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ABSTRACT: NADH-quinone (Q) oxidoreductase is a large and complex redox proton pump, which utilizes the free energy derived from oxidation of NADH with lipophilic electron/proton carrier Q to translocate protons across the membrane to generate an electrochemical proton gradient. Although its molecular mechanism is largely unknown, recent biochemical, biophysical, and molecular biological studies have revealed that particular subunits and cofactors play an essential role in the energy-coupling reaction. Based on these latest experimental data, we exhaustively analyzed the sequence information available from evolutionarily related enzymes such as [NiFe] hydrogenases. We found significant and conserved sequence differences in the PSST/Nqo6/NuoB, 49kDa/Nqo4/NuoD, and ND1/Nqo8/NuoH subunit homologs between complex I/NDH-1 and [NiFe] hydrogenases. The alterations, especially in the postulated ligand motif for cluster N2 in the PSST/Nqo6/NuoB subunits, appear to be evolutionarily important in determining the physiological function of complex I/NDH-1. These observations led us to propose a hypothetical evolutionary scheme: during the course of evolution, drastic changes have occurred in the putative cluster N2 binding site in the PSST/Nqo6/NuoB subunit and the progenitors of complex I/NDH-1 have concurrently become to utilize a lipophilic electron/proton carrier such as Q as its physiological substrate. This scheme provides new insights into the structure and function relationship of complex I/NDH-1 and may help us understand its energy-coupling mechanism.
Journal of Bioenergetics 07/2001; 33(3):213-22. · 2.81 Impact Factor
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ABSTRACT: The mitochondrial bioenergetics field has experienced an exciting breakthrough with the recent structure determination of several key membrane complexes. The latest addition to this line of structures, that of quinol-fumarate reductase, provides new insights into the mechanism of energy transduction.
Structure 03/2000; 8(2):R23-32. · 6.35 Impact Factor
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ABSTRACT: Molecular properties of the NQO9 subunit of Paracoccus denitrificans NDH-1, which is predicted to contain 2x[4Fe-4S] clusters, were investigated using recombinant expression techniques and EPR spectroscopy. The full-length form of NQO9 subunit co-expressed with thioredoxin in Escherichia coli at ambient temperature was found dominantly in the cytoplasmic membrane with low amplification. Genetic deletion of relatively hydrophobic and less conserved N-terminal stretches (30 or 40 amino acid residues long) of the NQO9 subunit resulted in the overexpression of the truncated soluble form of the subunit in a high yield in the cytoplasm. The purified soluble form of the NQO9 subunit contained only a small quantity of Fe and S(2-) (2.0-2.2 mol each per mol of subunit). However, the iron-sulfur content was considerably increased by in vitro reconstitution. The reconstituted NQO9 subunit contained 7.6-7.7 mol each of Fe and S(2-) per molecule and exhibited optical absorption spectra similar to those of 2x[4Fe-4S] ferredoxins. Two sets of relatively broad axial-type EPR signals with different temperature dependence and power saturation profile were detected in the dithionite-reduced preparations at a low temperature range (8-18 K). Due to a negative shift (<600 mV) of the apparent redox midpoint potential of the iron-sulfur clusters in the soluble form of the truncated NQO9 subunit, the following two possible cases could not be discriminated: (i) two sets of EPR signals arise from two distinct species of tetranuclear iron-sulfur clusters with two intrinsically different spectral parameters g(, perpendicular) = 2.05, approximately 1.93, and g(parallel, perpendicular) = 2.08, approximately 1.90, and respective slow (P((1)/(2)) = 8 milliwatts) and fast (P((1)/(2)) = 342 milliwatts) spin relaxation; (ii) two clusters exhibit similar intrinsic EPR spectra (g(parallel, perpendicular) = 2.05, approximately 1.93) with slow spin relaxation. When both clusters in the same subunit are concomitantly paramagnetic, their spin-spin interactions cause a shift of spectra to g(parallel, perpendicular) = 2.08, approximately 1.90, with enhanced spin relaxation. In either case, our EPR data provide the first experimental evidence for the presence of two [4Fe-4S] iron-sulfur clusters in the NQO9 subunit.
