Carbonic anhydrase subunits of the mitochondrial NADH dehydrogenase complex (complex I) in plants

The mitochondrial nicotinamide adenine dinucleotide, reduced (NADH) dehydrogenase complex (complex I) of plants has a molecular mass of about 1000 kDa and is composed of more than 40 distinct protein subunits. About three quarter of these subunits are homologous to complex I subunits of heterotrophic eukaryotes, whereas the remaining subunits are unique to plants. Among them are three to five structurally related proteins that resemble an archaebacterial γ-type carbonic anhydrase (γCA). The γCA subunits are attached to the membrane arm of complex I on the matrix-exposed side and form an extra spherical domain. At the same time, they span the inner mitochondrial membrane and are essential for assembly of the protein complex. Expression of the genes encoding γCA subunits is reduced if plants are cultivated in the presence of elevated CO₂ concentration. The functional role of these subunits within plant mitochondria is currently unknown but might be related to photorespiration. We propose that the complex I–integrated γCAs are involved in mitochondrial HCO₃⁻ formation to allow efficient recycling of inorganic carbon for CO₂ fixation in chloroplasts under high light conditions.     

A structural investigation of complex I and I+III2 supercomplex from Zea mays at 11-13 A resolution: assignment of the carbonic anhydrase domain and evidence for structural heterogeneity within complex I

The projection structures of complex I and the I+III2 supercomplex from the C4 plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 A. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric gamma-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the gamma-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant "type II" particle has a 15 A shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant "type I" particle. The I+III2 supercomplex was found to be a rigid structure which did not break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of "type I". This would mean that the "type II" particles are not involved in the supercomplex formation and, hence, could have a different physiological role.     

Insights into the composition and assembly of the membrane arm of plant complex I through analysis of subcomplexes in Arabidopsis mutant lines

NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.3) is the largest complex of the mitochondrial respiratory chain. In eukaryotes, it is composed of more than 40 subunits that are encoded by both the nuclear and mitochondrial genomes. Plant Complex I differs from the enzyme described in other eukaryotes, most notably due to the large number of plant-specific subunits in the membrane arm of the complex. The elucidation of the assembly pathway of Complex I has been a long-standing research aim in cellular biochemistry. We report the study of Arabidopsis mutants in Complex I subunits using a combination of Blue-Native PAGE and immunodetection to identify stable subcomplexes containing Complex I components, along with mass spectrometry analysis of Complex I components in membrane fractions and two-dimensional diagonal Tricine SDS-PAGE to study the composition of the largest subcomplex. Four subcomplexes of the membrane arm of Complex I with apparent molecular masses of 200, 400, 450, and 650 kDa were observed. We propose a working model for the assembly of the membrane arm of Complex I in plants and assign putative roles during the assembly process for two of the subunits studied.     

L-galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I

Mitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation.     

Projection structure of the membrane domain of Escherichia coli respiratory complex I at 8 A resolution

Respiratory complex I (NADH:ubiquinone oxidoreductase) is an L-shaped multisubunit protein assembly consisting of a hydrophobic membrane arm and a hydrophilic peripheral arm. It catalyses the transfer of two electrons from NADH to quinone coupled to the translocation of four protons across the membrane. Although we have solved recently the crystal structure of the peripheral arm, the structure of the complete enzyme and the coupling mechanism are not yet known. The membrane domain of Escherichia coli complex I consists of seven different subunits with total molecular mass of 258 kDa. It is significantly more stable than the whole enzyme, which allowed us to obtain well-ordered two-dimensional crystals of the domain, belonging to the space group p22(1)2(1). Comparison of the projection map of negatively stained crystals with previously published low-resolution structures indicated that the characteristic curved shape of the membrane domain is remarkably well conserved between bacterial and mitochondrial enzymes, helping us to interpret projection maps in the context of the intact complex. Two pronounced stain-excluding densities at the distal end of the membrane domain are likely to represent the two large antiporter-like subunits NuoL and NuoM. Cryo-electron microscopy on frozen-hydrated crystals allowed us to calculate a projection map at 8 A resolution. About 60 transmembrane alpha-helices, both perpendicular to the membrane plane and tilted, are present within one membrane domain, which is consistent with secondary structure predictions. A possible binding site and access channel for quinone are found at the interface with the peripheral arm. Tentative assignment of individual subunits to the features of the map has been made. The location of subunits NuoL and NuoM at substantial distance from the peripheral arm, which contains all the redox centres of the complex, indicates that conformational changes are likely to play a role in the mechanism of coupling between electron transfer and proton pumping.     

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.     

