NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone
Franz SchulerTakahiro YanoSalvatore Di BernardoTakao YagiVictoria YankovskayaThomas P. SingerJohn E. Casida
157
Citation
39
Reference
10
Related Paper
Citation Trend
Abstract:
The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3 ) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[ 3 H]pyridaben ([ 3 H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron–sulfur cluster N2 to quinone.Keywords:
Photoaffinity labeling
Thermus thermophilus
Affinity labeling
Affinity label
Submitochondrial particle
Photoaffinity labeling
Affinity label
Affinity labeling
Cite
Citations (0)
AFFINITY labeling for studying the amino acid topography of specific binding sites in biologically important proteins has emerged as an important biochemical technique during the past three decades. Hormone-specific proteins have been the subjects of affinity labeling during the past two decades, beginning with the analysis of the amino acids which constitute the catalytically active site of the enzymes involved in steroid biosynthesis. More recently, hormone-specific receptor and transport proteins have been characterized, and the amino acid compositions of their binding sites have been described by affinity labeling. The reagents used for affinity labeling are analogs of hormones which produce active site-directed irreversible inhibition in the proteins which they attack. Therefore, they have been regarded as potential new drugs for controlling fertility, treating endocrine disorders, and for treating hormone-sensitive cancers. The three major categories of affinity labeling hormone analogs have been classified according to the reagent groups they possess and the mechanisms by which they inhibit the proteins. Affinity alkylating analogs possess a reagent group which at all times is reactive toward certain amino acids. Photoaffinity labeling ana- logs are only reactive while they absorb light energy, and in the energized state they can react with most amino acids. Autoinactivating substrates have thus far only been used with enzymes because they are reactive toward amino acids only after an enzyme has converted the substrates to a reactive form. All three categories of affinity labeling hormone analogs cause irreversible inhibition when they react with an amino acid at a binding site of a hormone-specific protein.
Affinity labeling
Photoaffinity labeling
Affinity label
Cite
Citations (50)
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPhotoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5Anthony De Waal, Luitzen De Jong, Aloysius F. Hartog, and Albertus KempCite this: Biochemistry 1985, 24, 23, 6493–6499Publication Date (Print):November 1, 1985Publication History Published online1 May 2002Published inissue 1 November 1985https://pubs.acs.org/doi/10.1021/bi00344a028https://doi.org/10.1021/bi00344a028research-articleACS PublicationsRequest reuse permissionsArticle Views48Altmetric-Citations17LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Altmetrics
Photoaffinity labeling
Cite
Citations (28)
A systemic approach has been taken in the preparation and evaluation of photoaffinity labeling agents for the estrogen receptor from rat and lamb uterus. Several derivatives of estradiol and the nonsteroidal estrogen, hexestrol, containing photoreactive diazocarbonyl or azide functions have been synthesized. The receptor binding affinity of these compounds and their capacity to photointeract with the estrogen binding site (inactivate) can be assayed inderectly by competition assays. Several of the compounds that showed both resonably high binding affinities and inactivation efficiencies have been prepared in high specific activity, tritium-labeled form. Direct binding measurements with these derivatives in unpurified rat uterine receptor preparations, show that while these compounds bind to the receptor, they also show considerable nonspecific binding to nonreceptor proteins. Irradiation of these derivatives in rat uterine cytosol preparations results in incorporation of large amounts of radioactivity into protein in a covalent fashion. The amount of nonspecific labeling is so large, however, that estrogen site specificity (indicated by protection with unlabeled estradiol) cannot be demonstrated. More recently, we have used a partially purified receptor preparation from lamb uterus. The receptor in this preparation has been disaggregated by mild trypsinization and can be electrophoresed in native form. Electrophoretic analysis of the proteins in photolabeled preparations show some covalent incorporation into the receptor region of the gel with one derivative but not with another. The effectiveness of the photoaffinity labeling reagents prepared thus far is assessed, and suggestions are made for the design of new, more effective reagents.
Affinity label
Photoaffinity labeling
Affinity labeling
Hexestrol
Trypsinization
Cite
Citations (3)
The architecture of quinone/inhibitor-access channel in proton-translocating NADH-quinone oxidoreductase (respiratory complex I) was modeled by X-ray crystallography and cryo-EM, however, it remains debatable whether the channel model reflects the physiologically relevant state present throughout the catalytic cycle. Using photoreactive [125I]amilorides, we demonstrated that amiloride-type inhibitors bind to the interfacial region of multiple subunits (49-kDa, ND1, PSST, and 39-kDa subunits), which is difficult to reconcile with the current channel model. This report describes the procedures for photoaffinity labeling of bovine submitochondrial particles by photoreactive [125I]amilorides. The protocol could be widely applicable for the characterization of various biologically active compounds, whose target protein remains to be identified or characterized.
Submitochondrial particle
Photoaffinity labeling
Affinity label
Cite
Citations (6)
Acetogenin
Submitochondrial particle
Photoaffinity labeling
Affinity label
Cite
Citations (4)
NADH:ubiquinone oxidoreductase (complex I) is the entry enzyme of mitochondrial oxidative phosphorylation. To obtain the structural information on inhibitor/quinone binding sites, we synthesized [ 3 H]benzophenone‐asimicin ([ 3 H]BPA), a photoaffinity analogue of asimicin, which belongs to the acetogenin family known as the most potent complex I inhibitor. We found that [ 3 H]BPA was photo‐crosslinked to ND2, ND1 and ND5 subunits, by the three dimensional separation (blue‐native/doubled SDS–PAGE) of [ 3 H]BPA‐treated bovine heart submitochondrial particles. The cross‐linking was blocked by rotenone. This is the first finding that ND2 was photo‐crosslinked with a potent complex I inhibitor, suggesting its involvement in the inhibitor/quinone‐binding.
Submitochondrial particle
Photoaffinity labeling
Inhibitor protein
Rotenone
Anthraquinones
Affinity label
NADH dehydrogenase
Cite
Citations (53)
Photoaffinity labeling
Affinity labeling
Affinity label
Cite
Citations (2)
8-Azido-adenosine 5'-triphosphate (n8(3)ATP) appeared to be a suitable photoaffinity label for the protein kinase dependent on adenosine 3':5'-monophosphate (cAMP). It competes with ATP for the high-affinity ATP site in the undissociated form of the kinase and in the phosphotransferase reaction catalyzed by the catalytic subunit. Furthermore, it is accepted as a substrate in the phosphotransfer reaction. n8(3)ATP incorporated into the holoenzyme is covalently bound irradiation. Protection experiments with ATP indicated that this covalent attachment occurs in the high-affinity ATP site of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that n8(3)ATP is bound to the catalytic subunit. After irradiation the enzyme was dissociated by cAMP. Proportional to the incorporated [gamma-32P]n8(3)ATP, a loss in phosphotransferase activity was found. These results support our model that both ATP sites coincide with respect to their adenine binding part. Thus binding of the regulatory subunit to the catalytic subunit would then transform the low-affinity catalytically active ATP site into a high-affinity inactive site.
Affinity label
Photoaffinity labeling
Phosphotransferases
Affinity labeling
Adenosine triphosphate
Adenosine monophosphate
Cite
Citations (30)
Photoaffinity labeling
Affinity label
Affinity labeling
Cyclic peptide
Cite
Citations (6)