Association of apolipoprotein A5 variants with LDL particle size and triglyceride in Japanese Americans

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the entry point for electrons into the respiratory chains of many bacteria and mitochondria of most eucaryotes. It couples electron transfer with the translocation of protons across the membrane, thus providing the proton motive force essential for energy-consuming processes. Electron microscopy revealed the 'L'-shaped structure of the bacterial and mitochondrial complex with two arms arranged perpendicular to each other. Recently, we showed that the Escherichia coli complex I takes on another stable conformation with the two arms arranged side by side resulting in a horseshoe-shaped structure. This model reflects the evolution of complex I from pre-existing modules for electron transfer and proton translocation.     

A novel, enzymatically active conformation of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

Electron microscopy has demonstrated the unusual L-shaped structure of the respiratory complex I consisting of two arms, which are arranged perpendicular to each other. We found that the Escherichia coli complex I has an additional stable conformation, with the two arms arranged side by side, resulting in a horseshoe-shaped structure. The structure of both conformations was determined by means of electron microscopy of gold thioglucose-stained single particles. They were distinguished from each other by titration of the complex with polyethylene glycol and by means of analytical ultracentrifugation. The transition between the two conformations is induced by the ionic strength of the buffer and is reversible. Only the horseshoe-shaped complex I exhibits enzyme activity in detergent solution, which is abolished by the addition of salt. Therefore, it is proposed that this structure is the native conformation of the complex in the membrane.     

A ferredoxin bridge connects the two arms of plant mitochondrial complex I

Mitochondrial complex I is the main site for electron transfer to the respiratory chain and generates much of the proton gradient across the inner mitochondrial membrane. Complex I is composed of two arms, which form a conserved L-shape. We report the structures of the intact, 47-subunit mitochondrial complex I from Arabidopsis thaliana and the 51-subunit complex I from the green alga Polytomella sp., both at around 2.9 Å resolution. In both complexes, a heterotrimeric γ-carbonic anhydrase domain is attached to the membrane arm on the matrix side. Two states are resolved in A. thaliana complex I, with different angles between the two arms and different conformations of the ND1 (NADH dehydrogenase subunit 1) loop near the quinol binding site. The angle appears to depend on a bridge domain, which links the peripheral arm to the membrane arm and includes an unusual ferredoxin. We propose that the bridge domain participates in regulating the activity of plant complex I.

An unusual ferredoxin completes a protein bridge that links the two arms of plant mitochondrial complex I and adjusts their angle in an open or closed conformation.     

Spin labeling of the Escherichia coli NADH ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex with a peripheral arm involved in electron transfer and a membrane arm most likely involved in proton translocation. It was proposed that the quinone binding site is located at the joint of the two arms. Most likely, proton translocation in the membrane arm is enabled by the energy of the electron transfer reaction in the peripheral arm transmitted by conformational changes. For the detection of the conformational changes and the localization of the quinone binding site, we set up a combination of site-directed spin labeling and EPR spectroscopy. Cysteine residues were introduced to the surface of the Escherichia coli complex I. The spin label (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) was exclusively bound to the engineered positions. Neither the mutation nor the labeling had an effect on the NADH:decyl-ubiquinone oxidoreductase activity. The characteristic signals of the spin label were detected by EPR spectroscopy, which did not change by reducing the preparation with NADH. A decyl-ubiquinone derivative with the spin label covalently attached to the alkyl chain was synthesized in order to localize the quinone binding site. The distance between a MTSL labeled complex I variant and the bound quinone was determined by continuous-wave (cw) EPR allowing an inference on the location of the quinone binding site. The distances between the labeled quinone and other complex I variants will be determined in future experiments to receive further geometry information by triangulation.     

Complex I: A Chimaera of a Redox and Conformation-Driven Proton Pump?

From phylogenetic sequence analysis, it can be concluded that the proton-pumping NADH:ubiquinone oxidoreductase (complex I) has evolved from preexisting modules for electron transfer and proton translocation. It is built up by a peripheral NADH dehydrogenase module, an amphipatic hydrogenase module, and a membrane-bound transporter module. These modules, or at least part of them, are also present in various other bacterial enzymes. It is assumed that they fulfill a similar function in complex I and related enzymes. Based on the function of the individual modules, it is possible to speculate about the mechanism of complex I. The hydrogenase module might work as a redox-driven proton pump, while the transporter module might act as a conformation-driven proton pump. This implies that complex I contains two energy-coupling sites. The NADH dehydrogenase module seems to be involved in electron transfer and not in proton translocation.     

