the two active sites. Here, we describe experiments to determine whether complex I, isolated from bovine heart mitochondria, operates via a Q-cycle mechanism analogous to that observed in the cytochrome bc1 complex. No evidence for the 'reductant-induced oxidation' of ubiquinol could be detected; therefore no support for a Q-cycle mechanism was obtained. Unexpectedly, in the presence of NADH, complex I inhibited by either rotenone or piericidin A was found to catalyse the exchange of redox states between different quinone and quinol species, providing a possible route for future investigations into the mechanism of energy transduction.     

Interactions of quinone with the iron-sulfur protein of the bc(1) complex: is the mechanism spring-loaded?

Since available structures of native bc(1) complexes show a vacant Q(o)-site, occupancy by substrate and product must be investigated by kinetic and spectroscopic approaches. In this brief review, we discuss recent advances using these approaches that throw new light on the mechanism. The rate-limiting reaction is the first electron transfer after formation of the enzyme-substrate complex at the Q(o)-site. This is formed by binding of both ubiquinol (QH(2)) and the dissociated oxidized iron-sulfur protein (ISP(ox)). A binding constant of approximately 14 can be estimated from the displacement of E(m) or pK for quinone or ISP(ox), respectively. The binding likely involves a hydrogen bond, through which a proton-coupled electron transfer occurs. An enzyme-product complex is also formed at the Q(o)-site, in which ubiquinone (Q) hydrogen bonds with the reduced ISP (ISPH). The complex has been characterized in ESEEM experiments, which detect a histidine ligand, likely His-161 of ISP (in mitochondrial numbering), with a configuration similar to that in the complex of ISPH with stigmatellin. This special configuration is lost on binding of myxothiazol. Formation of the H-bond has been explored through the redox dependence of cytochrome c oxidation. We confirm previous reports of a decrease in E(m) of ISP on addition of myxothiazol, and show that this change can be detected kinetically. We suggest that the myxothiazol-induced change reflects loss of the interaction of ISPH with Q, and that the change in E(m) reflects a binding constant of approximately 4. We discuss previous data in the light of this new hypothesis, and suggest that the native structure might involve a less than optimal configuration that lowers the binding energy of complexes formed at the Q(o)-site so as to favor dissociation. We also discuss recent results from studies of the bypass reactions at the site, which lead to superoxide (SO) production under aerobic conditions, and provide additional information about intermediate states.     

The bc(1) complex of the iron-grown acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans functions in the reverse but not in the forward direction. Is there a second bc(1) complex?

Acidithiobacillus ferrooxidans is an acidophilic chemolithotrophic bacterium that can grow in the presence of either a weak reductant, Fe(2+), or reducing sulfur compounds that provide more energy for growth than Fe(2+). Here we first review the latest findings about the uphill electron transfer pathway established in iron-grown A. ferrooxidans, which has been found to involve a bc(1) complex. We then provide evidence that this bc(1) complex cannot function in the forward direction (exergonic reaction), even with an appropriate substrate. A search for the sequence of the three redox subunits of the A. ferrooxidans bc(1) complex (strain ATCC 19859) in the complete genome sequence of the A. ferrooxidans ATCC 23270 strain showed the existence of two different bc(1) complexes in A. ferrooxidans. Cytochrome b and Rieske protein sequence comparisons allowed us to point out some sequence particularities of these proteins in A. ferrooxidans. Lastly, we discuss the possible reasons for the existence of two different "classical" bc(1) complexes and put forward some suggestions as to what role these putative complexes may play in this acidophilic chemolithotrophic bacterium.     

How well is enzyme function conserved as a function of pairwise sequence identity?

Enzyme function conservation has been used to derive the threshold of sequence identity necessary to transfer function from a protein of known function to an unknown protein. Using pairwise sequence comparison, several studies suggested that when the sequence identity is above 40%, enzyme function is well conserved. In contrast, Rost argued that because of database bias, the results from such simple pairwise comparisons might be misleading. Thus, by grouping enzyme sequences into families based on sequence similarity and selecting representative sequences for comparison, he showed that enzyme function starts to diverge quickly when the sequence identity is below 70%. Here, we employ a strategy similar to Rost's to reduce the database bias; however, we classify enzyme families based not only on sequence similarity, but also on functional similarity, i.e. sequences in each family must have the same four digits or the same first three digits of the enzyme commission (EC) number. Furthermore, instead of selecting representative sequences for comparison, we calculate the function conservation of each enzyme family and then average the degree of enzyme function conservation across all enzyme families. Our analysis suggests that for functional transferability, 40% sequence identity can still be used as a confident threshold to transfer the first three digits of an EC number; however, to transfer all four digits of an EC number, above 60% sequence identity is needed to have at least 90% accuracy. Moreover, when PSI-BLAST is used, the magnitude of the E-value is found to be weakly correlated with the extent of enzyme function conservation in the third iteration of PSI-BLAST. As a result, functional annotation based on the E-values from PSI-BLAST should be used with caution. We also show that by employing an enzyme family-specific sequence identity threshold above which 100% functional conservation is required, functional inference of unknown sequences can be accurately accomplished. However, this comes at a cost: those true positive sequences below this threshold cannot be uniquely identified.     

