Structural basis for a complex I mutation that blocks pathological ROS production.

Mitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia-reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the "deactive" state, usually formed only after prolonged inactivity. Despite its tendency to adopt the "deactive" state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.

Complex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recent cryo-EM analyses have produced near-complete models of all 45 subunits in the bovine, ovine and porcine complexes and have identified two states relevant to complex I in ischemia-reperfusion injury. Here, we describe the 3.3-Å structure of complex I from mouse heart mitochondria, a biomedically relevant model system, in the 'active' state. We reveal a nucleotide bound in subunit NDUFA10, a nucleoside kinase homolog, and define mechanistically critical elements in the mammalian enzyme. By comparisons with a 3.9-Å structure of the 'deactive' state and with known bacterial structures, we identify differences in helical geometry in the membrane domain that occur upon activation or that alter the positions of catalytically important charged residues. Our results demonstrate the capability of cryo-EM analyses to challenge and develop mechanistic models for mammalian complex I.

The structure of eukaryotic and prokaryotic complex I.

The structures of the NADH dehydrogenases from Bos taurus and Aquifex aeolicus have been determined by 3D electron microscopy, and have been analyzed in comparison with the previously determined structure of Complex I from Yarrowia lipolytica. The results show a clearly preserved domain structure in the peripheral arm of complex I, which is similar in the bacterial and eukaryotic complex. The membrane arms of both eukaryotic complexes show a similar shape but also significant differences in distinctive domains. One of the major protuberances observed in Y. lipolytica complex I appears missing in the bovine complex, while a protuberance not found in Y. lipolytica connects in bovine complex I a domain of the peripheral arm to the membrane arm. The structural similarities of the peripheral arm agree with the common functional principle of all complex Is. The differences seen in the membrane arm may indicate differences in the regulatory mechanism of the enzyme in different species.

Single particle analysis confirms distal location of subunits NuoL and NuoM in Escherichia coli complex I.

Respiratory complex I catalyses the transfer of electrons from NADH to quinone coupled to the translocation of protons across the membrane. The mechanism of coupling and the structure of the complete enzyme are not known. The membrane domain of the complex contains three similar antiporter-like subunits NuoL/M/N, probably involved in proton pumping. We have previously shown that subunits NuoL/M can be removed from the rest of the complex, suggesting their location at the distal end of the membrane domain. Here, using electron microscopy and single particle analysis, we show that subunits NuoL and M jointly occupy a distal half of the membrane domain, separated by about 10nm from the interface with the peripheral arm. This indicates that coupling mechanism of complex I is likely to involve long range conformational changes.

Three-dimensional structure of NADH-dehydrogenase from Neurospora crassa by electron microscopy and conical tilt reconstruction.

NADH-dehydrogenase (Complex I) is the first complex of the mitochondrial respiratory chain. It is an amphipatic molecule located in the inner mitochondrial membrane and is composed of at least 35 unique subunits encoded by both mitochondrial and nuclear DNA. The whole complex was isolated in detergent from the fungus Neurospora crassa. It is very stable in its isolated form and was analysed as such by electron microscopy. Its mass, determined by dark-field scanning electron microscopy was estimated as 1.12 MDa. The complex was imaged by transmission electron microscopy, by negative staining and by cryo-electron microscopy. A three-dimensional model, with a resolution estimated at 35 A, was calculated from images of negatively stained complexes by the random conical tilt reconstruction technique. This model confirms the general L-shape of the molecule, with arms of equal length and corroborates the hypothesis of a subdivision of the whole complex into three functional domains. Immuno-labelling of the 49 kDA subunit of the peripheral arm allowed its localization within the complex. This is a first step in the subunit mapping of Complex I and the understanding of its activity.

Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I).

Two related forms of the respiratory chain NADH dehydrogenase (NADH:ubiquinone reductase or complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large form that consists of 25 subunits encoded by nuclear DNA and six to seven subunits encoded by mitochondrial DNA. Cells grown in the presence of chloramphenicol, however, make a smaller form comprising only 13 subunits, all encoded by nuclear DNA. When the large enzyme is dissected by chaotropic agents (such as NaBr), all those subunits of the large form that are missing in the small form can be isolated as a distinct, so-called hydrophobic fragment. The small enzyme and the hydrophobic fragment make up, with regard to their redox groups, subunit composition and function, two complementary parts of the large-form NADH dehydrogenase. Averaging of electron microscope images of single particles of the large enzyme was carried out, revealing an unusual L-shaped structure with two domains or "arms" arranged at right angles. The hydrophobic fragment obtained by the NaBr treatment corresponds in size and appearance to one of these arms. A three-dimensional reconstruction from images of negatively stained membrane crystals of the large-form NADH dehydrogenase shows a peripheral domain, protruding from the membrane, with weak unresolved density within the membrane. This peripheral domain was removed by washing the crystals in situ with 2 M-NaBr, exposing a large membrane-buried domain, which was reconstructed in three dimensions. A three-dimensional reconstruction of the small enzyme from negatively stained membrane crystals, also described here, shows only a peripheral domain. These results suggest that the membrane protruding arm of the large form corresponds to the small enzyme, whereas the arm lying within the membrane can be identified as the hydrophobic fragment. The two parts of NADH dehydrogenase that can be defined by the separate genetic origin of (most of) their subunits, their independent assembly, and their distinct contributions to the electron pathway can thus be assigned to the two arms of the L-shaped complex I.

Electron microscopy and image analysis of the complexes I and V of the mitochondrial respiratory chain.

The results of Section IV can be summarized in a simple ATP synthase model. This model implies that either the alpha or the beta subunits must be closer to the membrane. The work of Gao and Bauerlein (1987) indicates that the alpha subunits are closer to the membrane. Although the overall structure is more or less clear, important questions need to be clarified. First, the number and the arrangement of the subunits in the F0 part must be known. Second, the exact shape of F1, and particularly the shape of the large subunits needs to be elucidated. On the basis of fluorescence resonance energy transfer measurements by McCarty and Hammes (1987), a model was presented showing large oblong subunits. Such 'banana-shaped' subunits, which are also presented in the many phantasy models (e.g. Walker et al., 1982), are very unlikely in view of the electron microscopical results, although the large subunits do not need to be exactly spherical. The third and most interesting central question is on the changes in the structure that take place during the different steps in the synthesis of ATP. It can now be taken as proven that the energy transmitted to the ATP synthase is used to induce a conformational change in the latter enzyme, in such a way as to bring about the energy-requiring dissociation of already synthesized ATP (Penefsky, 1985 and reviewed in Slater, 1987). But the way in which the three parts of the ATP synthase are involved is completely unknown. It is rather puzzling that such a long distance exists between the catalytic sites, which are on the interface of the alpha and beta subunits and the F0 part where the proton movements occur, which, according to Mitchell's theory (1961), is the driving force for the synthesis of ATP. Perhaps alternative mechanisms such as the collision hypothesis formulated by Herweijer et al. (1985) are more realistic in describing the mechanism of ATP synthesis. It would bring the complexes I and V close together, not only in the artificial way treated in this paper, but in a useful way for energy conversion.