On the mechanism of respiratory complex I

The energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy and X-ray crystallography revealed the two-part structure of the enzyme complex. A peripheral arm extending into the aqueous phase catalyzes the electron transfer reaction. Accordingly, this arm contains the redox-active cofactors, namely one flavin mononucleotide (FMN) and up to ten iron-sulfur (Fe/S) clusters. A membrane arm embedded in the lipid bilayer catalyzes proton translocation by a yet unknown mechanism. The binding site of the substrate (ubi) quinone is located at the interface of the two arms. The oxidation of one NADH is coupled with the translocation of four protons across the membrane. In this review, the binding of the substrates, the intramolecular electron transfer, the role of individual Fe/S clusters and the mechanism of proton translocation are discussed in the light of recent data obtained from our laboratory.     

Conformation-driven and semiquinone-gated proton-pump mechanism in the NADH-ubiquinone oxidoreductase (complex I)

A novel mechanism for proton/electron transfer is proposed for NADH-quinone oxidoreductase (complex I) based on the following findings: (1) EPR signals of the protein-bound fast-relaxing semiquinone anion radicals (abbreviated as Q(Nf)-) are observable only in the presence of proton-transmembrane electrochemical potential; (2) Iron-sulfur cluster N2 and Q(Nf)- are directly spin-coupled; and (3) The projection of the interspin vector extends only 5A along the membrane normal [Yano, T., Dunham, W.R. and Ohnishi, T. (2005) Biochemistry, 44, 1744-1754]. We propose that the proton pump is operated by redox-driven conformational changes of the quinone binding protein. In the input state, semiquinone is reduced to quinol, acquiring two protons from the N (matrix) side of the mitochondrial inner membrane and an electron from the low potential (NADH) side of the respiratory chain. A conformational change brings the protons into position for release at the P (inter-membrane space) side of the membrane via a proton-well. Concomitantly, an electron is donated to the quinone pool at the high potential side of the coupling site. The system then returns to the original state to repeat the cycle. This hypothesis provides a useful frame work for further investigation of the mechanism of proton translocation in complex I.     

Role of subunit NuoL for proton translocation by respiratory complex I

The NADH:ubiquinone oxidoreductase, respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with a translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the electron transfer reaction and a membrane arm involved in proton translocation. The recently published X-ray structures of the complex revealed the presence of a unique 110 ? "horizontal" helix aligning the membrane arm. On the basis of this finding, it was proposed that the energy released by the redox reaction is transmitted to the membrane arm via a conformational change in the horizontal helix. The helix corresponds to the C-terminal part of the most distal subunit NuoL. To investigate its role in proton translocation, we characterized the electron transfer and proton translocation activity of complex I variants lacking either NuoL or parts of the C-terminal domain. Our data suggest that the H+/2e- stoichiometry of the ΔNuoL variant is 2, indicating a different stoichiometry for proton translocation as proposed from structural data. In addition, the same H+/e- stoichiometry is obtained with the variant lacking the C-terminal transmembraneous helix of NuoL, indicating its role in energy transmission.     

Architecture of complex I and its implications for electron transfer and proton pumping

Proton pumping NADH:ubiquinone oxidoreductase (complex I) is the largest and remains by far the least understood enzyme complex of the respiratory chain. It consists of a peripheral arm harbouring all known redox active prosthetic groups and a membrane arm with a yet unknown number of proton translocation sites. The ubiquinone reduction site close to iron-sulfur cluster N2 at the interface of the 49-kDa and PSST subunits has been mapped by extensive site directed mutagenesis. Independent lines of evidence identified electron transfer events during reduction of ubiquinone to be associated with the potential drop that generates the full driving force for proton translocation with a 4H⁺/2e⁻ stoichiometry. Electron microscopic analysis of immuno-labelled native enzyme and of a subcomplex lacking the electron input module indicated a distance of 35-60 Å of cluster N2 to the membrane surface. Resolution of the membrane arm into subcomplexes showed that even the distal part harbours subunits that are prime candidates to participate in proton translocation because they are homologous to sodium/proton antiporters and contain conserved charged residues in predicted transmembrane helices. The mechanism of redox linked proton translocation by complex I is largely unknown but has to include steps where energy is transmitted over extremely long distances. In this review we compile the available structural information on complex I and discuss implications for complex I function.     

Proton pumping by complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica reconstituted into proteoliposomes

The mechanism of energy converting NADH:ubiquinone oxidoreductase (complex I) is still unknown. A current controversy centers around the question whether electron transport of complex I is always linked to vectorial proton translocation or whether in some organisms the enzyme pumps sodium ions instead. To develop better experimental tools to elucidate its mechanism, we have reconstituted the affinity purified enzyme into proteoliposomes and monitored the generation of DeltapH and Deltapsi. We tested several detergents to solubilize the asolectin used for liposome formation. Tightly coupled proteoliposomes containing highly active complex I were obtained by detergent removal with BioBeads after total solubilization of the phospholipids with n-octyl-beta-D-glucopyranoside. We have used dyes to monitor the formation of the two components of the proton motive force,DeltapH and Deltapsi, across the liposomal membrane, and analyzed the effects of inhibitors, uncouplers and ionophores on this process. We show that electron transfer of complex I of the lower eukaryote Y. lipolytica is clearly linked to proton translocation. While this study was not specifically designed to demonstrate possible additional sodium translocating properties of complex I, we did not find indications for primary or secondary Na+ translocation by Y. lipolytica complex I.     

