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NADH:quinone oxidoreductase: the black box of the respiratory chain

Roth, Robert LU (2003)
Abstract (Swedish)
Popular Abstract in Swedish

Komplex I eller NADH:kinon oxidoreduktas är det största och mest komplexa av de fem membranbunda enzymkomplexen som utgör den mitokondriella andningskedjan. Enzymet finns i alla typer av organismer från bakterier till däggdjur. Enzymet katalyserar oxidationen av NADH, som producerats i citronsyracykeln, och reduktion av fettlösligt kinon i membranet. Flavin och ett antal järn-svavel kluster deltar i elektrontransporten genom enzymet, som är kopplad till proton pumpning över membranet. Enzymet kan också katalysera den omvända reaktionen, d.v.s. NAD+ reduktion som drivs av membranpotentialen. Till skillnad från de andra enzymkomplexen i andningskedjan, där man vet förhållandevis mycket om struktur... (More)
Popular Abstract in Swedish

Komplex I eller NADH:kinon oxidoreduktas är det största och mest komplexa av de fem membranbunda enzymkomplexen som utgör den mitokondriella andningskedjan. Enzymet finns i alla typer av organismer från bakterier till däggdjur. Enzymet katalyserar oxidationen av NADH, som producerats i citronsyracykeln, och reduktion av fettlösligt kinon i membranet. Flavin och ett antal järn-svavel kluster deltar i elektrontransporten genom enzymet, som är kopplad till proton pumpning över membranet. Enzymet kan också katalysera den omvända reaktionen, d.v.s. NAD+ reduktion som drivs av membranpotentialen. Till skillnad från de andra enzymkomplexen i andningskedjan, där man vet förhållandevis mycket om struktur och funktion har man för Komplex I enzymet ingen detaljerad information om strukturen, som endast har bestämts med låg upplösning och dessutom är den molekylära mekanismen bakom kopplingen mellan elektrontransport och proton pumpning är inte känd.



För att studera den funktionella mekanismen hos Komplex I och för att kunna formulera en hypotes för hur protonpumpningsmaskineriet fungerar som kan testas experimentiellt, är det nödvändigt att veta hur många inbindningsställen för kinon det finns. I den här studien har vi undersökt funktionen hos specifika inhibitorer som förhindrar inbindning av kinon till Komplex I. Vidare har vi syntetiserat azido-kinonanaloger för direkt inmärking av inbindningställen. Dessa föreningar är stabila i mörker med reagerar med polypeptider vid belysning med UV-ljus. Tre av azido-kinonanalogerna fungerade som substrat för Komplex I och visade normal inhibitorkänslighet. Två av föreningarna blockerade Komplex I vid belysning, vilket visar att specifik inmärkning ägde rum. Vi har också konstruerat fusionsproteiner för att bestämma transmembrantopologin på en av de proteinsubenheter som är en kandidat för att binda kinon.



Det Komplex I som finns i däggdjurs mitokondrier består av 46 olika proteinsubenheter medan det bakteriella enzymet endast består av 14 subenheter. Detta enklare Komplex I från bakterier är ett attraktivt modellsystem eftersom biokemiska och biofysikaliska metoder kan användas i kombination med molekylärbiologiska tekniker. Med hur generellt giltiga blir resultaten när man studerar ett modellsystem? I det här arbetet har vi gjort en detaljerad, parallell studie av de katalytiska aktiviterna och känsligheten mot inhibitorer hos det bakteriella och det mammala Komplex I enzymet. Det finns väldigt lite kunskap om vilken funktion de 32 extra subenheterna i mammalt Komplex I har. För vissa subenheter, som till exempel acyl carrier protein (ACP), finns det möjligen specifika funktionella roller. Men ofta anses de extra subenheternas betydelse vara strukturell eller att isolera enzymet och förhindra bildandet av fria radikaler. Med bioinformatiska metoder har vi visat att de flesta av dessa extra Komplex I subenheter måste ha funnits på plats i Komplex I i den ursprungliga eukaryota cell som sedan gav upphov till växter, djur och svampar. Förutom ACP har vi också hittat homologer till fyra andra av de extra Komplex I-subenheterna i bakteriers genom. Särskilt intressant är att vi bara hittar sådana homologa protein i alfaproteobakterier, som är den grupp av bakterier som mitokondrien utvecklats ifrån. (Less)
Abstract
Complex I or NADH:quinone oxidoreductase the largest, most complex and least understood of the five membrane-bound enzyme complexes constituting the mitochondrial respiratory chain. The enzyme is present in all types of organisms, from bacteria to mammals. The enzyme catalyses the oxidation of NADH produced by the citric acid cycle and reduction of lipid soluble quinone in the membrane. Flavin and a number of iron-sulfur clusters take part in the electron transfer through the enzyme, that is coupled to proton translocation across the membrane. The enzyme is also capable of catalysing the reverse reaction, i.e. DmH+ supported NAD+ reduction. No high resolution structure exists for this enzyme, and the mechanism of energy coupling is not... (More)
Complex I or NADH:quinone oxidoreductase the largest, most complex and least understood of the five membrane-bound enzyme complexes constituting the mitochondrial respiratory chain. The enzyme is present in all types of organisms, from bacteria to mammals. The enzyme catalyses the oxidation of NADH produced by the citric acid cycle and reduction of lipid soluble quinone in the membrane. Flavin and a number of iron-sulfur clusters take part in the electron transfer through the enzyme, that is coupled to proton translocation across the membrane. The enzyme is also capable of catalysing the reverse reaction, i.e. DmH+ supported NAD+ reduction. No high resolution structure exists for this enzyme, and the mechanism of energy coupling is not understood.



