Exzellenzcluster Makromolekulare Komplexe
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Mitochondrial complex I (NADH:ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30–37 °C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide:oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.
Production of reactive oxygen species (ROS) by the mitochondrial respiratory chain is considered to be one of the major causes of degenerative processes associated with oxidative stress. Mitochondrial ROS has also been shown to be involved in cellular signaling. It is generally assumed that ubisemiquinone formed at the ubiquinol oxidation center of the cytochrome bc(1) complex is one of two sources of electrons for superoxide formation in mitochondria. Here we show that superoxide formation at the ubiquinol oxidation center of the membrane-bound or purified cytochrome bc(1) complex is stimulated by the presence of oxidized ubiquinone indicating that in a reverse reaction the electron is transferred onto oxygen from reduced cytochrome b(L) via ubiquinone rather than during the forward ubiquinone cycle reaction. In fact, from mechanistic studies it seems unlikely that during normal catalysis the ubisemiquinone intermediate reaches significant occupancies at the ubiquinol oxidation site. We conclude that cytochrome bc(1) complex-linked ROS production is primarily promoted by a partially oxidized rather than by a fully reduced ubiquinone pool. The resulting mechanism of ROS production offers a straightforward explanation of how the redox state of the ubiquinone pool could play a central role in mitochondrial redox signaling.
Proton pumping respiratory complex I is a major player in mitochondrial energy conversion. Yet little is known about the molecular mechanism of this large membrane protein complex. Understanding the details of ubiquinone reduction will be prerequisite for elucidating this mechanism. Based on a recently published partial structure of the bacterial enzyme, we scanned the proposed ubiquinone binding cavity of complex I by site-directed mutagenesis in the strictly aerobic yeast Yarrowia lipolytica. The observed changes in catalytic activity and inhibitor sensitivity followed a consistent pattern and allowed us to define three functionally important regions near the ubiquinone-reducing iron-sulfur cluster N2. We identified a likely entry path for the substrate ubiquinone and defined a region involved in inhibitor binding within the cavity. Finally, we were able to highlight a functionally critical structural motif in the active site that consisted of Tyr-144 in the 49-kDa subunit, surrounded by three conserved hydrophobic residues.
Motivation: Complexome profiling combines native gel electrophoresis with mass spectrometry to obtain the inventory, composition and abundance of multiprotein assemblies in an organelle. Applying complexome profiling to determine the effect of a mutation on protein complexes requires separating technical and biological variations from the variations caused by that mutation.
Results: We have developed the COmplexome Profiling ALignment (COPAL) tool that aligns multiple complexome profiles with each other. It includes the abundance profiles of all proteins on two gels, using a multi-dimensional implementation of the dynamic time warping algorithm to align the gels. Subsequent progressive alignment allows us to align multiple profiles with each other. We tested COPAL on complexome profiles from control mitochondria and from Barth syndrome (BTHS) mitochondria, which have a mutation in tafazzin gene that is involved in remodeling the inner mitochondrial membrane phospholipid cardiolipin. By comparing the variation between BTHS mitochondria and controls with the variation among either, we assessed the effects of BTHS on the abundance profiles of individual proteins. Combining those profiles with gene set enrichment analysis allows detecting significantly affected protein complexes. Most of the significantly affected protein complexes are located in the inner mitochondrial membrane (mitochondrial contact site and cristae organizing system, prohibitins), or are attached to it (the large ribosomal subunit).
Availability and implementation: COPAL is written in python and is available from http://github.com/cmbi/copal.
We have analyzed the distribution of mitochondrial contact site and cristae organizing system (MICOS) complex proteins and mitochondrial intermembrane space bridging complex (MIB) proteins over (sub)complexes and over species. The MICOS proteins are associated with the formation and maintenance of mitochondrial cristae. Indeed, the presence of MICOS genes in genomes correlates well with the presence of cristae: all cristae containing species have at least one MICOS gene and cristae-less species have none. Mic10 is the most widespread MICOS gene, while Mic60 appears be the oldest one, as it originates in the ancestors of mitochondria, the proteobacteria. In proteobacteria the gene occurs in clusters with genes involved in heme synthesis while the protein has been observed in intracellular membranes of the alphaproteobacterium Rhodobacter sphaeroides. In contrast, Mic23 and Mic27 appear to be the youngest MICOS proteins, as they only occur in opisthokonts. The remaining MICOS proteins, Mic10, Mic19, Mic25 and Mic12, the latter we show to be orthologous to human C19orf70/QIL1, trace back to the root of the eukaryotes. Of the remaining MIB proteins, also DNAJC11 shows a high correlation with the presence of cristae. In mitochondrial protein complexome profiles, the MIB complex occurs as a defined complex and as separate subcomplexes, potentially reflecting various assembly stages. We find three main forms of the complex: A) The MICOS complex, containing all the MICOS proteins, B) a membrane bridging subcomplex, containing in addition SAMM50, MTX2 and the previously uncharacterized MTX3, and C) the complete MIB complex containing in addition DNAJC11 and MTX1.
