Mitochondrial complex I impairment and differential carbon monoxide sensitivity of cytochrome c oxidase in wild type and CMS II mutants of Nicotiana sylvestris

Plant mitochondria unlike their animal counterpart have some unique features with highly branched respiratory chain. The present work was undertaken in order to investigate the effect of loss/dysfunction of plant mitochondrial complex I on the relative flux of electrons through alternative oxidase (AOX) and cytochrome oxidase. Loss of a major subunit of mitochondrial complex I in cytoplasmic male sterile II (CMS II) mutant of Nicotiana sylvestris caused respiratory redox perturbations, as evident from the differential CO sensitivity of cytochrome oxidase. The leaf segments of CMS II mutant when exposed to CO under dark aerobic condition were insensitive to the inhibition of cytochrome oxidase, as against the wild type (WT). The differential CO response of WT and CMS II mutants appeared to be due to differences in the redox state of cytochrome a3 (cyt a3), the terminal electron acceptor during in situ respiration. Cyt a3 appeared to be more in its oxidized form in CMS II and hence unable to form cyt a3-CO complex. Pre-treatment of CMS II leaves with 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation increased the CO response. The slight increase in rotenone-insensitive respiration of CMS II could be attributed partly to enhanced flux of electrons through cytochrome pathway to compensate for the loss of phosphorylation site and partly through AOX, which was induced by nitrate.     

Mitochondria-driven changes in leaf NAD status exert a crucial influence on the control of nitrate assimilation and the integration of carbon and nitrogen metabolism

The Nicotiana sylvestris mutant, CMS, lacks the mitochondrial gene nad7 and functional complex I, and respires using low-affinity NADH (alternative) mitochondrial dehydrogenases. Here, we show that this adjustment of respiratory pathways is associated with a profound modification of foliar carbon-nitrogen balance. CMS leaves are characterized by abundant amino acids compared to either wild-type plants or CMS in which complex I function has been restored by nuclear transformation with the nad7 cDNA. The metabolite profile of CMS leaves is enriched in amino acids with low carbon/nitrogen and depleted in starch and 2-oxoglutarate. Deficiency in 2-oxoglutarate occurred despite increased citrate and malate and higher capacity of key anaplerotic enzymes, notably the mitochondrial NAD-dependent isocitrate dehydrogenase. The accumulation of nitrogen-rich amino acids was not accompanied by increased expression of enzymes involved in nitrogen assimilation. Partitioning of (15)N-nitrate into soluble amines was enhanced in CMS leaf discs compared to wild-type discs, especially in the dark. Analysis of pyridine nucleotides showed that both NAD and NADH were increased by 2-fold in CMS leaves. The growth retardation of CMS relative to the wild type was highly dependent on photoperiod, but at all photoperiod regimes the link between high contents of amino acids and NADH was observed. Together, the data provide strong evidence that (1) NADH availability is a critical factor in influencing the rate of nitrate assimilation and that (2) NAD status plays a crucial role in coordinating ammonia assimilation with the anaplerotic production of carbon skeletons.     

Expression of Ndi1p, an alternative NADH:ubiquinone oxidoreductase, increases mitochondrial membrane potential in a C. elegans model of mitochondrial disease

The NADH:ubiquinone oxidoreductase or complex I of the mitochondrial respiratory chain is an intricate enzyme with a vital role in energy metabolism. Mutations affecting complex I can affect at least three processes; they can impair the oxidation of NADH, reduce the enzyme's ability to pump protons for the generation of a mitochondrial membrane potential and increase the production of damaging reactive oxygen species. We have previously developed a nematode model of complex I-associated mitochondrial dysfunction that features hallmark characteristics of mitochondrial disease, such as lactic acidosis and decreased respiration. We have expressed the Saccharomyces cerevisiae NDI1 gene, which encodes a single subunit NADH dehydrogenase, in a strain of Caenorhabditis elegans with an impaired complex I. Expression of Ndi1p produces marked improvements in animal fitness and reproduction, increases respiration rates and restores mitochondrial membrane potential to wild type levels. Ndi1p functionally integrates into the nematode respiratory chain and mitigates the deleterious effects of a complex I deficit. However, we have also shown that Ndi1p cannot substitute for the absence of complex I. Nevertheless, the yeast Ndi1p should be considered as a candidate for gene therapy in human diseases involving complex I.     

