Oxidation-reduction and reactive oxygen species homeostasis in mutant plants with respiratory chain complex I dysfunction

Mutations in a mitochondrial or nuclear gene encoding respiratory chain complex I subunits lead to decreased or a total absence of complex I activity. Plant mutants with altered or lost complex I activity adapt their respiratory metabolism by inducing alternative pathways of the respiratory chain and changing energy metabolism. Apparently, complex I is a crucial component of the oxidation-reduction (redox) regulatory system in photosynthetic cells, and alternative NAD(P)H dehydrogenases of the mitochondrial electron transport chain (mtETC) cannot fully compensate for its impairment. In most cases, dysfunction of complex I is associated with lowered or unchanged hydrogen peroxide (H(2)O(2)) concentrations, but increased superoxide (O(2)(-)) levels. Higher production of reactive oxygen species (ROS) by mitochondria in the mosaic (MSC16) cucumber mutant may be related to retrograde signalling. Different effects of complex I dysfunction on H(2)O(2) and O(2)(-) levels in described mutants might result from diverse regulation of processes involved in H(2)O(2) and O(2)(-) production. Often, dysfunction of complex I did not lead to oxidative stress, but increased the capacity of the antioxidative system and enhanced stress tolerance. The new cellular homeostasis in mutants with dysfunction of complex I allows growth and development, reflecting the plasticity of plant metabolism.     

The respiratory chain complex thresholds in mitochondria of a Drosophila subobscura mutant strain

Analysis of a mutant strain of Drosophila subobscura revealed that most (80%) mitochondrial genomes have undergone a large scale deletion (5 kb) in the coding region. Compared with the wild-type strain, complex I and III activities are, respectively, reduced by 50% and 30% in the mutant. However, the ATP synthesis capacities remain unchanged. In order to elucidate how the ATP synthesis is maintained at a normal level, despite a significant decrease in complex I and III activities, we progressively inhibited respiratory chain complex activities, respiration rate and ATP synthesis. Complex I, III and IV activities were inhibited by rotenone, antimycin and KCN, respectively. Threshold curves were thus determined for each complex. Our results demonstrated that in the mutant strain, both mitochondrial respiration and ATP synthesis had decreased when complex I activity was inhibited by more than 20%, whereas 70% inhibition is required to induce similar changes in the wild-type. The complex I inhibition pattern of the wild-type was restored by a backcross (mutant female/wild-type male). The complex III activity threshold is below 20% in both strains, and we observed some difference in antimycin sensitivity, suggesting a modification of the complex enzymatic properties in the mutant. In contrast, threshold values of 70% were measured for complex IV inhibition. Our data suggest that the difference in the complex I threshold curves between the wild-type and mutant strains could partially account for the absence of pathological phenotype in the mutant.     

Primary structure of the nuclear-encoded 18.3 kDa subunit of NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria.

The primary structure of the nuclear-encoded 18.3 kDa subunit of the respiratory chain NADH: ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA and the N-terminus of the protein. The cDNA contains an open reading frame for a protein of 206 amino acids. The mature protein consists of 173 amino acids and has a molar mass of 18,341 Da. The precursor protein includes a characteristic mitochondrial import sequence with a typical matrix peptidase processing site.     

Phosphorylation pattern of the NDUFS4 subunit of complex I of the mammalian respiratory chain

The NDUFS4 subunit of complex I of the mammalian respiratory chain has a fully conserved carboxy-terminus with a canonical RVSTK phosphorylation site. Immunochemical analysis with specific antibodies shows that the serine in this site of the protein is natively present in complex I in both the phosphorylated and non-phosphorylated state. Two-dimensional IEF/SDS–PAGE electrophoresis, ³²P labelling and immunodetection show that “in vitro” PKA phosphorylates the serine in the C-terminus of the NDUFS4 subunit in isolated bovine complex I. ³²P labelling and TLC phosphoaminoacid mapping show that PKA phosphorylates serine and threonine residues in the purified heterologous human NDUFS4 protein.     

The disulfide relay system of mitochondria is connected to the respiratory chain

All proteins of the intermembrane space of mitochondria are encoded by nuclear genes and synthesized in the cytosol. Many of these proteins lack presequences but are imported into mitochondria in an oxidation-driven process that relies on the activity of Mia40 and Erv1. Both factors form a disulfide relay system in which Mia40 functions as a receptor that transiently interacts with incoming polypeptides via disulfide bonds. Erv1 is a sulfhydryl oxidase that oxidizes and activates Mia40, but it has remained unclear how Erv1 itself is oxidized. Here, we show that Erv1 passes its electrons on to molecular oxygen via interaction with cytochrome c and cytochrome c oxidase. This connection to the respiratory chain increases the efficient oxidation of the relay system in mitochondria and prevents the formation of toxic hydrogen peroxide. Thus, analogous to the system in the bacterial periplasm, the disulfide relay in the intermembrane space is connected to the electron transport chain of the inner membrane.     