X-ray structure of the dimeric cytochrome bc(1) complex from the soil bacterium Paracoccus denitrificans at 2.7-Å resolution

The respiratory cytochrome bc(1) complex is a fundamental enzyme in biological energy conversion. It couples electron transfer from ubiquinol to cytochrome c with generation of proton motive force which fuels ATP synthesis. The complex from the α-proteobacterium Paracoccus denitrificans, a model for the medically relevant mitochondrial complexes, lacked structural characterization. We show by LILBID mass spectrometry that truncation of the organism-specific, acidic N-terminus of cytochrome c(1) changes the oligomerization state of the enzyme to a dimer. The fully functional complex was crystallized and the X-ray structure determined at 2.7-? resolution. It has high structural homology to mitochondrial complexes and to the Rhodobacter sphaeroides complex especially for subunits cytochrome b and ISP. Species-specific binding of the inhibitor stigmatellin is noteworthy. Interestingly, cytochrome c(1) shows structural differences to the mitochondrial and even between the two Rhodobacteraceae complexes. The structural diversity in the cytochrome c(1) surface facing the ISP domain indicates low structural constraints on that surface for formation of a productive electron transfer complex. A similar position of the acidic N-terminal domains of cytochrome c(1) and yeast subunit QCR6p is suggested in support of a similar function. A model of the electron transfer complex with membrane-anchored cytochrome c(552), the natural substrate, shows that it can adopt the same orientation as the soluble substrate in the yeast complex. The full structural integrity of the P. denitrificans variant underpins previous mechanistic studies on intermonomer electron transfer and paves the way for using this model system to address open questions of structure/function relationships and inhibitor binding.     

The dimeric Mcm8–9 complex of Xenopus laevis likely has a conserved function for resistance to DNA damage

The success of Imatinib mesylate (STI571, Gleevec) in treating chronic myeloid leukemia (CML) is, to date, the crowning achievement of targeted molecular therapy in cancer. Nearly 90% of newly diagnosed patients treated with Imatinib in the chronic phase of the disease achieve a complete cytogenetic response. However, more than 95% of these patients retain detectable levels of BCR-ABL mRNA and patients discontinuing Imatinib therapy almost invariably relapse, demonstrating that an Imatinib insensitive population of leukemia-initiating cells (LICs) persists in nearly all patients. These findings underscore the need for treatments specifically targeting the leukemia-initiating population of CML cells. While mounting evidence suggests that the LIC in the chronic phase of CML is the BCR-ABL positive hematopoietic stem cell, several recent publications suggest that during CML blast crisis, a granulocyte-macrophage progenitor (GMP) population also acquires LIC properties through activation of the β-catenin pathway. Characterization of these cells and evaluation of their sensitivity to Imatinib is critical to our understanding and treatment of CML blast crisis.     

The ligand-binding site of bovine beta-lactoglobulin: evidence for a function?

Ever since the fortuitous observation that beta-lactoglobulin (beta-Lg), the major whey protein in the milk of ruminants, bound retinol, the details of the binding have been controversial. beta-Lg is a lipocalin, like plasma retinol-binding protein, so that ligand association was expected to make use of the central cavity in the protein. However, an early crystallographic analysis and some of the more recent solution studies indicated binding elsewhere. We have now determined the crystal structures of the complexes of the trigonal form of beta-Lg at pH 7.5 with bound retinol (R=21.4% for 7329 reflections between 20 and 2.4 A resolution, R(free)=30.6%) and with bound retinoic acid (R=22.7% for 7813 reflections between 20 and 2.34 A resolution, R(free)=29.8%). Both ligands are found to occupy the central calyx in a manner similar to retinol binding in retinol-binding protein. We find no evidence of binding at the putative external binding site in either of these structural analyses. Further, competition between palmitic acid and retinol reveals only palmitate bound to the protein. An explanation is provided for the lack of ligand binding to the orthorhombic crystal form also obtained at pH 7.5. Finally, the possible function of beta-Lg is discussed in the light of its species distribution and similarity to other lipocalins.     

Proton-coupled electron transfer at the Q(o) site: what type of mechanism can account for the high activation barrier?