Pancreatic mitochondrial complex I exhibits aberrant hyperactivity in diabetes

It is well established that NADH/NAD⁺ redox balance is heavily perturbed in diabetes, and the NADH/NAD⁺ redox imbalance is a major source of oxidative stress in diabetic tissues. In mitochondria, complex I is the only site for NADH oxidation and NAD⁺ regeneration and is also a major site for production of mitochondrial reactive oxygen species (ROS). Yet how complex I responds to the NADH/NAD⁺ redox imbalance and any potential consequences of such response in diabetic pancreas have not been investigated. We report here that pancreatic mitochondrial complex I showed aberrant hyperactivity in either type 1 or type 2 diabetes. Further studies focusing on streptozotocin (STZ)-induced diabetes indicate that complex I hyperactivity could be attenuated by metformin. Moreover, complex I hyperactivity was accompanied by increased activities of complexes II to IV, but not complex V, suggesting that overflow of NADH via complex I in diabetes could be diverted to ROS production. Indeed in diabetic pancreas, ROS production and oxidative stress increased and mitochondrial ATP production decreased, which can be attributed to impaired pancreatic mitochondrial membrane potential that is responsible for increased cell death. Additionally, cellular defense systems such as glucose 6-phosphate dehydrogenase, sirtuin 3, and NQO1 were found to be compromised in diabetic pancreas. Our findings point to the direction that complex I aberrant hyperactivity in pancreas could be a major source of oxidative stress and β cell failure in diabetes. Therefore, inhibiting pancreatic complex I hyperactivity and attenuating its ROS production by various means in diabetes might serve as a promising approach for anti-diabetic therapies.     

Deletion of the last two exons of the mitochondrialnad7 gene results in lack of the NAD7 polypeptide in aNicotiana sylvestris CMS mutant

InNicotiana sylvestris, two cytoplasmic male sterile (CMS) mutants obtained by protoplast culture show abnormal developmental features of both vegetative and reproductive organs, and mitochondrial gene reorganization following homologous recombination between 65 bp repeated sequences. A mitochondrial region of 16.2 kb deleted from both CMS mutants was found to contain the last two exons of thenad7 gene coding for a subunit of the mitochondrial respiratory chain complex I, which is encoded in the nucleus in fungi and animals but was recently found to be encoded by the mitochondrial genome in wheat. Although theN. sylvestris nad7 gene shows strong homology with its wheat counterpart, it contains only three introns instead of four. Polymerase chain reaction (PCR) experiments indicated that the parental gene organization, including the completenad7 gene, is probably maintained at a substoichiometric level in the CMS mutants, but this proportion is too low to have a significant physiological role, as confirmed by expression studies showing the lack of detectable amounts of the NAD7 polypeptide. Consequently, absence of NAD7 is not lethal to plant cells but a deficiency of complex I could be involved in the abnormal CMS phenotype.     

Interorganellar gene transfer in bryophytes: the functional nad7 gene is nuclear encoded in Marchantia polymorpha

The nad7 gene, encoding subunit 7 of NADH dehydrogenase, is mitochondrially encoded in seed plants. In the liverwort, Marchantia polymorpha, only a pseudogene is located in the mitochondrial genome. We have now identified the functional nad7 gene copy in the nuclear genome of Marchantia, coding for a polypeptide of 468 amino acids. The nuclear-encoded nad7 has lost the two group II introns present in the mitochondrial pseudogene copy. Instead, a typical nuclear intron is found to split an exon encoding the presumptive mitochondrial targeting signal peptide and the mature subunit 7 of NADH dehydrogenase. These results suggest that RNA-mediated gene transfer from the mitochondrial into the nuclear genome occurs not only in seed plants but also in bryophytes.     