The location of NuoL and NuoM subunits in the membrane domain of the Escherichia coli complex I: implications for the mechanism of proton pumping

The molecular organization of bacterial NADH: ubiquinone oxidoreductase (complex I or NDH-1) is not established, apart from a rough separation into dehydrogenase, connecting and membrane domains. In this work, complex I was purified from Escherichia coli and fragmented by replacing dodecylmaltoside with other detergents. Exchange into decyl maltoside led to the removal of the hydrophobic subunit NuoL from the otherwise intact complex. Diheptanoyl phosphocholine led to the loss of NuoL and NuoM subunits, whereas other subunits remained in the complex. The presence of N,N-dimethyldodecylamine N-oxide or Triton X-100 led to further disruption of the membrane domain into fragments containing NuoL/M/N, NuoA/K/N, and NuoH/J subunits. Among the hydrophilic subunits, NuoCD was most readily dissociated from the complex, whereas NuoB was partially dissociated from the peripheral arm assembly in N,N-dimethyldodecylamine N-oxide. A model of subunit arrangement in bacterial complex I based on these data is proposed. Subunits NuoL and NuoM, which are homologous to antiporters and are implicated in proton pumping, are located at the distal end of the membrane arm, spatially separated from the redox centers of the peripheral arm. This is consistent with proposals that the mechanism of proton pumping by complex I is likely to involve long range conformational changes.     

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.     

A partially assembled complex I in NAD4-deficient mitochondria of maize

The proton-translocating NADH:ubiquinone oxidoreductase (respiratory complex I) consists of at least 32 subunits in higher plants, nine of which are mitochondrially encoded (NAD 1–7, NAD4L, NAD9). Complex I (CI) has been analyzed from a mitochondrial mutant of maize, NCS2, that carries a deletion for the 3′ end of the nad4 gene. Mitochondria from highly defective, near-homoplasmic mutant plants have only trace amounts of the normal complex I. Instead, a reduced amount of a smaller complex, which also exhibits NADH dehydrogenase activity, is detected on ‘blue-native’ polyacrylamide gels. Subunits of 76 kDa, 40 kDa and 55 kDa, as well as NAD7 and NAD9, have been identified in the subcomplex by their cross-reactivity with heterologous antisera. The corresponding subunits in Neurospora are localized in a ‘peripheral arm’ of CI, which is known to assemble independently of a ‘membrane arm’. The maize NCS2 CI subcomplex is loosely bound to the membrane and is missing several subunits that could be membrane components. Thus, the mutant CI subcomplex may consist of a peripheral arm. A reduction in the steady-state levels of NAD7 and NAD9 in NCS2 mitochondria occurs despite normal rates of biosynthesis and there is a concomitant decrease of the nuclear encoded 76 kDa subunit. The reduction in CI-associated NADH dehydrogenase activity in the nad4 -deficient NCS2 mutant mitochondria is not associated with a compensatory increase in the activities or amounts of the putative ‘exogenous’ NAD(P)H dehydrogenases that are found in plant mitochondria.     

Isolation,characterization and electron microscopic single particle analysis of the NADH:ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 degrees C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 degrees C. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 degrees C, with a half-life of about 10 h at 80 degrees C. The activity shows a linear Arrhenius plot at 50-85 degrees C with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90 degrees ) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.     

Inactivation of Genes Encoding Subunits of the Peripheral and Membrane Arms of Neurospora Mitochondrial Complex I and Effects on Enzyme Assembly

We have isolated and characterized the nuclear genes encoding the 12.3-kD subunit of the membrane arm and the 29.9-kD subunit of the peripheral arm of complex I from Neurospora crassa. The former gene was known to be located in linkage group I and the latter is now assigned to linkage group IV of the fungal genome. The genes were separately transformed into different N. crassa strains and transformants with duplicated DNA sequences were isolated. Selected transformants were then mated with other strains to generate repeat-induced point mutations in both copies of the genes present in the nucleus of the parental transformant. From the progeny of the crosses, we were then able to recover two individual mutants lacking the 12.3- and 29.9-kD proteins in their mitochondria, mutants nuo12.3 and nuo29.9, respectively. Several other subunits of complex I are present in the mutant organelles, although with altered stoichiometries as compared with those in the wild-type strain. Based on the analysis of Triton-solubilized mitochondrial complexes in sucrose gradients, neither mutant is able to fully assemble complex I. Our results indicate that mutant nuo12.3 separately assembles the peripheral arm and most of the membrane arm of the enzyme. Mutant nuo29.9 seems to accumulate the membrane arm of complex I and being devoid of the peripheral part. This implicates the 29.9-kD protein in an early step of complex I assembly.     

