The stoichiometry of the iron‐sulphur clusters 1a, 1b and 2 of NADH:Q oxidoreductase as present in beef‐heart submitochondrial particles

Albracht, S.P.J.; Leeuwerik, Frans J.; Swol, Bart van

doi:10.1016/0014-5793(79)81114-x

Cited by 43 publications

(21 citation statements)

References 13 publications

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“…A Hearshen et al 1981) were as follows: for the N1b signal, g 1,2,3 =2.023, 1.941, 1.924 and widths (1,2,3)=1.05, 1.25, 2.1 mT; for the N2 signal, g 1,2,3 =2.0538, 1.925, 1.925 and widths(1,2,3)=1.0, 1.6, 1.6 mT; for the N3 signal, g 1,2,3 = 2.037, 1.9263, 1.863 and widths(1,2,3)=2.5, 1.4, 3.0 mT; for the N4 signal, g 1,2,3 =2.103, 1.938, 1.884 and widths(1,2,3)=2.4, 1.9, 1.9 mT. In spectrum B the relative intensity of the N1b signal was taken half that of the other signals, this in accordance with extensive determinations of the relative spin concentration of the N1b signal (Albracht et al , 1979Van Belzen et al 1992). The red vertical arrows indicate how the redox behaviour of the four signals is measured via the amplitudes of their main lines at 1.94 (N1b), 1.92 (N2), 1.88 (N4) and 1.86 (N3).…”

Section: Resultssupporting

confidence: 78%

“…In Open circles, not treated by DCCD; circles with dot, treated with 0.125 mM DCCD for 30 min; circles with horizontal bar, treated with 1 mM DCCD for 30 min; circles with vertical bar, treated with 2 mM DCCD for 30 min; circles with + sign, treated with 4 mM DCCD for 30 min; circles with x sign, treated with 4 mM DCCD for 60 min. The absolute spin concentration represented by the N2 EPR signal from the SMP was determined (Albracht et al 1979;Van Belzen et al 1990) and the Complex I concentration was taken as half that value turn, NADPH alone induces a 50% bleaching not affected by rotenone; subsequent addition of NADH extends the bleaching to 100%. Such observations were also made with SMP (Hatefi and Hanstein 1973).…”

Section: Discussionmentioning

confidence: 99%

“…(Ohnishi 1975;Beinert and Albracht 1982;Ohnishi 1998)). As, according to repeated measurements in the Amsterdam group, the N2 signal in the bovine enzyme represents a spin concentration twice that of the N1b signal Albracht et al 1979;Van Belzen et al 1992), it follows that it is caused by two clusters with (at X-band and even at Q-band (Albracht 1974;Albracht 1984)) indistinguishable EPR signals.…”

Section: Q 10mentioning

confidence: 95%

“…In the Amsterdam group the total spin concentration represented by the EPR spectrum of Hatefi's Complex I reduced with NADH was found to be seven times the spin concentration of the signal of cluster N1b Albracht et al 1979;Van Belzen et al 1992;Finel et al 1994;Albracht and De Jong 1997). This means that the spectrum receives contributions from one [2Fe-2S] cluster (N1b) and six [4Fe-4S] clusters; thus all clusters, except N1a, are EPR detectable.…”

Section: Uncertainties In the Assignments Of G-value Sets To The Fourmentioning

confidence: 99%

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The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

Albracht

2010

J Bioenerg Biomembr

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Bovine NADH:ubiquinone oxidoreductase (Complex I) is the first complex in the mitochondrial respiratory chain. It has long been assumed that it contained only one FMN group. However, as demonstrated in 2003, the intact enzyme contains two FMN groups. The second FMN was proposed to be located in a conserved flavodoxin fold predicted to be present in the PSST subunit. The longknown reaction of Complex I with NADPH differs in many aspects from that with NADH. It was proposed that the second flavin group was specifically involved in the reaction with NADPH. The X-ray structure of the hydrophilic domain of Complex I from Thermus thermophilus (Sazanov and Hinchliffe 2006, Science 311, 1430-1436 disclosed the positions of all redox groups of that enzyme and of the subunits holding them. The PSST subunit indeed contains the predicted flavodoxin fold although it did not contain FMN. Inspired by this structure, the present paper describes a re-evaluation of the enigmatic reactions of the bovine enzyme with NADPH. Published data, as well as new freeze-quench kinetic data presented here, are incompatible with the general opinion that NADPH and NADH react at the same site. Instead, it is proposed that these pyridine nucleotides react at opposite ends of the 90Å long chain of prosthetic groups in Complex I. Ubiquinone is proposed to react with the Fe-S clusters in the TYKY subunit deep inside the hydrophilic domain. A new model for electron transfer in Complex I is proposed. In the accompanying paper this model is compared with the one advocated in current literature.

