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Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo?electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier?like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak. Purine nucleotides inhibit uncoupling protein 1 in a proton-impermeable intermediary state by a pH-dependent mechanism.

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH-OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X-ray crystallographic analysis, we report the structure of NADH-OH bound in the active site of a soluble fragment of complex I at 2.0 Å resolution. We have identified key amino acid residues that are specific and essential for binding NADH-OH. Furthermore, the structure sheds light on the specificity of NADH-OH towards the unique Rossmann-fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH-OH acts as a lead-structure for the synthesis of a novel class of ROS suppressors.

SummaryStructural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription were previously facilitated by the use of synthetic oligonucleotides. Here we report structures of initiation complexes de novo converted from pre-initiation complex (PIC) through catalytic activities and stalled at different template positions. Contrary to previous models, the closed-to-open promoter transition was accompanied by a large positional change of the general transcription factor TFIIH which became in closer proximity to TFIIE for the active delivery of the downstream DNA to the pol II active center. The initially-transcribing complex (ITC) reeled over 80 base pairs of the downstream DNA by scrunching, while retaining the fixed upstream contact, and underwent the transition to elongation when it encountered promoter-proximal pol II from a preceding round of transcription. TFIIH is therefore conducive to promoter melting, TSS scanning, and promoter escape, extending far beyond synthesis of a short transcript.

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria.

Respiratory electron transport complexes are organized as individual entities or combined as large super-complexes (SC). The Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the first structure of a bc1-cbb3 type SC (CIII2CIV, 5.2Å resolution) and three conformers of native CIII2 (3.3Å resolution) of functional relevance. The SC contains all catalytic subunits and cofactors as well as two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and conveys electrons to CIV differently than cyt c2. For the first time, this work establishes the structural characteristics of membrane-confined and membrane-external electron transport pathways of SCs in Gram-negative bacteria.

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria., Respiratory chains generate the proton motive force used for ATP synthesis. Cryo-EM structures of functional respiratory CIII2CIV supercomplex and native CIII2 from Rhodobacter capsulatus provide insight into CIII2CIV assembly and respiratory electron transport pathways in Gram-negative bacteria.

The respiratory electron transport complexes convey electrons from nutrients to oxygen and generate a proton-motive force used for energy (ATP) production in cells. These enzymes are conserved among organisms, and organized as individual complexes or combined forming large super-complexes (SC). Bacterial electron transport pathways are more branched than those of mitochondria and contain multiple variants of such complexes depending on their growth modes. The Gram-negative species deploy a mitochondrial-like cytochromebc1(Complex III, CIII2), and may have bacteria-specificcbb3-type cytochromecoxidases (Complex IV, CIV) in addition to, or instead of, the canonicalaa3-type CIV. Electron transfer between these complexes is mediated by two different carriers: the soluble cytochromec2which is similar to mitochondrial cytochromecand the membrane-anchored cytochromecywhich is unique to bacteria. Here, we report the first cryo-EM structure of a respiratorybc1-cbb3type SC (CIII2CIV, 5.2Å resolution) and several conformers of native CIII2(3.3Å resolution) from the Gram-negative bacteriumRhodobacter capsulatus. The SC contains all catalytic subunits and cofactors of CIII2and CIV, as well as two extra transmembrane helices attributed to cytochromecyand the assembly factor CcoH. Remarkably, some of the native CIII2are structural heterodimers with different conformations of their [2Fe-2S] cluster-bearing domains. The unresolved cytochromecdomain ofcysuggests that it is mobile, and it interacts with CIII2CIV differently than cytochromec2. Distance requirements for electron transfer suggest that cytochromecyand cytochromec2donate electrons to hemecp1and hemecp2of CIV, respectively. For the first time, the CIII2CIV architecture and its electronic connections establish the structural features of two separate respiratory electron transport pathways (membrane-confined and membrane-external) between its partners in Gram-negative bacteria.

The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649–15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII(2), yielding a small amount of active CI+CIII(2) super-complex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII(2) supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH(2)-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII(2), but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII(2) and CIV(2) and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.