Analysis of the subunit composition of complex I from bovine heart mitochondria

Complex I purified from bovine heart mitochondria is a multisubunit membrane-bound assembly. In the past, seven of its subunits were shown to be products of the mitochondrial genome, and 35 nuclear encoded subunits were identified. The complex is L-shaped with one arm in the plane of the membrane and the other lying orthogonal to it in the mitochondrial matrix. With mildly chaotropic detergents, the intact complex has been resolved into various subcomplexes. Subcomplex Ilambda represents the extrinsic arm, subcomplex Ialpha consists of subcomplex Ilambda plus part of the membrane arm, and subcomplex Ibeta is another substantial part of the membrane arm. The intact complex and these three subcomplexes have been subjected to extensive reanalysis. Their subunits have been separated by three independent methods (one-dimensional SDS-PAGE, two-dimensional isoelectric focusing/SDS-PAGE, and reverse phase high pressure liquid chromatography (HPLC)) and analyzed by tryptic peptide mass fingerprinting and tandem mass spectrometry. The masses of many of the intact subunits have also been measured by electrospray ionization mass spectrometry and have provided valuable information about post-translational modifications. The presence of the known 35 nuclear encoded subunits in complex I has been confirmed, and four additional nuclear encoded subunits have been detected. Subunits B16.6, B14.7, and ESSS were discovered in the SDS-PAGE analysis of subcomplex Ilambda, in the two-dimensional gel analysis of the intact complex, and in the HPLC analysis of subcomplex Ibeta, respectively. Despite many attempts, no sequence information has been obtained yet on a fourth new subunit (mass 10,566+/-2 Da) also detected in the HPLC analysis of subcomplex Ibeta. It is unlikely that any more subunits of the bovine complex remain undiscovered. Therefore, the intact enzyme is a complex of 46 subunits, and, assuming there is one copy of each subunit in the complex, its mass is 980 kDa.     

Glucose oxidation catalyzed by liposomal glucose oxidase in the presence of catalase-containing liposomes

A catalase-containing liposome (CAL) was prepared and characterized in terms of stability during storage and catalysis of the decomposition of hydrogen peroxide (H2O2) that was initially added or produced in the oxidation of glucose catalyzed by the glucose oxidase-containing liposomes (GOL). The reactors used were a test tube and an external loop airlift bubble column as the static liquid and circulating liquid flow systems, respectively. The free catalase (CA) at low concentrations was unstable during storage at 4 degrees C as a result of dissociation of the tetrameric CA subunits. On the other hand, the deactivation of the CA activity in the CAL was depressed because of the high CA concentration in the CAL liposome. The CAL effectively catalyzed the repeated decompositions at 25 degrees C with 10 mM H2O2 added initially, whereas the free CA was significantly deactivated during the repeated reactions. The high stability of the CAL was attributed to the moderately depressed reactivity, which was essentially derived from the diffusion limitation of the CAL membrane to H2O2 in the liquid bulk. In the GOL-catalyzed prolonged oxidation of 10 mM glucose at 40 degrees C in the static liquid in a test tube, both the free CA and CAL could continuously catalyze the decomposition of H2O2 produced. This was because the glucose oxidation rate was small due to the limited reactivity of the GOL to glucose with its low permeability through the GOL membrane. In the glucose oxidation catalyzed by the GOL with the free CA or the CAL in the airlift, much larger oxidation rates were observed compared to those in the test tube because the permeability of the GOL membrane to glucose was increased in the gas-liquid two phase flow in the airlift. The GOL/CAL system in the airlift operated in an acidic condition, which was preferable to the GO activity, gave the largest oxidation rate with negligible accumulation of the H2O2 produced. On the other hand, the GOL/free CA system gave an oxidation rate smaller than that of the GOL/CAL system even under the acidic condition due to an unfavorable interaction of the free CA molecules with the GOL membranes leading to the decreased reactivity of the GOL.     

Composition of complex I from Neurospora crassa and disruption of two "accessory" subunits

Respiratory chain complex I of the fungus Neurospora crassa contains at least 39 polypeptide subunits, of which 35 are conserved in mammals. The 11.5 kDa and 14 kDa proteins, homologues of bovine IP15 and B16.6, respectively, are conserved among eukaryotes and belong to the membrane domain of the fungal enzyme. The corresponding genes were separately inactivated by repeat-induced point-mutations, and null-mutant strains of the fungus were isolated. The lack of either subunit leads to the accumulation of distinct intermediates of the membrane arm of complex I. In addition, the peripheral arm of the enzyme seems to be formed in mutant nuo14 but, interestingly, not in mutant nuo11.5. These results and the analysis of enzymatic activities of mutant mitochondria indicate that both polypeptides are required for complex I assembly and function.     

