ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.     

Characterization of two different acyl carrier proteins in complex I from Yarrowia lipolytica

Acyl carrier proteins of mitochondria (ACPMs) are small (∼ 10 kDa) acidic proteins that are homologous to the corresponding central components of prokaryotic fatty acid synthase complexes. Genomic deletions of the two genes ACPM1 and ACPM2 in the strictly aerobic yeast Yarrowia lipolytica resulted in strains that were not viable or retained only trace amounts of assembled mitochondrial complex I, respectively. This suggested different functions for the two proteins that despite high similarity could not be complemented by the respective other homolog still expressed in the deletion strains. Remarkably, the same phenotypes were observed if just the conserved serine carrying the phosphopantethein moiety was exchanged with alanine. Although this suggested a functional link to the lipid metabolism of mitochondria, no changes in the lipid composition of the organelles were found. Proteomic analysis revealed that both ACPMs were tightly bound to purified mitochondrial complex I. Western blot analysis revealed that the affinity tagged ACPM1 and ACPM2 proteins were exclusively detectable in mitochondrial membranes but not in the mitochondrial matrix as reported for other organisms. Hence we conclude that the ACPMs can serve all their possible functions in mitochondrial lipid metabolism and complex I assembly and stabilization as subunits bound to complex I.     

Functional sulfurtransferase is associated with mitochondrial complex I from Yarrowia lipolytica, but is not required for assembly of its iron-sulfur clusters

Here, we report that in the obligate aerobic yeast Yarrowia lipolytica, a protein exhibiting rhodanese (thiosulfate:cyanide sulfurtransferase) activity is associated with proton pumping NADH:ubiquinone oxidoreductase (complex I). Complex I is a key enzyme of the mitochondrial respiratory chain that contains eight iron-sulfur clusters. From a rhodanese deletion strain, we purified functional complex I that lacked the additional protein but was fully assembled and displayed no functional defects or changes in EPR signature. In contrast to previous suggestions, this indicated that the sulfurtransferase associated with Y. lipolytica complex I is not required for assembly of its iron-sulfur clusters.     

用噬菌体展示筛选Gal-α-1,3-Gal的模拟肽

猪心移植是解决心脏移植供体少的有效方法.由于猪心血管内皮细胞表面抗原半乳糖-α-1,3-半乳糖（Gal-α-1,3-Gal）,能与人体内预存的天然抗体结合产生超急性排斥反应（HAR）,而无法应用于临床.为了解决这一难题, 应用噬菌体展示技术,筛选出一个能与抗B型血单克隆抗体（mAB anti-B) 特异性结合的阳性噬菌体克隆,应用酶联免疫吸附测定(ELISA)和竞争性ELISA结果表明,所获得的阳性噬菌体克隆能特异性地与mAB anti-B结合,并且这种结合可被蜜二糖（具有Gal-α-1,6-Glc的结构)所竞争抑制.由此推测,筛选到的噬菌体阳性克隆很可能就结合在蜜二糖与mAB anti-B结合的位点.同时,进行了阳性噬菌体克隆的抑制猪红细胞凝集活性试验,试验结果表明,此阳性克隆不仅可抑制Gal-α-1,3-Gal与抗体的结合, 也可抑制Gal-α-1,3-Gal与西非单叶豆凝集素（GS-I-B4）的结合.此噬菌体阳性克隆测序后,得小肽序列为CCWLLRQPVRFVRSIRS.鉴于以上的结果,认为此小肽可以作为Gal-α-1,3-Gal的模拟肽,同时有望开发成抗猪器官异种移植超急性排斥反应的新药.     

The role of the invariant glutamate 95 in the catalytic site of Complex I from Escherichia coli

Replacement of glutamate 95 for glutamine in the NADH- and FMN-binding NuoF subunit of E. coli Complex I decreased NADH oxidation activity 2.5-4.8 times depending on the used electron acceptor. The apparent K_m for NADH was 5.2 and 10.4 μM for the mutant and wild type, respectively. Analysis of the inhibitory effect of NAD⁺ on activity showed that the E95Q mutation caused a 2.4-fold decrease of K_i^NAD+ in comparison to the wild type enzyme. ADP-ribose, which differs from NAD⁺ by the absence of the positively charged nicotinamide moiety, is also a competitive inhibitor of NADH binding. The mutation caused a 7.5-fold decrease of K_i^ADP-ribose relative to wild type enzyme. Based on these findings we propose that the negative charge of Glu95 accelerates turnover of Complex I by electrostatic interaction with the negatively charged phosphate groups of the substrate nucleotide during operation, which facilitates release of the product NAD⁺. The E95Q mutation was also found to cause a positive shift of the midpoint redox potential of the FMN, from − 350 mV to − 310 mV, which suggests that the negative charge of Glu95 is also involved in decreasing the midpoint potential of the primary electron acceptor of Complex I.     

Shoot HAR1 mediates nitrate inhibition of nodulation in Lotus japonicus

Nitrate is a major environmental factor in the inhibition of nodulation. In a model legume Lotus japonicus, a CLV1-like receptor kinase, HAR1, mediates nitrate inhibition and autoregulation of nodulation. Autoregulation of nodulation involves root-to-shoot-to-root long-distance communication, and HAR1 functions in shoots. However, it remains elusive where HAR1 functions in the nitrate inhibition of nodulation. We performed grafting experiments with the har1 mutant under various nitrate conditions, and found that shoot HAR1 is critical for the inhibition of nodulation at 10 mM nitrate. Combined with our recent finding that the nitrate-induced CLE-RS2 glycopeptide binds directly to the HAR1 receptor, this result suggests that CLE-RS2/HAR1 long-distance signaling plays an important role in the both nitrate inhibition and the autoregulation of nodulation.     

The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of . This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme.     

Characterization of a subcomplex of mitochondrial NADH:ubiquinone oxidoreductase (complex I) lacking the flavoprotein part of the N-module

Mitochondrial NADH:ubiquinone oxidoreductase is the largest and most complicated proton pump of the respiratory chain. Here we report the preparation and characterization of a subcomplex of complex I selectively lacking the flavoprotein part of the N-module. Removing the 51-kDa and the 24-kDa subunit resulted in loss of catalytic activity. The redox centers of the subcomplex could be reduced neither by NADH nor NADPH demonstrating that physiological electron input into complex I occurred exclusively via the N-module and that the NADPH binding site in the 39-kDa subunit and further potential nucleotide binding sites are isolated from the electron transfer pathway within the enzyme. Taking advantage of the selective removal of two of the eight iron-sulfur clusters of complex I and providing additional evidence by redox titration and site-directed mutagenesis, we could for the first time unambiguously assign cluster N1 of fungal complex I to mammalian cluster N1b.     

Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (Complex I)

Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.