In Rhodobacter sphaeroides, transfer of the first electron in quinol oxidation by the bc(1) complex shows kinetic features (a slow rate (approx. 1.5 x 10(3)/s), high activation energy (approx. 65 kJ/mol) and reorganization energy, lambda (2.5 V)) that are unexpected from Marcus theory and the distances shown by the structures. Reduction of the oxidized iron-sulfur protein occurs after formation of the enzyme-substrate complex, and involves a H-transfer in which the electron transfer occurs through the approx. 7 A of a bridging histidine forming a H-bond with quinol and a ligand to 2Fe-2S. The anomalous kinetic features can be explained by a mechanism in which the electron transfer is constrained by coupled transfer of the proton. We discuss this in the context of mutant strains with modified E(m,7) and pK for the iron-sulfur protein, and Marcus theory for proton-coupled electron transfer. We suggest that transfer of the second proton and electron involve movement of semiquinone in the Q(o) site, and rotation of the Glu of the conserved -PEWY- sequence. Mutational studies show a key role for the domain proximal to heme b(L). The effects of mutation at Tyr-302 (Tyr-279 in bovine sequence) point to a possible linkage between conformational changes in the proximal domain, and changes leading to closure of the iron-sulfur protein access channel at the distal domain.     

The cytochrome bc1 complex in the mitochondrial respiratory chain functions according to the Q cycle hypothesis of Mitchell: the proof using a stochastic approach?

The bc1 complex is a central complex in the mitochondrial respiratory chain. It links the electrons transfer from ubiquinol (or coenzyme Q) to cytochrome c and proton translocation across the inner mitochondrial membrane. It is widely agreed that the "Q-cycle mechanism" proposed by Mitchell correctly describes the bc1 complex working. It is based on an unexpected separation of the two electrons coming from the coenzyme Q bound at the Q0 site of the bc1 complex. Using the stochastic approach of Gillespie and the known spatial structure of bc1 complexes with the kinetic parameters described by Moser and Dutton we demonstrated the natural emergence of the Q-cycle mechanism and the quasi absence of short-circuits in the functional dimer of bc1 complex without the necessity to invoke any additional mechanism. This approach gives a framework which is well adapted to the modelling of all oxido-reduction reactions of the respiratory chain complexes, normal or mutant.     

The insulin receptor: a prototype for dimeric, allosteric membrane receptors?

The recent crystallographic structure of the insulin receptor (IR) extracellular domain has brought us closer to ending several decades of speculation regarding the stoichiometry and mechanism of insulin-receptor binding and negative cooperativity. It supports a bivalent crosslinking model whereby two sites on the insulin molecule alternately crosslink two partial-binding sites on each insulin-receptor half. Ligand-induced or -stabilized receptor dimerization or oligomerization is a general feature of receptor tyrosine kinases (RTKs), in addition to cytokine receptors, but the kinetic consequences of this mechanism have been less well studied in other RTKs than in the IR. Surprisingly, recent studies indicate that constitutive dimerization and negative cooperativity are also ubiquitous properties of G-protein-coupled receptors (GPCRs), which show allosteric mechanisms similar to those described for the IR.     

The 2 micron plasmid purloins the yeast cohesin complex: a mechanism for coupling plasmid partitioning and chromosome segregation?

The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locus. The periodicity of cohesin association and dissociation is nearly identical for the plasmid and the chromosomes. The timely disassembly of cohesin is a prerequisite for plasmid segregation. Cohesin-mediated pairing and unpairing likely provides a counting mechanism for evenly partitioning plasmids either in association with or independently of the chromosomes.     

How does microgravity affect the muscular and kinematic synergies in a complex movement?

The planning and the execution of voluntary movement relies on sensorimotor transformations in which representations of the external environment are integrated into motor programs. We studied executions of Whole Body Pointing movements, in normal and in transient microgravity (parabolic flights) conditions. Three processes could lead to adaptation to the new environmental condition: a radical change of terrestrial synergies, their partial modification or preservation. By applying a multivariate analysis on kinematic and electromyographic (EMG) data and by comparing the 1g and 0g conditions, our findings hint the hypothesis the descending information from vestibular system may be directed to change the synergies' modulation. An analogous analysis was performed on the kinematics: the invariance of intersegmental coordination among the segments' elevation angles suggests that these kinematic waveforms are used as reference signals to determine the appropriate muscle synergies in a subordinate and flexible manner in order to adapt to the novel mechanical constraints.     

How does the brain control its own activity? A new function for the basal ganglia

It has long been a problem in neuroscience to known how the brain controls its own activity, how it is able to control the level of CNS excitability and how it is able to select and act on some information as opposed to some other information. In this paper I propose a new theory in which the basal ganglia play a role in selecting information ("selective attention") and in controlling the general level of excitability of the CNS ("state control"), the two processes being to some extent interdependent. The basal ganglia achieve these functions by actions on the thalamic-frontal cortical axis and on the brainstem mesencephalic reticular formation.