Comparative genomic studies of the bacterial MFS-type copper importer CcoA, required for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis, revealed a widespread CcoA-like transporters (CalT) family, containing the conserved CcoA Cu-binding MxxxM and HxxxM motifs. Surprisingly, this family also included the RfnT-like proteins, earlier suggested to transport riboflavin. However, presence of the Cu-binding motifs in these proteins raised the possibility that they might be Cu transporters. To test this hypothesis, the genomic context of the corresponding genes was examined, and three of such genes from Ochrobactrum anthropi, Rhodopseudomonas palustris and Agrobacterium tumefaciens were expressed in Escherichia coli (ΔribB) and Rhodobacter capsulatus (ΔccoA) mutants. Copper and riboflavin uptake abilities of these strains were compared with those expressing R. capsulatus CcoA and Rhizobium leguminosarum RibN as bona fide copper and riboflavin importers, respectively. Overall data demonstrated that the “RfnT-like” CalT proteins are unable to efficiently transport riboflavin, but they import copper like CcoA. Nevertheless, even though expressed and membrane-localized in a R. capsulatus mutant lacking CcoA, these transporters were unable to accumulate Cu or complement for cbb3-Cox defect. This lack of functional exchangeability between the different subfamilies of CalT homologs suggests that MFS-type bacterial copper importers might be species-specific.

The ubiquinol:cytochrome (cyt) c oxidoreductase (or cyt bc1) is an important membrane protein complex in photosynthetic and respiratory energy transduction. In bacteria such as Rhodobacter capsulatus it is constituted of three subunits: the iron-sulfur protein, cyt b and cyt c1, which form two catalytic domains, the Qo (hydroquinone (QH2) oxidation) and Qi (quinone (Q) reduction) sites. At the Qo site, the pathways of bifurcated electron transfers emanating from QH2 oxidation are known, but the associated proton release routes are not well defined. In energy transducing complexes, Zn2+ binding amino acid residues often correlate with proton uptake or release pathways. Earlier, using combined EXAFS and structural studies, we identified Zn coordinating residues of mitochondrial and bacterial cyt bc1. In this work, using the genetically tractable bacterial cyt bc1, we substituted each of the proposed Zn binding residues with non-protonatable side chains. Among these mutants, only the His291Leu substitution destroyed almost completely the Qo site catalysis without perturbing significantly the redox properties of the cofactors or the assembly of the complex. In this mutant, which is unable to support photosynthetic growth, the bifurcated electron transfer reactions that result from QH2 oxidation at the Qo site, as well as the associated proton(s) release, were dramatically impaired. Based on these findings, on the putative role of His291 in liganding Zn, and on its solvent exposed and highly conserved position, we propose that His291 of cyt b is critical for proton release associated to QH2 oxidation at the Qo site of cyt bc1.

In many Gram-negative bacteria, including Rhodobacter capsulatus, cytochrome c maturation (Ccm) is carried out by a membrane-integral machinery composed of nine proteins (CcmA to I). During this process, the periplasmic thiol-disulfide oxidoreductase DsbA is thought to catalyze the formation of a disulfide bond between the Cys residues at the apocytochrome c heme-binding site (CXXCH). Subsequently, a Ccm-specific thioreductive pathway involving CcmG and CcmH reduces this disulfide bond to allow covalent heme ligation. Currently, the sequence of thioredox reactions occurring between these components and apocytochrome c and the identity of their active Cys residues are unknown. In this work, we first investigated protein-protein interactions among the apocytochrome c, CcmG, and the heme-ligation components CcmF, CcmH, and CcmI. We found that they all interact with each other, forming a CcmFGHI-apocytochrome c complex. Using purified wild-type CcmG, CcmH, and apocytochrome c, as well as their respective Cys mutant variants, we determined the rates of thiol-disulfide exchange reactions between selected pairs of Cys residues from these proteins. We established that CcmG can efficiently reduce the disulfide bond of apocytochrome c and also resolve a mixed disulfide bond formed between apocytochrome c and CcmH. We further show that Cys-45 of CcmH and Cys-34 of apocytochrome c are most likely to form this mixed disulfide bond, which is consistent with the stereo-specificity of the heme-apocytochrome c ligation reaction. We conclude that CcmG confers efficiency, and CcmH ensures stereo-specificity during Ccm and present a comprehensive model for thioreduction reactions that lead to heme-apocytochrome c ligation.