Theory and simulation of proton-coupled electron transfer, hydrogen-atom transfer, and proton translocation in proteins

A theory of proton coupled electron transfer (PCET) is reviewed with application to charge transfer steps in the photosystem II oxygen-evolving complex (PSII/OEC). The relation between PCET when it is a concerted electron proton transfer (ETPT) process and hydrogen-atom transfer (HAT) reactions is discussed. Signatures expected for HAT reactions in terms of the size of the kinetic isotope effect and overall magnitude of the rate constant are discussed in the context of PSII/OEC. The formal similarity of ETPT to proton transfer and translocation is used to introduce a combined quantum mechanical (for the transferring protons) and molecular dynamics for the heavy-atom degrees of freedom approach. The method is used to examine double proton transfer in cytochrome c oxidase where two waters and a glutamate (Glu286) that is implicated in the proton translocation mechanism form a cyclic hydrogen bonded structure. Protonation of the glutamate is found to occur in agreement with experimental results.     

Asp563 of the horizontal helix of subunit NuoL is involved in proton translocation by the respiratory complex I

The NADH:ubiquinone oxidoreductase couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. It contains a 110 Å long helix running parallel to the membrane part of the complex. Deletion of the helix resulted in a reduced H⁺/e^? stoichiometry indicating its direct involvement in proton translocation. Here, we show that the mutation of the conserved amino acid D563^L, which is part of the horizontal helix of the Escherichia coli complex I, leads to a reduced H⁺/e^? stoichiometry. It is discussed that this residue is involved in transferring protons to the membranous proton translocation site.     

Electronic wiring of a multi-redox site membrane protein in a biomimetic surface architecture

Bioelectronic coupling of multi-redox-site membrane proteins was accomplished with cytochrome c oxidase (CcO) as an example. A biomimetic membrane system was used for the oriented immobilization of the CcO oxidase on a metal electrode. When the protein is immobilized with the CcO binding side directed toward the electrode and reconstituted in situ into a lipid bilayer, it is addressable by direct electron transfer to the redox centers. Electron transfer to the enzyme via the spacer, referred to as electronic wiring, shows an exceptionally high rate constant. This allows a kinetic analysis of all four consecutive electron transfer steps within the enzyme to be carried out. Electron transfer followed by rapid scan cyclic voltametry in combination with surface-enhanced resonance Raman spectroscopy provides mechanistic and structural information about the heme centers. Probing the enzyme under turnover conditions showed mechanistic insights into proton translocation coupled to electron transfer. This bioelectronic approach opens a new field of activity to investigate complex processes in a wide variety of membrane proteins.     

Mössbauer spectroscopy on respiratory complex I: the iron-sulfur cluster ensemble in the NADH-reduced enzyme is partially oxidized

In mitochondria, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. It contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters that transfer the electrons to quinone. Understanding electron transfer in complex I requires spectroscopic and structural data to be combined to reveal the properties of individual clusters and of the ensemble. EPR studies on complex I from Bos taurus have established that five clusters (positions 1, 2, 3, 5, and 7 along the seven-cluster chain extending from the flavin) are (at least partially) reduced by NADH. The other three clusters, positions 4 and 6 plus a cluster on the other side of the flavin, are not observed in EPR spectra from the NADH-reduced enzyme: they may remain oxidized, have unusual or coupled spin states, or their EPR signals may be too fast relaxing. Here, we use M?ssbauer spectroscopy on (57)Fe-labeled complex I from the mitochondria of Yarrowia lipolytica to show that the cluster ensemble is only partially reduced in the NADH-reduced enzyme. The three EPR-silent clusters are oxidized, and only the terminal 4Fe cluster (position 7) is fully reduced. Together with the EPR analyses, our results reveal an alternating profile of higher and lower potential clusters between the two active sites in complex I; they are not consistent with the consensus picture of a set of isopotential clusters. The implications for intramolecular electron transfer along the extended chain of cofactors in complex I are discussed.     