The acyl-carrier protein in Neurospora crassa mitochondria is a subunit of NADH:ubiquinone reductase (complex I).

We determined the primary structure of a 9.6-kDa subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa mitochondria and found a close relationship between this subunit and the bacterial or chloroplast acyl-carrier protein. The degree of sequence identity amounts to 80% in a region of 19 residues around the serine to which the phosphopantetheine is bound. The N-terminal presequence of the subunit has the characteristic features of a mitochondrial import sequence. We cultivated the auxotroph pan-2 mutant of N. crassa in the presence of [14C]pantothenate and recovered all radioactivity incorporated into mitochondrial protein in the 9.6-kDa subunit of complex I. We cultivated N. crassa in the presence of chloramphenicol to accumulate the nuclear-encoded peripheral arm of complex I. This pre-assembled arm also contains the 9.6-kDa subunit. These results demonstrate that an acyl-carrier protein with pantothenate as prosthetic group is a constituent part of complex I in N. crassa.     

Molecular characterization and mutational analysis of the human B17 subunit of the mitochondrial respiratory chain complex I

Bovine NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain consists of about 36 nuclear-encoded subunits. We review the current knowledge of the 15 human complex I subunits cloned so far, and report the 598-bp cDNA sequence, the chromosomal localization and the tissue expression of an additional subunit, the B17 subunit. The cDNA open reading frame of B17 comprises 387 bp and encodes a protein of 128 amino acids (calculated M _r 15.5 kDa). There is 82.7% and 78.1% homology, respectively, at the cDNA and amino acid level with the bovine counterpart. The gene of the B17 subunit has been mapped to chromosome 2. Multiple-tissue dot-blots showed ubiquitous expression of the mRNA with relatively higher expression in tissues known for their high energy demand. Of these, kidney showed the highest expression. Mutational analysis of the subunit revealed no mutations or polymorphisms in 20 patients with isolated enzymatic complex I deficiency in cultured skin fibroblasts. Received: 5 February 1998 / Accepted: 6 April 1998     

Decylubiquinol impedes mitochondrial respiratory chain complex I activity

In this study, indirect immunofluorescence labeling was used to examine the cellular dynamic distribution of Thr11 phosphorylated H3 at mitosis in MCF-7 cells. The Thr11 phosphorylation was observed beginning at prophase at centromeres. Upon progression of mitosis, fluorescence signal was enhanced in the central region of the metaphase plate and maintained till anaphase at centromeres. During telophase, the fluorescent signal of Thr11 phosphorylated H3 disappears from centromeres, but the signal appears again at the midbody during cytokinesis, which suggests that the modified histones may take part in the formation of the midbody and play a crucial role in cytokinesis. Chromatin immunoprecipitation (ChIP) was used to confirm that Thr11 phosphorylated H3 is specifically associated with centromere DNA at prophase to metaphase, which is coincident with the results observed by immunofluorescence. In conclusion, there was a precise spatial and temporal correlation between H3 phosphorylation of Thr11 and stages of chromatin condensation. The timing of Thr11 phosphorylation and dephosphorylation in mitosis were similar to that reported for Ser10 phosphorylation of H3. The Thr11 phosphorylated H3 localized at centromeres during mitosis, which was different from the Ser10 phosphorylated H3 localized at telomere regions and Thr3 phosphorylated H3 localized along the chromosome arms. The results suggest that the Thr11 phosphorylation of histone H3 may play a specific role which was different from Ser10 and Thr3 phosphorylation in mitosis.     

The respiratory chain components of higher plant mitochondria

Tightly coupled mitochondria have been prepared from a variety of plant sources: white potato (Solanum tuberosum), Jerusalem artichoke (Heliantus tuberosus), cauliflower buds (Brassica oleracea), and mung bean hypocotyls (Phaseolus aureus). Mitochondria with no appreciable coupling were also prepared from skunk cabbage spadices (Symplocarpus foetidus).

Room temperature difference spectra show that these mitochondria are very similar in the qualitative and quantitative composition of their electron carriers. The different cytochromes are present in the amounts of 0.1 to 0.3 mμmole per mg of mitochondrial protein. The molar ratios of the different electron carriers are, on the average: 0.7:0.7:1.0:3 to 4:10 to 15 respectively for cytochrome aa₃, cytochromes b, cytochromes c, flavoproteins, and pyridine nucleotides.