Direct localization of the 51 and 24 kDa subunits of mitochondrial complex I by three-dimensional difference imaging

Complex I is the largest complex in the respiratory chain, and the least understood. We have determined the 3D structure of complex I from Yarrowia lipolytica lacking the flavoprotein part of the N-module, which consists of the 51 kDa (NUBM) and the 24 kDa (NUHM) subunits. The reconstruction was determined by 3D electron microscopy of single particles. A comparison to our earlier reconstruction of the complete Y. lipolytica complex I clearly assigns the two flavoprotein subunits to an outer lobe of the peripheral arm of complex I. Localizing the two subunits allowed us to fit the X-ray structure of the hydrophilic fragment of complex I from Thermus thermophilus. The fit that is most consistent with previous immuno-electron microscopic data predicts that the ubiquinone reducing catalytic center resides in the second peripheral lobe, while the 75 kDa subunit is placed near the previously seen connection between the peripheral arm and the membrane arm protrusions.     

Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6

Background

The quaternary structure of eukaryotic NADH:ubiquinone oxidoreductase (complex I), the largest complex of the oxidative phosphorylation, is still mostly unresolved. Furthermore, it is unknown where transiently bound assembly factors interact with complex I. We therefore asked whether the evolution of complex I contains information about its 3D topology and the binding positions of its assembly factors. We approached these questions by correlating the evolutionary rates of eukaryotic complex I subunits using the mirror-tree method and mapping the results into a 3D representation by multidimensional scaling.

Results

More than 60% of the evolutionary correlation among the conserved seven subunits of the complex I matrix arm can be explained by the physical distance between the subunits. The three-dimensional evolutionary model of the eukaryotic conserved matrix arm has a striking similarity to the matrix arm quaternary structure in the bacterium Thermus thermophilus (rmsd=19 ?) and supports the previous finding that in eukaryotes the N-module is turned relative to the Q-module when compared to bacteria. By contrast, the evolutionary rates contained little information about the structure of the membrane arm. A large evolutionary model of 45 subunits and assembly factors allows to predict subunit positions and interactions (rmsd = 52.6 ?). The model supports an interaction of NDUFAF3, C8orf38 and C2orf56 during the assembly of the proximal matrix arm and the membrane arm. The model further suggests a tight relationship between the assembly factor NUBPL and NDUFA2, which both have been linked to iron-sulfur cluster assembly, as well as between NDUFA12 and its paralog, the assembly factor NDUFAF2.

Conclusions

The physical distance between subunits of complex I is a major correlate of the rate of protein evolution in the complex I matrix arm and is sufficient to infer parts of the complex??s structure with high accuracy. The resulting evolutionary model predicts the positions of a number of subunits and assembly factors.     

Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica

Here we present a first assessment of the subunit inventory of mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica. A total of 37 subunits were identified. In addition to the seven central, nuclear coded, and the seven mitochondrially coded subunits, 23 accessory subunits were found based on 2D electrophoretic and mass spectroscopic analysis in combination with sequence information from the Y. lipolytica genome. Nineteen of the 23 accessory subunits are clearly conserved between Y. lipolytica and mammals. The remaining four accessory subunits include NUWM, which has no apparent homologue in any other organism and is predicted to contain a single transmembrane domain bounded by highly charged extramembraneous domains. This structural organization is shared among a group of 7 subunits in the Y. lipolytica and 14 subunits in the mammalian enzyme. Because only five of these subunits display significant evolutionary conservation, their as yet unknown function is proposed to be structure- rather than sequence-specific. The NUWM subunit could be assigned to a hydrophobic subcomplex obtained by fragmentation and sucrose gradient centrifugation. Its position within the membrane arm was determined by electron microscopic single particle analysis of Y. lipolytica complex I decorated with a NUWM-specific monoclonal antibody.     

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.     

Challenges in elucidating structure and mechanism of proton pumping NADH:ubiquinone oxidoreductase (complex I)

Proton pumping NADH:ubiquinone oxidoreductase (complex I) is the most complicated and least understood enzyme of the respiratory chain. All redox prosthetic groups reside in the peripheral arm of the L-shaped structure. The NADH oxidation domain harbouring the FMN cofactor is connected via a chain of iron–sulfur clusters to the ubiquinone reduction site that is located in a large pocket formed by the PSST- and 49-kDa subunits of complex I. An access path for ubiquinone and different partially overlapping inhibitor binding regions were defined within this pocket by site directed mutagenesis. A combination of biochemical and single particle analysis studies suggests that the ubiquinone reduction site is located well above the membrane domain. Therefore, direct coupling mechanisms seem unlikely and the redox energy must be converted into a conformational change that drives proton pumping across the membrane arm. It is not known which of the subunits and how many are involved in proton translocation. Complex I is a major source of reactive oxygen species (ROS) that are predominantly formed by electron transfer from FMNH₂. Mitochondrial complex I can cycle between active and deactive forms that can be distinguished by the reactivity towards divalent cations and thiol-reactive agents. The physiological role of this phenomenon is yet unclear but it could contribute to the regulation of complex I activity in-vivo.