Insights from the structure of the yeast cytochrome bc1 complex: crystallization of membrane proteins with antibody fragments

The ubiquinol:cytochrome c oxidoreductase (EC 1.20.2.2, QCR or cytochrome bc1 complex) is a component of respiratory and photosynthetic electron transfer chains in mitochondria and bacteria. The complex transfers electrons from quinol to cytochrome c. Electron transfer is coupled to proton translocation across the lipid bilayer, thereby generating an electrochemical proton gradient, which conserves the free energy of the redox reaction. The yeast complex was crystallized with antibody Fv fragments, a promising technique to obtain well-ordered crystals from membrane proteins. The high-resolution structure of the yeast protein reveals details of the catalytic sites of the complex, which are important for electron and proton transfer.     

Proton motive function of the terminal antiporter-like subunit in respiratory complex I

In the aerobic respiratory chains of many organisms, complex I functions as the first electron input. By reducing ubiquinone (Q) to ubiquinol, it catalyzes the translocation of protons across the membrane as far as ~200 Å from the site of redox reactions. Despite significant amount of structural and biochemical data, the details of redox coupled proton pumping in complex I are poorly understood. In particular, the proton transfer pathways are extremely difficult to characterize with the current structural and biochemical techniques. Here, we applied multiscale computational approaches to identify the proton transfer paths in the terminal antiporter-like subunit of complex I. Data from combined classical and quantum chemical simulations reveal for the first time structural elements that are exclusive to the subunit, and enables the enzyme to achieve coupling between the spatially separated Q redox reactions and proton pumping. By studying long time scale protonation and hydration dependent conformational dynamics of key amino acid residues, we provide novel insights into the proton pumping mechanism of complex I.     

The respiratory complex I of bacteria, archaea and eukarya and its module common with membrane-bound multisubunit hydrogenases

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first of the respiratory complexes providing the proton motive force which is essential for energy consuming processes like the synthesis of ATP. Homologues of this complex exist in bacteria, archaea, in mitochondria of eukaryotes and in chloroplasts of plants. The bacterial and mitochondrial complexes function as NADH dehydrogenase, while the archaeal complex works as F420H2 dehydrogenase. The electron donor of the cyanobacterial and plastidal complex is not yet known. Despite the different electron input sites, 11 polypeptides constitute the structural framework for proton translocation and quinone binding in the complex of all three domains of life. Six of them are also present in a family of membrane-bound multisubunit [NiFe] hydrogenases. It is discussed that they build a module for electron transfer coupled to proton translocation.     

New developments on the functions of coenzyme Q in mitochondria

The notion of a mobile pool of coenzyme Q (CoQ) in the lipid bilayer has changed with the discovery of respiratory supramolecular units, in particular the supercomplex comprising complexes I and III; in this model, the electron transfer is thought to be mediated by tunneling or microdiffusion, with a clear kinetic advantage on the transfer based on random collisions. The CoQ pool, however, has a fundamental function in establishing a dissociation equilibrium with bound quinone, besides being required for electron transfer from other dehydrogenases to complex III. The mechanism of CoQ reduction by complex I is analyzed regarding recent developments on the crystallographic structure of the enzyme, also in relation to the capacity of complex I to generate superoxide. Although the mechanism of the Q-cycle is well established for complex III, involvement of CoQ in proton translocation by complex I is still debated. Some additional roles of CoQ are also examined, such as the antioxidant effect of its reduced form and the capacity to bind the permeability transition pore and the mitochondrial uncoupling proteins. Finally, a working hypothesis is advanced on the establishment of a vicious circle of oxidative stress and supercomplex disorganization in pathological states, as in neurodegeneration and cancer.     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a₃/Cu_B centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the “prepumped” proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different E_m values of the electron acceptor in the binuclear site.     

A Drosophila model of mitochondrial disease caused by a complex I mutation that uncouples proton pumping from electron transfer

Mutations affecting mitochondrial complex I, a multi-subunit assembly that couples electron transfer to proton pumping, are the most frequent cause of heritable mitochondrial diseases. However, the mechanisms by which complex I dysfunction results in disease remain unclear. Here, we describe a Drosophila model of complex I deficiency caused by a homoplasmic mutation in the mitochondrial-DNA-encoded NADH dehydrogenase subunit 2 (ND2) gene. We show that ND2 mutants exhibit phenotypes that resemble symptoms of mitochondrial disease, including shortened lifespan, progressive neurodegeneration, diminished neural mitochondrial membrane potential and lower levels of neural ATP. Our biochemical studies of ND2 mutants reveal that complex I is unable to efficiently couple electron transfer to proton pumping. Thus, our study provides evidence that the ND2 subunit participates directly in the proton pumping mechanism of complex I. Together, our findings support the model that diminished respiratory chain activity, and consequent energy deficiency, are responsible for the pathogenesis of complex-I-associated neurodegeneration.KEY WORDS: Mitochondria, Drosophila, Mitochondrial disease, Respiratory chain, Leigh syndrome, Neurodegeneration