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Query: UNIPROT:O14944 (EPR)
 13,097 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enzymically active subcomplexes were purified from bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I) by sucrose-gradient centrifugation in the presence of detergents. These subcomplexes, named I lambda, IS, and I lambda S, catalyse ferricyanide and ubiquinone-1 (Q-1) reduction by NADH at similar rates to complex I, but do not catalyse the reduction of decylubiquinone. In addition, the Q-1 reductase activity of all the subcomplexes is insensitive to rotenone. Chemical and EPR analyses of the subcomplexes show that FMN and all the Fe-S clusters of complex I are present, but that the line shape of cluster 2 is modified. The smallest subcomplex, I lambda S, contains only approximately 13 subunits, as compared to approximately 22 in the previously described subcomplex I alpha [Finel, M., Skehel, J. M., Albracht, S. J. P., Fearnley, I. M. & Walker, J. E. (1992) Biochemistry 31, 11425-11434], but it retains the 75-, 51-, 49-, 30-, 24-, 23- (TYKY) and 20-kDa (PSST) subunits, which are suggested to form a functional core that comprises the EPR-detectable Fe-S clusters 1-4, and FMN. The structural and functional implications of such an arrangement are discussed.
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PMID:Isolation and characterisation of subcomplexes of the mitochondrial NADH:ubiquinone oxidoreductase (complex I). 795 54

NADH-quinone 1 oxidoreductase (Complex I) isolated from bovine heart mitochondria was, until recently, the major source for the study of this most complicated energy transducing device in the mitochondrial respiratory chain. Complex I has been shown to contain 43 subunits and possesses a molecular mass of about 1 million. Recently, Complex I genes have been cloned and sequenced from several bacterial sources including Escherichia coli, Paracoccus denitrificans, Rhodobacter capsulatus and Thermus thermophilus HB-8. These enzymes are less complicated than the bovine enzyme, containing a core of 13 or 14 subunits homologous to the bovine heart Complex I. From this data, important clues concerning the subunit location of both the substrate binding site and intrinsic redox centers have been gleaned. Powerful molecular genetic approaches used in these bacterial systems can identify structure/function relationships concerning the redox components of Complex I. Site-directed mutants at the level of bacterial chromosomes and over-expression and purification of single subunits have allowed detailed analysis of the amino acid residues involved in ligand binding to several iron-sulfur clusters. Therefore, it has become possible to examine which subunits contain individual iron-sulfur clusters, their location within the enzyme and what their ligand residues are. The discovery of g=2.00 EPR signals arising from two distinct species of semiquinone (SQ) in the activated bovine heart submitochondrial particles (SMP) is another line of recent progress. The intensity of semiquinone signals is sensitive to DeltamicroH+ and is diminished by specific inhibitors of Complex I. To date, semiquinones similar to those reported for the bovine heart mitochondrial Complex I have not yet been discovered in the bacterial systems. This mini-review describes three aspects of the recent progress in the study of the redox components of Complex I: (A) the location of the substrate (NADH) binding site, flavin, and most of the iron-sulfur clusters, which have been identified in the hydrophilic electron entry domain of Complex I; (B) experimental evidence indicating that the cluster N2 is located in the amphipathic domain of Complex I, connecting the promontory and membrane parts. Very recent data is also presented suggesting that the cluster N2 may have a unique ligand structure with an atypical cluster-ligation sequence motif located in the NuoB (NQO6/PSST) subunit rather than in the long advocated NuoI (NQO9/TYKY) subunit. The latter subunit contains the most primordial sequence motif for two tetranuclear clusters; (C) the discovery of spin-spin interactions between cluster N2 and two distinct Complex I-associated species of semiquinone. Based on the splitting of the g1 signal of the cluster N2 and concomitant strong enhancement of the semiquinone spin relaxation, one semiquinone species was localized 8-11 A from the cluster N2 within the inner membrane on the matrix side (N-side). Spin relaxation of the other semiquinone species is much less enhanced, and thus it was proposed to have a longer distance from the cluster N2, perhaps located closer to the other side (P-side) surface of the membrane. A brief introduction of EPR technique was also described in Appendix A of this mini-review.
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PMID:Iron-sulfur clusters/semiquinones in complex I. 959 87

Proton-translocating NADH:ubiquinone oxidoreductase (complex I) is the largest and least understood enzyme of the respiratory chain. Complex I from bovine mitochondria consists of more than forty different polypeptides. Subunit PSST has been suggested to carry iron-sulfur center N-2 and has more recently been shown to be involved in inhibitor binding. Due to its pH-dependent midpoint potential, N-2 has been proposed to play a central role both in ubiquinone reduction and proton pumping. To obtain more insight into the functional role of PSST, we have analyzed site-directed mutants of conserved acidic residues in the PSST homologous subunit of the obligate aerobic yeast Yarrowia lipolytica. Mutations D136N and E140Q provided functional evidence that conserved acidic residues in PSST play a central role in the proton translocating mechanism of complex I and also in the interaction with the substrate ubiquinone. When Glu(89), the residue that has been suggested to be the fourth ligand of iron-sulfur center N-2 was changed to glutamine, alanine, or cysteine, the EPR spectrum revealed an unchanged amount of this redox center but was shifted and broadened in the g(z) region. This indicates that Glu(89) is not a ligand of N-2. The results are discussedin the light of structural similarities to the homologous [NiFe] hydrogenases.
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PMID:Function of conserved acidic residues in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica. 1081 5

