Engineering the Respiratory Complex I to Energy-converting NADPH:Ubiquinone Oxidoreductase

Morina, Klaudia; Schulte, Marius; Hubrich, Florian; Steimle, Stefan; Stolpe, Stefan; Friedrich, Thorsten

doi:10.1074/jbc.m111.274571

Cited by 16 publications

(29 citation statements)

References 49 publications

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“…Our data yield a NADH binding rate ( k on ) of 3.1±0.6×10 7 m −1 s −1 in good agreement with k cat / K M values (1.5–4.0×10 7 m −1 s −1 ) from steady-state measurements[9,17] (Supporting Information) and an estimate for the half-life of hydride transfer of 20±5 μs that is consistent with the short distance for hydride transfer of 3.2 Å between the C 4N of the NADH nicotinamide ring and N 5 of the FMN isoalloxazine ring, which are in stacking interaction. [4a]…”

supporting

confidence: 81%

“…Our data yield a NADH binding rate (k on ) of 3.1 AE 0.6 10 7 m À1 s À1 in good agreement with k cat /K M values (1.5-4.0 10 7 m À1 s À1 ) from steady-state measurements [9,17] (Supporting Information) and an estimate for the half-life of hydride transfer of 20 AE 5 ms that is consistent with the short distance for hydride transfer of 3.2 between the C 4N of the NADH nicotinamide ring and N 5 of the FMN isoalloxazine ring, which are in stacking interaction. [4a] The reaction with NADH comprises three sequential turnovers, yielding full reduction of FMN, N2, N1a, N1b, and N4 according to the following series of events: NADH reduces FMN to FMNH À through hydride transfer followed by rapid protonation to FMNH 2 .…”

supporting

confidence: 79%

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Electron Tunneling Rates in Respiratory Complex I Are Tuned for Efficient Energy Conversion

et al. 2015

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Respiratory complex I converts the free energy of ubiquinone reduction by NADH into a proton motive force, a redox reaction catalyzed by flavin mononucleotide(FMN) and a chain of seven iron–sulfur centers. Electron transfer rates between the centers were determined by ultrafast freeze-quenching and analysis by EPR and UV/Vis spectroscopy. The complex rapidly oxidizes three NADH molecules. The electron-tunneling rate between the most distant centers in the middle of the chain depends on the redox state of center N2 at the end of the chain, and is sixfold slower when N2 is reduced. The conformational changes that accompany reduction of N2 decrease the electronic coupling of the longest electron-tunneling step. The chain of iron–sulfur centers is not just a simple electron-conducting wire; it regulates the electron-tunneling rate synchronizing it with conformation-mediated proton pumping, enabling efficient energy conversion. Synchronization of rates is a principle means of enhancing the specificity of enzymatic reactions.

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confidence: 81%

supporting

confidence: 79%

Electron Tunneling Rates in Respiratory Complex I Are Tuned for Efficient Energy Conversion

et al. 2015

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“…27. The NADH oxidase activity and the NADH/ferricyanide oxidoreductase activity were measured as already described5455. Protein concentrations were determined according to the biuret method.…”

Section: Methodsmentioning

confidence: 99%

Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

Arias-Cartin¹,

Ceccaldi²,

Schoepp‐Cothenet³

et al. 2016

Sci Rep

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A major gap of knowledge in metalloproteins is the identity of the prefolded state of the protein before cofactor insertion. This holds for molybdoenzymes serving multiple purposes for life, especially in energy harvesting. This large group of prokaryotic enzymes allows for coordination of molybdenum or tungsten cofactors (Mo/W-bisPGD) and Fe/S clusters. Here we report the structural data on a cofactor-less enzyme, the nitrate reductase respiratory complex and characterize the conformational changes accompanying Mo/W-bisPGD and Fe/S cofactors insertion. Identified conformational changes are shown to be essential for recognition of the dedicated chaperone involved in cofactors insertion. A solvent-exposed salt bridge is shown to play a key role in enzyme folding after cofactors insertion. Furthermore, this salt bridge is shown to be strictly conserved within this prokaryotic molybdoenzyme family as deduced from a phylogenetic analysis issued from 3D structure-guided multiple sequence alignment. A biochemical analysis with a distantly-related member of the family, respiratory complex I, confirmed the critical importance of the salt bridge for folding. Overall, our results point to a conserved cofactors insertion mechanism within the Mo/W-bisPGD family.

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“…Aus unseren Daten berechnet sich eine NADH‐Bindungskonstante ( k on ) von 3.1±0.6×10 7 M −1 s −1 , die in guter Übereinstimmung mit k cat / K M ‐Werten (1.5–4.0×10 7 M −1 s −1 ) aus Gleichgewichtsmessungen ist 9. 17 Daraus kann die Halbwertszeit des Hydridtransfers zu 20±5 μs abgeschätzt werden, was wiederum im Einklang mit dem kurzen Abstand von 3.2 Å zwischen dem C 4N des Nikotinamids und dem N 5 des Isoalloxazinrings ist, die zueinander in Stapelwechselwirkung stehen 4a…”

Section: Kinetische Und Gleichgewichtskonstanten[a]unclassified

Die Elektronentunnelraten im Atmungskettenkomplex I sind auf eine effiziente Energiewandlung abgestimmt

2015

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[6] 95 ms [7] und 275 ms [8] berechnet. Die des Transfers zwischen den Zentren N1b und 4Fe[75]C wurden zu t 1/2 = 5-10 ms und die der übrigen Schritte zu t 1/2 = 25-400 ns berechnet. [6][7][8] Kürzlich wurde die Elektronenübertragung im Komplex I mittels EPR-Spektroskopie an schnell eingefrorenen Proben untersucht.[9] Dabei wurde eine schnelle Reduktion der Zentren N2 und N1a mit t 1/2 % 60 ms durch das erste NADH beobachtet.[9] Die Reduktion der Zentren N1b und N4 durch das zweite NADH war mit t 1/2 % 1 ms deutlich langsamer, was auf die langsame Dissoziation des NAD + vom Komplex zurückgeführt wurde. [9] Wir haben die Reaktion von NADH mit einer hochreinen Probe des E. coli Komplex I [10] in Gegenwart und Abwesenheit des spezifischen Inhibitors Piericidin A, der in der QTasche bindet, anhand der Redoxzustände des FMN und der FeS-Zentren quantitativ analysiert. Durch methodische Unterschiede beim "Freeze-Quench" [11] unterscheiden sich sowohl die experimentellen Daten als auch deren Interpretation von der vorherigen Verçffentlichung.[9] Die Funktion des distalen Zentrums N2 für die Synchronisierung von Elektronentunneln und die Protonentranslokation wird aufgezeigt.Zuerst wurde die Anzahl der von Komplex I oxidierten NADH-Moleküle bestimmt (Abbildung S1 in den Hintergrundinformationen) und die Gleichgewichtsverteilung der

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Engineering the Respiratory Complex I to Energy-converting NADPH:Ubiquinone Oxidoreductase

Cited by 16 publications

References 49 publications

Electron Tunneling Rates in Respiratory Complex I Are Tuned for Efficient Energy Conversion

Electron Tunneling Rates in Respiratory Complex I Are Tuned for Efficient Energy Conversion

Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

Die Elektronentunnelraten im Atmungskettenkomplex I sind auf eine effiziente Energiewandlung abgestimmt

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