Mitochondrial complex I has a key role in cellular energy metabolism, generating a major portion of the proton motive force that drives aerobic ATP synthesis. The hydrophilic arm of the L-shaped ~1 MDa membrane protein complex transfers electrons from NADH to ubiquinone, providing the energy to drive proton pumping at distant sites in the membrane arm. The critical steps of energy conversion are associated with the redox chemistry of ubiquinone. We report the cryo-EM structure of complete mitochondrial complex I from the aerobic yeast Yarrowia lipolytica both in the deactive form and after capturing the enzyme during steady-state activity. The site of ubiquinone binding observed during turnover supports a two-state stabilization change mechanism for complex I.
Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the largest enzyme of the mitochondrial respiratory chain and a significant source of reactive oxygen species (ROS). We hypothesized that during energy conversion by complex I, electron transfer onto ubiquinone triggers the concerted rearrangement of three protein loops of subunits ND1, ND3, and 49-kDa thereby generating the power-stoke driving proton pumping. Here we show that fixing loop TMH1-2ND3 to the nearby subunit PSST via a disulfide bridge introduced by site-directed mutagenesis reversibly disengages proton pumping without impairing ubiquinone reduction, inhibitor binding or the Active/Deactive transition. The X-ray structure of mutant complex I indicates that the disulfide bridge immobilizes but does not displace the tip of loop TMH1-2ND3. We conclude that movement of loop TMH1-2ND3 located at the ubiquinone-binding pocket is required to drive proton pumping corroborating one of the central predictions of our model for the mechanism of energy conversion by complex I proposed earlier.
Proton-pumping complex I of the mitochondrial respiratory chain is among the largest and most complex membrane protein complexes. The enzyme contributes substantially to oxidative energy-conversion in eukaryotic cells. Its malfunctions are implicated in many hereditary and degenerative disorders. Here, we report the X-ray structure of mitochondrial complex I at 3.6- 3.9 Å resolution describing in detail the central subunits that execute the bioenergetic function. A continuous axis of basic and acidic residues running centrally through the membrane arm connects the ubiquinone reduction site in the hydrophilic arm to four putative proton-pumping units. The binding position for a substrate analogous inhibitor and blockage of the predicted ubiquinone binding site provide a model for the ‘deactive’ form of the enzyme. The proposed transition into the active form is based on a concerted structural rearrangement at the ubiquinone reduction site rendering support for a two-state stabilization-change mechanism of protonpumping.
The mitochondrial kinase PINK1 and the ubiquitin ligase Parkin are participating in quality control after CCCP- or ROSinduced mitochondrial damage, and their dysfunction is associated with the development and progression of Parkinson’s disease. Furthermore, PINK1 expression is also induced by starvation indicating an additional role for PINK1 in stress response. Therefore, the effects of PINK1 deficiency on the autophago-lysosomal pathway during stress were investigated. Under trophic deprivation SH-SY5Y cells with stable PINK1 knockdown showed downregulation of key autophagic genes, including Beclin, LC3 and LAMP-2. In good agreement, protein levels of LC3-II and LAMP-2 but not of LAMP-1 were reduced in different cell model systems with PINK1 knockdown or knockout after addition of different stressors. This downregulation of autophagic factors caused increased apoptosis, which could be rescued by overexpression of LC3 or PINK1. Taken together, the PINK1-mediated reduction of autophagic key factors during stress resulted in increased cell death, thus defining an additional pathway that could contribute to the progression of Parkinson’s disease in patients with PINK1 mutations.
Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8m delta) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na+/H+ antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8m delta. Nevertheless, nb8m delta still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.