Journal of Biological Chemistry 11/1999; 274(40):28598-605. · 4.77 Impact Factor
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Biochemical Society Transactions 09/1999; 27(4):586-91. · 3.71 Impact Factor
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ABSTRACT: The EPR and thermodynamic properties of semiquinone (SQ) species stabilized by mammalian succinate:quinone reductase (SQR) in situ in the mitochondrial membrane and in the isolated enzyme have been well documented. The equivalent semiquinones in bacterial membranes have not yet been characterized, either in SQR or quinol:fumarate reductase (QFR) in situ. In this work, we describe an EPR-detectable QFR semiquinone using Escherichia coli mutant QFR (FrdC E29L) and the wild-type enzyme. The SQ exhibits a g = 2.005 signal with a peak-to-peak line width of approximately 1.1 milliteslas at 150 K, has a midpoint potential (E(m(pH 7.2))) of -56.6 mV, and has a stability constant of approximately 1.2 x 10(-2) at pH 7.2. It shows extremely fast spin relaxation behavior with a P(1/2) value of >500 milliwatts at 150 K, which closely resembles the previously described SQ species (SQ(s)) in mitochondrial SQR. This SQ species seems to be present also in wild-type QFR, but its stability constant is much lower, and its signal intensity is near the EPR detection limit around neutral pH. In contrast to mammalian SQR, the membrane anchor of E. coli QFR lacks heme; thus, this prosthetic group can be excluded as a spin relaxation enhancer. The trinuclear iron-sulfur cluster FR3 in the [3Fe-4S](1+) state is suggested as the dominant spin relaxation enhancer of the SQ(FR) spins in this enzyme. E. coli QFR activity and the fast relaxing SQ species observed in the mutant enzyme are sensitive to the inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO). In wild-type E. coli QFR, HQNO causes EPR spectral line shape perturbations of the iron-sulfur cluster FR3. Similar spectral line shape changes of FR3 are caused by the FrdC E29L mutation, without addition of HQNO. This indicates that the SQ and the inhibitor-binding sites are located in close proximity to the trinuclear iron-sulfur cluster FR3. The data further suggest that this site corresponds to the proximal quinone-binding site in E. coli QFR.
Journal of Biological Chemistry 09/1999; 274(37):26157-64. · 4.77 Impact Factor
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ABSTRACT: A model for energy conversion in Complex I is proposed that is a conservative expansion of Mitchell's Q-cycle using a simple mechanistic variation of that already established experimentally for Complex III. The model accommodates the following proposals. (1) The large number of flavin and iron-sulfur redox cofactors integral to Complex I form a simple but long electron transfer chain guiding submillisecond electron transfer from substrate NADH in the matrix to the [4Fe-4S] cluster N2 close to the matrix-membrane interface. (2) The reduced N2 cluster injects a single electron into a ubiquinone (Q) drawn from the membrane pool into a nearby Qnz site, generating an unstable transition state semiquinone (SQ). The generation of a SQ species is the primary step in the energy conversion process in Complex I, as in Complex III. In Complex III, the SQ at the Qo site near the cytosolic side acts as a strong reductant to drive electronic charge across the membrane profile via two hemes B to a Qi site near the matrix side. We propose that in Complex I, the SQ at the Qnz site near the matrix side acts as a strong oxidant to pull electronic charge across the membrane profile via a quinone (Qny site) from a Qnx site near the cytosolic side. The opposing locations of matrix side Qnz and cytosolic side Qo, together with the opposite action of Qnz as an oxidant rather than a reductant, renders the Complex I and III processes vectorially and energetically complementary. The redox properties of the Qnz and Qo site occupants can be identical. (3) The intervening Qny site of Complex I acts as a proton pumping