A Drosophila model of mitochondrial disease caused by a complex I mutation that uncouples proton pumping from electron transfer

Mutations affecting mitochondrial complex I, a multi-subunit assembly that couples electron transfer to proton pumping, are the most frequent cause of heritable mitochondrial diseases. However, the mechanisms by which complex I dysfunction results in disease remain unclear. Here, we describe a Drosophila model of complex I deficiency caused by a homoplasmic mutation in the mitochondrial-DNA-encoded NADH dehydrogenase subunit 2 (ND2) gene. We show that ND2 mutants exhibit phenotypes that resemble symptoms of mitochondrial disease, including shortened lifespan, progressive neurodegeneration, diminished neural mitochondrial membrane potential and lower levels of neural ATP. Our biochemical studies of ND2 mutants reveal that complex I is unable to efficiently couple electron transfer to proton pumping. Thus, our study provides evidence that the ND2 subunit participates directly in the proton pumping mechanism of complex I. Together, our findings support the model that diminished respiratory chain activity, and consequent energy deficiency, are responsible for the pathogenesis of complex-I-associated neurodegeneration.KEY WORDS: Mitochondria, Drosophila, Mitochondrial disease, Respiratory chain, Leigh syndrome, Neurodegeneration     

Subcomplex Iλ Specifically Controls Integrated Mitochondrial Functions in Caenorhabditis elegans

Complex I dysfunction is a common, heterogeneous cause of human mitochondrial disease having poorly understood pathogenesis. The extensive conservation of complex I composition between humans and Caenorhabditis elegans permits analysis of individual subunit contribution to mitochondrial functions at both the whole animal and mitochondrial levels. We provide the first experimentally-verified compilation of complex I composition in C. elegans, demonstrating 84% conservation with human complex I. Individual subunit contribution to mitochondrial respiratory capacity, holocomplex I assembly, and animal anesthetic behavior was studied in C. elegans by RNA interference-generated knockdown of nuclear genes encoding 28 complex I structural subunits and 2 assembly factors. Not all complex I subunits directly impact respiratory capacity. Subcomplex Iλ subunits along the electron transfer pathway specifically control whole animal anesthetic sensitivity and complex II upregulation, proportionate to their relative impairment of complex I-dependent oxidative capacity. Translational analysis of complex I dysfunction facilitates mechanistic understanding of individual gene contribution to mitochondrial disease. We demonstrate that functional consequences of complex I deficiency vary with the particular subunit that is defective.     

Pathway for Nitrate Assimilation in Corn (Zea mays L.) Leaves: Cellular Distribution of Enzymes and Energy Sources for Nitrate Reduction

The localization of enzymes responsible for nitrate assimilation and the generation of NADH for nitrate reduction were studied in corn (Zea mays L.) leaf blades. The techniques used effectively separated mesophyll and bundle sheath cells as judged by microscopic observations, enzymic assays, chlorophyll a/b ratios and photochemical activities. Nitrate reductase, nitrite reductase, and the nitrate content of leaf blades were localized primarily in the mesophyll cells, although some nitrite reductase was found in the bundle sheath cells. Glutamine synthetase, NAD-malate dehydrogenase, NAD-glyceraldehyde-3-phosphate dehydrogenase, and NADP-glutamate dehydrogenase were found in both types of cells, however, more NADP-glutamate dehydrogenase was found in the bundle sheath cells than in the mesophyll cells. These data indicate that the mesophyll cells are the major site for nitrate assimilation in the leaf blade because they contained an ample supply of nitrate and the enzymes considered essential for the assimilation of nitrate into amino acids. Because the specific activity of nitrate reductase was severalfold lower than the other enzymes involved in nitrate assimilation, nitrate reduction is indicated as the rate-limiting step in situ. A sequence of reactions is proposed for nitrate assimilation in the mesophyll cells of corn leaves as related to the C-4 pathway of photosynthesis.     