From low temperature difference spectra carried out under particular experimental conditions, it can be deduced that these mitochondria contain 3 b cytochromes whose α bands are located at 552, 557, and 561 mμ, and 2 c cytochromes, one of which, a c₁-like cytochrome, is firmly bound to the mitochondrial membrane. Cytochrome oxidase can be optically resolved into its 2 components a and a₃.

For all kinds of mitochondria, the rates of oxidation of succinate are similar as well as the turnover of cytochrome oxidase (50-70 sec⁻¹), regardless of the metabolic activities of the tissues. The number of mitochondria per cell appears to be the controlling factor of the intensity of tissue respiration.

     

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.     

Respiratory complex III is required to maintain complex I in mammalian mitochondria

A puzzling observation in patients with oxidative phosphorylation (OXPHOS) deficiencies is the presence of combined enzyme complex defects associated with a genetic alteration in only one protein-coding gene. In particular, mutations in the mtDNA encoded cytochrome b gene are associated either with combined complex I+III deficiency or with only complex III deficiency. We have reproduced the combined complex I+III defect in mouse and human cultured cell models harboring cytochrome b mutations. In both, complex III assembly is impeded and causes a severe reduction in the amount of complex I, not observed when complex III activity was pharmacologically inhibited. Metabolic labeling in mouse cells revealed that complex I was assembled, although its stability was severely hampered. Conversely, complex III stability was not influenced by the absence of complex I. This structural dependence among complexes I and III was confirmed in a muscle biopsy of a patient harboring a nonsense cytochrome b mutation.     

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.     

Inactivation of complex I of the respiratory chain of maize mitochondria incubated in vitro by elevated temperature

The functional stability of the ‘external’ NADH dehydrogenase and complexes I–IV of the respiratory chain of maize mitochondria was studied during mitochondria incubation in vitro at elevated temperatures. The increase in the incubation temperature from 0°C to 37°C significantly changed the stability of the respiratory chain. At 27°C and higher, the rate of oxidation of NAD-depended substrates decreased drastically, which is related to inactivation of complex I. Complexes II, III and IV of the respiratory chain and the ‘external’ NADH dehydrogenase were functionally stable at elevated temperatures. Moreover, the possibility of electron transport during oxidation of NAD-dependent substrates, in particular malate, bypasses complex I using rotenon insensitive NADH dehydrogenase.     

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.     

The first nuclear-encoded complex I mutation in a patient with Leigh syndrome.

Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest multiprotein enzyme complex of the respiratory chain. The nuclear-encoded NDUFS8 (TYKY) subunit of complex I is highly conserved among eukaryotes and prokaryotes and contains two 4Fe4S ferredoxin consensus patterns, which have long been thought to provide the binding site for the iron-sulfur cluster N-2. The NDUFS8 cDNA contains an open reading frame of 633 bp, coding for 210 amino acids. Cycle sequencing of amplified NDUFS8 cDNA of 20 patients with isolated enzymatic complex I deficiency revealed two compound heterozygous transitions in a patient with neuropathologically proven Leigh syndrome. The first mutation was a C236T (P79L), and the second mutation was a G305A (R102H). Both mutations were absent in 70 control alleles and cosegregated within the family. A progressive clinical phenotype proceeding to death in the first months of life was expressed in the patient. In the 19 other patients with enzymatic complex I deficiency, no mutations were found in the NDUFS8 cDNA. This article describes the first molecular genetic link between a nuclear-encoded subunit of complex I and Leigh syndrome.     

Relationships of respiratory chain and ATP-synthetase in energized mitochondria

The present study reveals that the previously described effect of ATP-synthetase inhibition concomitant with inhibition of respiratory chain functioning may be observed at different absolute values of delta psi on the mitochondrial membrane. This fact points out that the membrane potential is not a unique regulator in coupling of ATP-synthetase and respiratory chain activities. We found, using the double-inhibitor titration technique, that ATP-synthetase inhibition induces proportional inhibition of respiratory chain enzymes and vice versa respiratory chain inhibition induces proportional inhibition of ATP-synthetase. This effect is shown to exist only when osmolarity is close to 150-300 (mosM) (in the physiological range). The coupling effectivity (ADP/O) of mitochondria under these conditions is maximal. Under conditions of high osmolarity (400-600 mosM) the respiratory chain and ATP-synthetase behave as if they were coupled by bulk phase delta -mu H+, from the kinetic point of view.