element (akin to the proton pump of Complex IV), rather than the simple electron guiding hemes B of Complex III. Thus the transmembrane action of Complex I doubles to four (or more) the number of protons and charges translocated per NADH oxidized and Q reduced. The Qny site does not exchange with the pool and may even be covalently bound. (4) The Qnx site on the cytosol side of Complex I is complementary to the Qi site on the matrix side of Complex III and can have the same redox properties. The Qnx site draws QH2 from the membrane pool to be oxidized in two single electron steps. Besides explaining earlier observations and making testable predictions, this Complex I model re-establishes a uniformity in the mechanisms of respiratory energy conversion by using engineering principles common to Complexes III and IV: (1) all the primary energy coupling reactions in the different complexes use oxygen chemistry in the guise of dioxygen or ubiquinone, (2) these reactions are highly localized structurally, utilizing closely placed catalytic redox cofactors, (3) these reactions are also highly localized energetically, since virtually all the free energy defined by substrates is conserved in the form of transition state that initiates the transmembrane action and (4) all complexes possess apparently supernumerary oxidation-reduction cofactors which form classical electron transfer chains that operate with high directional specificity to guide electron at near zero free energies to and from the sites of localized coupling.
Biochimica et Biophysica Acta 06/1998; 1364(2):245-57. · 4.66 Impact Factor
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ABSTRACT: 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.
Biochimica et Biophysica Acta 06/1998; 1364(2):186-206. · 4.66 Impact Factor
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ABSTRACT: The iron-sulfur (Fe-S) protein subunit of the bc1 complex, known as the Rieske protein, contains a high-potential [2Fe-2S] cluster ligated by two nitrogen and two sulfur atoms to its apoprotein. Earlier work indicated that in Rhodobacter capsulatus these atoms are provided by two cysteine (C133 and C153) and two histidine (H135 and H156) residues, located at the carboxyl-terminal end of the protein [Davidson, E., Ohnishi, T., Atta-Asafo-Adjei, E., & Daldal, F. (1992) Biochemistry 31, 3342-3351]. These ligands are part of the conserved sequences C133THLGC138 (box I) and C153PCHGS158 (box II) and affect the properties of the Fe-S protein and its [2Fe-2S] cluster. In this work, the role of amino acid side chains at positions 134 and 136, adjacent to the cluster ligands in box I, was probed by using site-directed mutagenesis and biophysical analyses. These positions were substituted with R, D, H, and G to probe the effect of charged, polar, large, and small amino acid side chains on the properties of the [2Fe-2S] cluster. Of the mutants obtained T134R, -H, and -G were photosynthetically competent (Ps+) but contained Fe-S proteins with redox midpoint potentials (Em7) 50-100 mV lower than that of a wild type strain. In contrast, T134D was Ps- and contained no detectable [2Fe-2S] cluster, although it reverted frequently to Ps+ by substitution of D with N. On the other hand, all L136 mutants were Ps-, the EPR characteristics of their [2Fe-2S] cluster were perturbed, and they were unable to sense the Qpool redox state or to bind stigmatellin properly. The overall data indicated that replacement of the amino acid side chain at position 134 of the Fe-S protein affects mainly the Em7 and oxygen sensitivity of the [2Fe-2S] cluster without abolishing its function, while substitutions at position 136 perturb drastically its ability to monitor the Qpool redox state and its interaction with the Qo site inhibitor stigmatellin. These two distinct phenotypes of box I T134 and L136 mutants are discussed with regard to the recently published three-dimensional structure of the water soluble part of the bovine heart mitochondrial Rieske Fe-S protein.