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Section: Resultssupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Q 10mentioning

confidence: 95%

Section: Uncertainties In the Assignments Of G-value Sets To The Fourmentioning

confidence: 99%

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The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

Albracht

2010

J Bioenerg Biomembr

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“…Simulation was carried out as described earlier (Beinert & Albracht, 1982). Quantification of EPR signals was carried out by direct double integration of the experimental spectra (Albracht, 1984;Aasa & Vänngård, 1975) or by comparison with a well-fitting simulation (Albracht et al, 1979).…”

Section: Methodsmentioning

confidence: 99%

NADH:Ubiquinone Oxidoreductase of Vibrio alginolyticus: Purification, Properties, and Reconstitution of the Na⁺ Pump

Albracht

Belzen

et al. 1996

Biochemistry

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The NADH: ubiquinone oxidoreductase of Vibrio Alginolyticus: purification, properties and reconstitution of the Na+ pump. Pfenniger-Li, X.D.; Albracht, S.P.J.; van Belzen, R.; Dimroth, P. Published in: Biochemistry DOI:10.1021/bi953032lLink to publication Citation for published version (APA):Pfenniger-Li, X. D., Albracht, S. P. J., van Belzen, R., & Dimroth, P. (1996). The NADH: ubiquinone oxidoreductase of Vibrio Alginolyticus: purification, properties and reconstitution of the Na+ pump. Biochemistry, 35, 6233-6242. DOI: 10.1021/bi953032l General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. ABSTRACT: The Na + -activated NADH:ubiquinone oxidoreductase of Vibrio alginolyticus was extracted from the membranes with lauryldimethylamine-N-oxide and purified by two successive anion exchange columns. This preparation, yielding four major and several minor stained bands after SDS-PAGE, retained the NADH-dehydrogenase activity (with menadione as an artificial electron acceptor) and ubiquinone-1 (Q) reductase activity. On further fractionation of the enzyme, the Q-reductase activity essentially disappeared. Chemical analyses revealed the presence of FAD but not FMN, of non-heme iron and of acid-labile sulfur and tightly-bound ubiquinone-8 in the purified Q-reductase preparation. The participation of an iron-sulfur cluster of the [2Fe-2S] type in the electron translocation was demonstrated by the appearance of a typical EPR signal for this prosthetic group after the reduction of Q-reductase with NADH. A strong EPR signal typical for a radical observed upon reduction of the enzyme might arise from the formation of quinone radicals. In the absence of Na + , the path of the electrons apparently ends with the reduction of ubiquinone-1 to the semiquinone derivative which in the presence of O 2 becomes reoxidized with concomitant formation of superoxide radicals. In the presence of Na + , these oxygen radicals are not formed and the semiquinone is further reduced to the quinol derivative. These results indicate that the Na + -dependent step in the electron transfer catalyzed by NADH:ubiquinone oxidoreductase is the reduction of ubisemiquinone to ubiquinol. After reconstitution of the purified Q-reductase into proteoliposomes, NADH oxidation by ubiquinone-1 was coupled to Na + tra...

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A novel subfractionation approach for mitochondrial proteins: A three-dimensional mitochondrial proteome map

Hanson

Schulenberg²,

Patton³

et al. 2001

Electrophoresis

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As mitochondria play critical roles in both cell life and cell death, there is great interest in obtaining a human mitochondrial proteome map. Such a map could potentially be useful in diagnosing diseases, identifying targets for drug therapy, and in screening for unwanted drug side effects. In this paper, we present a novel approach to obtaining a human mitochondrial proteome map that combines sucrose gradient centrifugation with standard two-dimensional gel electrophoresis. The resulting three-dimensional separation of proteins allows us to address some of the problems encountered during previous attempts to obtain mitochondrial proteome maps such as resolution of proteins and solubility of hydrophobic proteins during isoelectric focusing. In addition, we show that this new approach provides functional information about protein complexes within the organelle that is not obtained with two-dimensional gel electrophoresis of whole mitochondria.

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The stoichiometry of the iron‐sulphur clusters 1a, 1b and 2 of NADH:Q oxidoreductase as present in beef‐heart submitochondrial particles

Cited by 43 publications

References 13 publications

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

NADH:Ubiquinone Oxidoreductase of Vibrio alginolyticus: Purification, Properties, and Reconstitution of the Na⁺ Pump

A novel subfractionation approach for mitochondrial proteins: A three-dimensional mitochondrial proteome map

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The stoichiometry of the iron‐sulphur clusters 1a, 1b and 2 of NADH:Q oxidoreductase as present in beef‐heart submitochondrial particles

Cited by 43 publications

References 13 publications

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

NADH:Ubiquinone Oxidoreductase of Vibrio alginolyticus: Purification, Properties, and Reconstitution of the Na+ Pump

A novel subfractionation approach for mitochondrial proteins: A three-dimensional mitochondrial proteome map

Contact Info

Product

Resources

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NADH:Ubiquinone Oxidoreductase of Vibrio alginolyticus: Purification, Properties, and Reconstitution of the Na⁺ Pump