Partial Purification and Characterization of Complex I, NADH:Ubiquinone Reductase, from the Inner Membrane of Beetroot Mitochondria

A NADH dehydrogenase was isolated from an inner membrane-enriched fraction of beetroot mitochondria (Beta vulgaris L.) by solubilization with sodium deoxycholate and purified using gel filtration and affinity chromatography. The NADH dehydrogenase preparation contained a minor ATPase contamination. Beetroot mitochondria were chosen as the isolation material for purifying the enzymes responsible for oxidizing matrix NADH due to the absence of the externally facing NADH dehydrogenase in the variety we have used. The purified NADH dehydrogenase complex catalyzed the reduction of various electron acceptors with NADH as the electron donor, was not sensitive to rotenone inhibition, and had a slow NADPH-ubiquinone 5 reductase activity. The isolated complex contained 14 major polypeptides. It was concluded that the dehydrogenase represented a form of the plant mitochondrial complex I and not the internally facing rotenone-insensitive NADH dehydrogenase found in plant mitochondria because of its complex structure, its cross-reactivity with antisera raised against bovine heart mitochondrial complex I, and the similarity of its kinetics and inhibitor responses to rotenone-sensitive NADH oxidation by beetroot submitochondrial particles.     

Root respiration associated with ammonium and nitrate absorption and assimilation by barley

We examined nitrate assimilation and root gas fluxes in a wild-type barley (Hordeum vulgare L. cv Steptoe), a mutant (nar1a) deficient in NADH nitrate reductase, and a mutant (nar1a;nar7w) deficient in both NADH and NAD(P)H nitrate reductases. Estimates of in vivo nitrate assimilation from excised roots and whole plants indicated that the nar1a mutation influences assimilation only in the shoot and that exposure to NO₃⁻ induced shoot nitrate reduction more slowly than root nitrate reduction in all three genotypes. When plants that had been deprived of nitrogen for several days were exposed to ammonium, root carbon dioxide evolution and oxygen consumption increased markedly, but respiratory quotient—the ratio of carbon dioxide evolved to oxygen consumed—did not change. A shift from ammonium to nitrate nutrition stimulated root carbon dioxide evolution slightly and inhibited oxygen consumption in the wild type and nar1a mutant, but had negligible effects on root gas fluxes in the nar1a;nar7w mutant. These results indicate that, under NH₄⁺ nutrition, 14% of root carbon catabolism is coupled to NH₄⁺ absorption and assimilation and that, under NO₃⁻ nutrition, 5% of root carbon catabolism is coupled to NO₃⁻ absorption, 15% to NO₃⁻ assimilation, and 3% to NH₄⁺ assimilation. The additional energy requirements of NO₃⁻ assimilation appear to diminish root mitochondrial electron transport. Thus, the energy requirements of NH₄⁺ and NO₃⁻ absorption and assimilation constitute a significant portion of root respiration.     

Regulation of NADH supply for nitrate reduction in green plants via photosynthesis and mitochondrial respiration.

The oxidation of NADH by mitochondrial respiration is inhibited by increased adenylate charge produced by photophosphorylation and under these conditions the reducing equivalents are used for nitrate reduction in the cytoplasm. Thus nitrate appears to function as an alternative electron acceptor to O₂ for NADH generated by the citric acid cycle dehydrogenases. This mechanism ensures that nitrate assimilation occurs only in light and thus the accumulation of toxic levels of nitrite in the dark is avoided.     

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.     

SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD

To date no models exist to study MnSOD deficiency in human cells. To address this deficiency, we created a SOD2-null human cell line that is completely devoid of detectable MnSOD protein expression and enzyme activity. We utilized the CRISPR/Cas9 system to generate biallelic SOD2 disruption in HEK293T cells. These SOD2-null cells exhibit impaired clonogenic activity, which was rescued by either treatment with GC4419, a pharmacological small-molecule mimic of SOD, or growth in hypoxia. The phenotype of these cells is primarily characterized by impaired mitochondrial bioenergetics. The SOD2-null cells displayed perturbations in their mitochondrial ultrastructure and preferred glycolysis as opposed to oxidative phosphorylation to generate ATP. The activities of mitochondrial complex I and II were both significantly impaired by the absence of MnSOD activity, presumably from disruption of the Fe/S centers in NADH dehydrogenase and succinate dehydrogenase subunit B by the aberrant redox state in the mitochondrial matrix of SOD2-null cells. By creating this model we provide a novel tool with which to study the consequences of lack of MnSOD activity in human cells.     

Trypanosoma cruzi maxicircle heterogeneity in Chagas disease patients from Brazil

The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form, IF). Each year, ∼3% of them develop lesions in the heart or gastrointestinal tract. Cardiomyopathy (CCHD) is the most severe manifestation of Chagas disease. The factors that determine the outcome of the infection are unknown, but certainly depend on complex interactions amongst the genetic make-up of the parasite, the host immunogenetic background and environment. In a previous study we verified that the maxicircle gene NADH dehydrogenase (mitochondrial complex I) subunit 7 (ND7) from IF isolates had a 455 bp deletion compared with the wild type (WT) ND7 gene from CCHD strains. We proposed that ND7 could constitute a valuable target for PCR assays in the differential diagnosis of the infective strain. In the present study we evaluated this hypothesis by examination of ND7 structure in parasites from 75 patients with defined pathologies, from Southeast Brazil. We also analysed the structure of additional mitochondrial genes (ND4/CR4, COIII and COII) since the maxicircle is used for clustering Trypanosoma cruzi strains into three clades/haplogroups. We conclude that maxicircle genes do not discriminate parasite populations which induce IF or CCHD forms. Interestingly, the great majority of the analysed isolates belong to T. cruzi II (discrete typing unit, (DTU) IIb) genotype. This scenario is at variance with the prevalence of hybrid (DTU IId) human isolates in Bolivia, Chile and Argentina. The distribution of WT and deleted ND7 and ND4 genes in T. cruzi strains suggests that mutations in the two genes occurred in different ancestrals in the T. cruzi II cluster, allowing the identification of at least three mitochondrial sub-lineages within this group. The observation that T. cruzi strains accumulate mutations in several genes coding for complex I subunits favours the hypothesis that complex I may have a limited activity in this parasite.     

Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids

NADH:ubiquinone oxidoreductase (complex I) is the largest multiprotein complex of the mitochondrial respiratory chain. His-tagged complex I purified from the strictly aerobic yeast Yarrowia lipolytica exhibited electron transfer rates from NADH to n-decylubiquinone of less than 2% when compared to turnover numbers calculated for native mitochondrial membranes from this organism. Reactivation was observed upon addition of asolectin, purified phospholipids and different phospholipid mixtures. Maximal activities of 6-7 μmol NADH min⁻¹ mg⁻¹ were observed following incubation with a mixture of 76% phosphatidylcholine, 19% phosphatidylethanolamine and 5% cardiolipin. For full reactivation, 400-500 phospholipid molecules per complex I were needed. This demonstrated that the inactivation of complex I from Y. lipolytica by general delipidation could be fully reversed simply by returning the phospholipids that had been removed during the purification procedure. Thus, our homogeneous and highly pure complex I preparation had retained its full catalytic potential and no specific, functionally essential component had been lost. As the purified enzyme was also found to contain only substoichiometric amounts of ubiquinone-9 (0.2-0.4 mol/mol), a functional requirement of this endogeneous ubiquinone could also be excluded.