Biochemistry 09/1997; 36(39):11675-84. · 3.42 Impact Factor
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ABSTRACT: The Rieske iron-sulfur (Fe-S) protein subunit of bc1 complexes contains in its carboxyl-terminal part two highly conserved hexapeptide motifs (box I and box II) that include the four amino acid ligands of its [2Fe-2S] cluster. In the preceding paper [Liebl, U., Sled, V., Brasseur, G., Ohnishi, T., & Daldal, F. (1997) Biochemistry 36, 11675-11684], the effects of mutations at two of the nonliganding residues [threonine (T) 134 and leucine (L) 136 in the Rhodobactercapsulatus Rieske Fe-S protein] of box I have been described. In this work, interactions between the occupants of the Qo site of the bc1 complex (UQ/UQH2 and the inhibitors stigmatellin and myxothiazol) and the [2Fe-2S] cluster of the Rieske Fe-S protein were probed by isolating photosynthesis-proficient (Ps+) revertants of the Ps- mutants L136R, -H, -D and -G. These revertants contained either a single substitution at the original position 136 or an additional mutation located in the amino-terminal part of the Fe-S protein at either position 44 or 46. The same-site revertants L136A and -Y grew well under photosynthetic conditions and contained highly active bc1 complexes but exhibited modified EPR spectra both in the presence and in the absence of stigmatellin. Unexpectedly, they were highly resistant to stigmatellin (StiR) and hypersensitive to myxothiazol (MyxHS) in vivo, demonstrating for the first time that mutations located in the Fe-S subunit confer resistance to stigmatellin. The [2Fe-2S] cluster of the same-site revertants responded weakly to the Qpool redox state and had redox midpoint potential (Em7) values (around 265 mV) lower than those of their wild type counterpart (about 310 mV). On the other hand, the second-site revertants L136H/V44L, L136G/V44F, and L136G/A46T, -V, or -P supported photosynthetic growth poorly, were StiR and MyxHS, and contained barely active bc1 complexes. Like the same-site revertants, they exhibited modified EPR spectra both in the presence and in the absence of stigmatellin and had perturbed Qo site occupancy. In addition, they contained substoichiometric amounts of the Fe-S protein with respect to the other subunits of the bc1 complex. The Em7 values of the [2Fe-2S] cluster of these double mutants were lower (around 245 mV) than that of the wild type strain but appreciably higher than those of their Ps- parents (about 200 mV for L136G). In order to define the molecular nature of the suppression mediated by the second-site mutations, the single mutants V44L and -F and A46T and -V were constructed in the absence of the original mutations at position 136. These mutants behaved like a wild type strain with respect to their Ps+ growth ability, inhibitor sensitivity, EPR spectra of their [2Fe-2S] cluster, and response to stigmatellin or to the Qpool redox state. But surprisingly, the Em7 values of their [2Fe-2S] cluster were much higher (about 385 mV) than that of a wild type strain. These findings demonstrated for the first time that the amino-terminal part of the Rieske Fe-S protein encompassing residues 44 and 46 is important not only for the structure and function of the Qo site of the bc1 complex but also for the properties of its [2Fe-2S] cluster.
Biochemistry 09/1997; 36(39):11685-96. · 3.42 Impact Factor
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ABSTRACT: The genes encoding the proton-translocating NADH-quinone oxidoreductase (NDH-1) of a thermophilic bacterium Thermus thermophilus HB-8 were cloned and sequenced. They constitute a cluster that is composed of 14 structural genes and contains no unidentified reading frames. All of the 14 structural genes, which are designated NQO1-14, encode subunits homologous to those of Paracoccus denitrificans NDH-1, respectively, and are arranged in the same order as other bacterial NDH-1 genes. T. thermophilus NDH-1 contains at most nine putative iron-sulfur cluster binding sites, eight of which are commonly found in other organisms. The T. thermophilus NQO2 subunit was expressed in Escherichia coli. The expressed subunit bears a single [2Fe-2S] cluster whose optical and EPR properties are very similar to those of N1a cluster in the P. denitrificans NQO2 subunit (Yano, T., Sled', V.D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). These results strongly suggest that the T. thermophilus NDH-1 is similar to other NDH-1 enzyme complexes in terms of subunit and cofactor composition. The T. thermophilus NQO2 subunit displayed much higher stability than the mesophilic equivalent and its iron-sulfur cluster remained intact even after incubation for 3 h at 65 degrees C under anaerobic conditions. With the advantage of thermostability, the T. thermophilus NDH-1 provides a great model system to investigate the structure-function relationship of the NDH-1 enzyme complexes.