To learn more about the functional mechanism of Complex I and to be able to formulate an experimentally testable hypothesis for how the proton pumping machinery works, it is essential to know the number and location of the quinone binding sites. In this work we have investigated the action of Complex I specific inhibitors that interfere with quinone binding. Furthermore, we have synthesised azido-quinone analogues for direct photo-labelling of the quinone binding sites in Complex I. Three azido-quinone analogues were accepted as substrates by Complex I and exhibited normal inhibitor sensitivities, whereas only two of the compounds inactivated Complex I upon illumination. We have also used fusion protein techniques to determine the transmembrane topology of one protein subunit, that is a candidate for harbouring a quinone binding area.



In mammalian mitochondria Complex I is composed of 46 different protein subunits whereas the bacterial enzyme consists of only 14 proteins. The simpler bacterial Complex I is an attractive model system, since biochemical and biophysical methods can be applied in combination with molecular biological techniques. But how generally applicable are the results? In this work we have made a detailed, in parallel comparison of the catalytic activities and inhibitor sensitivities of mammalian and bacterial Complex I. The role and function of the 32 accessory subunits in mammalian Complex I is poorly understood. Some subunits probably have a functional role, but often the accessory subunits are thought of as merely structural or insulatory. We have demonstrated that most, if not all, of the accessory Complex I subunits were present in the ancestral eukaryote evolving into plants, animals and fungi. We have found homologues to 4 accessory Complex I subunits in bacteria, but notably only in the a-proteobacteria, the group from which the ancestor of mitochondria arose. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Prof Brandt, Ulrich, Institut f. Biochemie I - Molekulare Bioenergetik, Universitaetsklinikum Frankfurt, ZBC,Frankfurt am Main, Federal Republic of Germany
organization
publishing date
type
Thesis
publication status
published
subject
keywords
NADH:quinone oxidoreductase, Complex I, electron transport, membrane protein, ubiquinone, respiratory chain, respiration, metabolism, Metabolism, Biokemi, Biochemistry
pages
184 pages
publisher
Robert Roth, Dept of Biochemistry, Lund University
defense location
Kemicentrum Hörsal B, Sölvegatan 39, Lund
defense date
2003-04-24 10:15
ISBN
91-628-5609-X
language
English
LU publication?
yes
id
4b368655-9a90-4a35-ab25-63c1e799c377 (old id 465700)
date added to LUP
2007-10-14 14:35:30
date last changed
2016-09-19 08:45:03
@phdthesis{4b368655-9a90-4a35-ab25-63c1e799c377,
  abstract     = {Complex I or NADH:quinone oxidoreductase the largest, most complex and least understood of the five membrane-bound enzyme complexes constituting the mitochondrial respiratory chain. The enzyme is present in all types of organisms, from bacteria to mammals. The enzyme catalyses the oxidation of NADH produced by the citric acid cycle and reduction of lipid soluble quinone in the membrane. Flavin and a number of iron-sulfur clusters take part in the electron transfer through the enzyme, that is coupled to proton translocation across the membrane. The enzyme is also capable of catalysing the reverse reaction, i.e. DmH+ supported NAD+ reduction. No high resolution structure exists for this enzyme, and the mechanism of energy coupling is not understood.<br/><br>
<br/><br>
To learn more about the functional mechanism of Complex I and to be able to formulate an experimentally testable hypothesis for how the proton pumping machinery works, it is essential to know the number and location of the quinone binding sites. In this work we have investigated the action of Complex I specific inhibitors that interfere with quinone binding. Furthermore, we have synthesised azido-quinone analogues for direct photo-labelling of the quinone binding sites in Complex I. Three azido-quinone analogues were accepted as substrates by Complex I and exhibited normal inhibitor sensitivities, whereas only two of the compounds inactivated Complex I upon illumination. We have also used fusion protein techniques to determine the transmembrane topology of one protein subunit, that is a candidate for harbouring a quinone binding area.<br/><br>
<br/><br>
In mammalian mitochondria Complex I is composed of 46 different protein subunits whereas the bacterial enzyme consists of only 14 proteins. The simpler bacterial Complex I is an attractive model system, since biochemical and biophysical methods can be applied in combination with molecular biological techniques. But how generally applicable are the results? In this work we have made a detailed, in parallel comparison of the catalytic activities and inhibitor sensitivities of mammalian and bacterial Complex I. The role and function of the 32 accessory subunits in mammalian Complex I is poorly understood. Some subunits probably have a functional role, but often the accessory subunits are thought of as merely structural or insulatory. We have demonstrated that most, if not all, of the accessory Complex I subunits were present in the ancestral eukaryote evolving into plants, animals and fungi. We have found homologues to 4 accessory Complex I subunits in bacteria, but notably only in the a-proteobacteria, the group from which the ancestor of mitochondria arose.},
  author       = {Roth, Robert},
  isbn         = {91-628-5609-X},
  keyword      = {NADH:quinone oxidoreductase,Complex I,electron transport,membrane protein,ubiquinone,respiratory chain,respiration,metabolism,Metabolism,Biokemi,Biochemistry},
  language     = {eng},
  pages        = {184},
  publisher    = {Robert Roth, Dept of Biochemistry, Lund University},
  school       = {Lund University},
  title        = {NADH:quinone oxidoreductase: the black box of the respiratory chain},
  year         = {2003},
}