We have cloned and inactivated, by repeat-induced point mutations, the nuclear gene encoding the 19.3 kDa subunit of complex I (EC 1.6.5.3) from Neurospora crassa, the homologue of the bovine PSST polypeptide. Mitochondria from mutant nuo19.3 lack the peripheral arm of complex I while its membrane arm accumulates. Transformation with wild-type cDNA rescues this phenotype and assembly of complex I is restored. To interfere with assembly of a proposed bound iron-sulphur cluster, site-directed mutants were constructed by introducing cDNA with altered codons for two adjacent cysteines, Cys-101 and Cys-102. The mutant complexes were purified and their enzymic activities and EPR and UV/visible spectra were analysed. Either of the mutations abolishes assembly of iron-sulphur cluster N2, showing that this redox group is bound to the 19.3 kDa protein. We also observed an interference with the reduction of redox group X, suggesting that cluster N2 is the electron donor to this high-potential redox group.
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PMID:Disruption of iron-sulphur cluster N2 from NADH: ubiquinone oxidoreductase by site-directed mutagenesis. 1204 48

In oxidative phosphorylation, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. Complex I contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters, in several subunits, to transfer the electrons to quinone. Understanding coupled electron transfer in complex I requires a detailed knowledge of the properties of individual clusters and of the cluster ensemble, and so it requires the correlation of spectroscopic and structural data: This has proved a challenging task. EPR studies on complex I from Bos taurus have established that EPR signals N1b, N2 and N3 arise, respectively, from the 2Fe cluster in the 75 kDa subunit, and from 4Fe clusters in the PSST and 51 kDa subunits (positions 2, 7, and 1 along the seven-cluster chain extending from the flavin). The other clusters have either evaded detection or definitive signal assignments have not been established. Here, we combine double electron-electron resonance (DEER) spectroscopy on B. taurus complex I with the structure of the hydrophilic domain of Thermus thermophilus complex I. By considering the magnetic moments of the clusters and the orientation selectivity of the DEER experiment explicitly, signal N4 is assigned to the first 4Fe cluster in the TYKY subunit (position 5), and N5 to the all-cysteine ligated 4Fe cluster in the 75 kDa subunit (position 3). The implications of our assignment for the mechanisms of electron transfer and energy transduction by complex I are discussed.
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PMID:Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron-electron resonance. 2013 38

The first purification of bovine NADH:ubiquinone oxidoreductase (Complex I) was reported nearly half a century ago (Hatefi et al. J Biol Chem 237:1676-1680, 1962). The pathway of electron-transfer through the enzyme is still under debate. A major obstacle is the assignment of EPR signals to the individual iron-sulfur clusters in the subunits. The preceding paper described a working model based on the kinetics with NADPH. This model is at variance with current views in the field. The present paper provides a critical overview on the possible causes for the discrepancies. It is concluded that the stability of all purified preparations described thus far, including Hatefi's Complex I, is compromised due to removal of the enzyme from the protective membrane environment. In addition, most preparations described during the last two decades are purified by methods involving synthetic detergents and column chromatography. This results in delipidation, loss of endogenous quinones and loss of reactions with (artificial) quinones in a rotenone-sensitive way. The Fe:FMN ratio's indicate that FMN-a is absent, but that all Fe-S clusters may be present. In contrast to the situation in bovine SMP and Hatefi's Complex I, three of the six expected [4Fe-4S] clusters are not detected in EPR spectra. Qualitatively, the overall EPR lineshape of the remaining three cubane signals may seem similar to that of Hatefi's Complex I, but quantitatively it is not. It is further proposed that point mutations in any of the TYKY, PSST, 49-kDa or 30-kDa subunits, considered to make up the delicate structural heart of Complex I, may have unpredictable effects on any of the other subunits of this quartet. The fact that most point mutations led to inactive enzymes makes a correct interpretation of such mutations even more ambiguous. In none of the Complex-I-containing membrane preparations from non-bovine origin, the pH dependencies of the NAD(P)H-->O(2) reactions and the pH-dependent reduction kinetics of the Fe-S clusters with NADPH have been determined. This excludes a proper discussion on the absence or presence of FMN-a in native Complex I from other organisms.
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PMID:The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited: II. Comparison of the proposed working hypothesis with literature data. 2063 77