Journal of Biological Chemistry 03/1997; 272(7):4201-11. · 4.77 Impact Factor
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ABSTRACT: Continuous wave electron nuclear double resonance (CW ENDOR) spectra of [delta-15N,epsilon(-14)N]histidine-labeled phthalate dioxygenase (PDO) from Pseudomonas cepacia were recorded and found to be virtually identical to those previously recorded from [delta,epsilon-15N2]histidine-labeled protein [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. Thus, the two histidine residues, previously shown to ligate one of the irons in the cluster [cf. Gurbiel et al. 1989)], both coordinate the metal at the N(delta) position of their imidazole rings. Pulsed ENDOR studies showed that the "remote", noncoordinating nitrogen of the histidine imidazole ring could be observed from the Rieske protein in a sample of Rhodobacter capsulatus cytochrome bc1 complex uniformly labeled with 15N but not in a sample of PDO labeled with [delta-15N,epsilon-14N]histidine, but this atom was easily observed with a sample of Rh. capsulatus cytochrome bc1 complex that had been uniformly labeled with 15N; this confirmed the conclusion from the CW ENDOR studies that ligation is exclusively via N(delta) for both ligands in the PDO center. Modifications in the algorithms previously used to simulate 14N ENDOR spectra permitted us to compute spectra without any constraints on the relative orientation of hyperfine and quadrupole tensors. This new algorithm was used to analyze current and previously published spectra, and slightly different values for the N-Fe-N angle and imidazole ring rotation angles are presented [cf. Gurbiel et al. (1989) Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., and Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. This analysis has permitted us to refine the proposed structure of the [2Fe-2S] Rieske-type cluster and rationalize some of the properties of these novel centers. Although the spectra of cytochrome bc1 complex from Rh. capsulatus are of somewhat lower resolution than those obtained with samples of PDO, our analysis nevertheless permits the conclusion that the geometry of the cluster is essentially the same for all Rieske and Rieske-type proteins. Structural constraints inferred from the spectroscopic results permitted us to apply the principles of distance geometry to arrive at possible three-dimensional models of the active site structure of Rieske protein from Rh. capsulatus. Results from this test case indicate that similar procedures should be generally useful in metalloprotein systems. We also recorded the pulsed and CW ENDOR spectra of 57Fe-labeled PDO, and the resulting data were used to derive the full hyperfine tensors for both Fe(III) and Fe(II) ions, including their orientations relative to the g tensor. The A tensor of the ferric ion is nominally isotropic, while the A tensor of the ferrous ion is axial, having A(parallel) > A(perpendicular); both tensors are coincident with the observed g tensor, with A(parallel) of the ferrous ion lying along the maximum g-value, g1. These results were examined using refinements of existing theories of spin-coupling in [2Fe-2S]+ clusters, and it is concluded that current theories are not adequate to fully describe the experimental results.
Biochemistry 06/1996; 35(24):7834-45. · 3.42 Impact Factor
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ABSTRACT: A water-soluble fragment of the bc1 complex from bovine heart mitochondria was isolated containing the intact Rieske [2Fe-2S] cluster. The fragment consists of the last 129 amino acid residues of the Rieske iron-sulfur protein and has a molecular mass of 14592 Da including two iron atoms. The absorption, visible CD, and EPR spectra of the fragment are indistinguishable from those of the membrane-bound iron-sulfur protein. The redox potential as determined by EPR-monitored redox titration was + 306 mV. The far-ultraviolet CD spectrum is indicative of a protein with little regular secondary structure, while significant alpha-helix content was detected in the membrane anchor of the complete iron-sulfur protein. The fragment could be crystallized using poly(ethylene glycol) 6000 as precipitant. Needle-shaped single crystals have been grown by the hanging-drop vapor diffusion technique. These crystals belong to the space group P21 and diffract well beyond 0.2 nm resolution. Phase determination using the multiple-wavelength anomalous-scattering technique is underway.