The proton-pumping NADH:ubiquinone oxidoreductase, respiratory complex I, couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. In Escherichia coli the complex is made up of 13 different subunits encoded by the so-called nuo-genes. Mutants, in which each of the nuo-genes was individually disrupted by the insertion of a resistance cartridge were unable to assemble a functional complex I. Each disruption resulted in the loss of complex I-mediated activity and the failure to extract a structurally intact complex. Thus, all nuo-genes are required either for the assembly or the stability of a functional E. coli complex I. The three subunits comprising the soluble NADH dehydrogenase fragment of the complex were detected in the cytoplasm of several nuo-mutants as one distinct band after BN-PAGE. It is discussed that the fully assembled NADH dehydrogenase fragment represents an assembly intermediate of the E. coli complex I. A partially assembled complex I bound to the membrane was detected in the nuoK and nuoL mutants, respectively. Overproduction of the ΔNuoL variant resulted in the accumulation of two populations of a partially assembled complex in the cytoplasmic membranes. Both populations are devoid of NuoL. One population is enzymatically active, while the other is not. The inactive population is missing cluster N2 and is tightly associated with the inducible lysine decarboxylase. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

PMID: 26147723; Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn(2+) ions are capable of binding to acidic residues, often found within such specific pathways, thereby leading to a blockage. Here we probed Zn(2+) inhibition of the proton-pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy. Numerous conformational changes were identified including those that arise from the reorganization of the membrane arm upon electron transfer in the peripheral arm of the protein. Signals at very high wavenumbers (1781 and 1756 cm(-1)) point to the perturbation of acidic residues in a highly hydrophobic environment upon Zn(2+) binding. In variant D563N(L), which lacks part of the proton pumping activity (residue located on the horizontal amphipathic helix), the spectral signature of Zn(2+) binding is changed. Our data support a role for this residue in proton translocation.

Summary The NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the redox reaction and a membrane arm catalyzing proton translocation. The membrane arm is almost completely aligned by a 110 A unique horizontal helix that is discussed to transmit conformational changes induced by the redox reaction in a piston-like movement to the membrane arm driving proton translocation. Here, we analyzed such a proposed movement by cysteine-scanning of the helix of the Escherichia coli complex I. The accessibility of engineered cysteine residues and the flexibility of individual positions were determined by labeling the preparations with a fluorescent marker and a spin-probe, respectively, in the oxidized and reduced states. The differences in fluorescence labeling and the rotational flexibility of the spin probe between both redox states indicate only slight conformational changes at distinct positions of the helix but not a large movement.

The NADH:ubiquinone oxidoreductase, respira- tory complex I, couples the transfer ofelectrons from NADHto ubiquinone with a translocation of protons across the mem- brane. The complex consists of a peripheral arm catalyzing the electron transfer reaction and a membrane arm involved in protontranslocation.TherecentlypublishedX-raystructuresof the complex revealed the presence of a unique 110 A "hor- izontal" 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 conforma- tional 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 þ /2estoichiometry of the ΔNuoL variantis2,indicatingadifferentstoichiometryforprotontranslocationasproposedfromstructuraldata.Inaddition,thesameH þ / estoichiometry is obtained with the variant lacking the C-terminal transmembraneous helix of NuoL, indicating its role in energy transmission.

The respiratory complex I couples the electron transfer from NADH to ubiquinone with a translocation of protons across the membrane. Its nucleotide-binding site is made up of a unique Rossmann fold to accommodate the binding of the substrate NADH and of the primary electron acceptor flavin mononucleotide. Binding of NADH includes interactions of the hydroxyl groups of the adenosine ribose with a conserved glutamic acid residue. Structural analysis revealed that due to steric hindrance and electrostatic repulsion, this residue most likely prevents the binding of NADPH, which is a poor substrate of the complex. We produced several variants with mutations at this position exhibiting up to 200-fold enhanced catalytic efficiency with NADPH. The reaction of the variants with NAD(P)H is coupled with proton translocation in an inhibitor-sensitive manner. Thus, we have created an energy-converting NADPH:ubiquinone oxidoreductase, an activity so far not found in nature. Remarkably, the oxidation of NAD(P)H by the variants leads to an enhanced production of reactive oxygen species.

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 A "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.

The NADH:ubiquinone oxidoreductase couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. It contains a 110Å 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.