European Journal of Biochemistry 05/1996; 237(1):71-5. · 3.58 Impact Factor
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ABSTRACT: This study reports the expression of the flavoprotein (FP) subcomplex of the proton-translocating NADH-quinone oxidoreductase (NDH-1) from Paracoccus denitrificans, which is composed of the NQO1 (50 kDa) and the NQO2 (25 kDa) subunits. The two subunits are co-expressed in Escherichia coli using a double expression plasmid system. The expressed subunits form a water-soluble heterodimer complex with 1:1 stoichiometry. The expressed complex contained one [2Fe 2S] cluster but almost no FMN or [4Fe 4S] cluster. The two latter prosthetic groups could be partially reconstituted with FMN, Na2S, and (NH4)2Fe(SO4)2 in vitro under anaerobic conditions. The reconstituted FP subcomplex showed EPR signals from two distinct species of iron-sulfur cluster. One resonance transition originates from a [2Fe-2S] cluster with g values of gx,y,z = 1.92, 1.95, and 2.00 and slow spin relaxation, which was tentatively assigned to the cluster N1a. These EPR properties are very similar to those reported for the NQO2 subunit expressed alone (Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). The other originates from a [4Fe 4S] cluster with g values of gx,y, z = 1.87, 1.94, and 2.04 and fast relaxing behavior, which are reminiscent of the cluster N3 in the membrane bound enzyme complex. After reconstitution with FMN, the FP subcomplex catalyzed electron transfer from NADH and from deamino-NADH to a variety of electron acceptors. The enzymatic properties of the FP subcomplex, reconstituted with FMN and iron-sulfur, correspond to those of the isolated P. denitrificans NADH-dehydrogenase complex.
Journal of Biological Chemistry 04/1996; 271(10):5907-13. · 4.77 Impact Factor
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ABSTRACT: Two distinct species of Complex I-associated ubisemiquinones (SQNf and SQNs) were detected by cryogenic EPR analysis of tightly coupled submitochondrial particles oxidizing NADH or succinate under steady-state conditions. The g = 2.00 signals from both fast-relaxing SQNf (P1/2 = 170 mW at 40 K) and slow-relaxing SQNs (P1/2 = 0.7 mW) are sensitive to uncouplers, rotenone and thermally induced deactivation of Complex I. At higher temperatures the SQNf signal is broadened and only the SQNs signal is seen (P1/2 = 7 mW at 105 K). The spin-spin interaction between SQNf and the iron-sulfur cluster N2 was detected as split peaks of the g parallel 2.5 signal with a coupling constant of 1.65 mT, revealing their mutual distance of 8-11 A. The data obtained are consistent with a model in which N2 and two interacting bound ubisemiquinone species are spatially arranged within the hydrophobic domain of Complex I, participating in the vectorial proton translocation.
FEBS Letters 09/1995; 370(1-2):83-7. · 3.54 Impact Factor
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ABSTRACT: The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 dissimilar subunits which are designated NQO1-14 and contains one noncovalently bound FMN and at least five EPR-visible iron-sulfur clusters (N1a, N1b, N2, N3, and N4) as prosthetic groups. Comparison of the deduced primary structures of the subunits with consensus sequences for the cofactor binding sites has predicted that NQO1, NQO2, NQO3, NQO9, and probably NQO6 subunits are cofactor binding subunits. Previously, we have reported that the NQO2 (25 kDa) subunit was overexpressed as a water-soluble protein in Escherichia coli and was found to ligate a single [2Fe-2S] cluster with rhombic symmetry (gx,y,z = 1.92, 1.95, and 2.00) (Yano, T., Sled', V.D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). In the present study, the NQO3 (66 kDa) subunit, which is equivalent to the 75-kDa subunit of bovine heart Complex I, was overexpressed in E. coli. The expressed NQO3 subunit was found predominantly in the cytoplasmic phase and was purified by ammonium sulfate fractionation and anion-exchange chromatography. The chemical analyses and UV-visible and EPR spectroscopic studies showed that the expressed NQO3 subunit contains at least two distinct iron-sulfur clusters: a [2Fe-2S] cluster with axial EPR signals (g perpendicular, parallel = 1.934 and 2.026, and L perpendicular parallel = 1.8 and 3.0 millitesla) and a [4Fe-4S] cluster with rhombic symmetry (gx,y,z = 1.892, 1.928, and 2.063, and Lx,y,z = 2.40, 1.55, and 1.75 millitesla). The midpoint redox potentials of [2Fe-2S] and [4Fe-4S] clusters at pH 8.6 are -472 and -391 mV, respectively. The tetranuclear cluster in the isolated NQO3 subunit is sensitive toward oxidants and converts into [3Fe-4S] form. The assignment of these iron-sulfur clusters to those identified in the P. denitrificans NDH-1 enzyme complex and the possible functional role of the NQO3 subunit is discussed.
Journal of Biological Chemistry 09/1995; 270(31):18264-70. · 4.77 Impact Factor
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ABSTRACT: The proton-translocating NADH:ubiquinone oxidoreductase (complex I) was isolated from Escherichia coli by chromatographic steps performed in the presence of an alkylglucoside detergent at pH 6.0. The complex is obtained in a monodisperse state with a molecular mass of approximately 550,000 Da and is composed of 14 subunits. The subunits were assigned to the 14 genes of the nuo operon, partly based on their N-terminal sequences and partly on their apparent molecular masses. The preparation contains one noncovalently bound FMN/molecule. At least two binuclear (N1b and N1c) and three tetranuclear (N2, N3 and N4) iron-sulfur clusters were detected by EPR in the preparation when reduced with NADH. Their EPR characteristics remained mostly unaltered during the isolation process. After reconstitution in phospholipid membranes, the preparation catalyses piericidin-A-sensitive electron transfer from NADH to ubiquinone-2 with Km values similar to those of complex I in cytoplasmic membranes but with only 10% of the Vmax value. The isolated complex I was cleaved into three fragments when the pH was raised from 6.0 to 7.5 and the detergent exchanged to Triton X-100. One of these fragments is a water-soluble NADH dehydrogenase fragment which is composed of three subunits bearing at least four iron-sulfur clusters (N1b, N1c, N3 and N4) that can be reduced with NADH, one of them bearing FMN. The second, amphipathic, fragment, which is presumed to connect the NADH dehydrogenase fragment with the membrane, contains four subunits and at least one EPR-detectable iron-sulfur cluster whose spectral properties are reminiscent of the eucaryotic cluster N2. The third membrane fragment is composed of seven homologues of the mitochondrially encoded subunits of the eucaryotic complex I. This subunit arrangement coincidences to some extent with the order of the genes on the nuo operon. A topological model of the E. coli complex I is proposed.
European Journal of Biochemistry 07/1995; 230(2):538-48. · 3.58 Impact Factor
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ABSTRACT: Succinate:quinone reductases (SQRs) and quinol:fumarate reductases (QFRs) each contain a bi-, a tri- and a tetra-nuclear iron-sulfur cluster. The C-terminal half of the iron-sulfur protein subunit of these enzymes shows two fully conserved motifs of cysteine residues, stereotypical for ligands of [3Fe-4S] and [4Fe-4S] clusters. To analyze the functional role of the trinuclear cluster S3 in Bacillus subtilis SQR, a fourth cysteine residue was introduced into the putative ligation motif to that cluster. A corresponding mutation in Escherichia coli QFR results in a tri- to tetranuclear conversion (Manodori et al. (1992) Biochemistry 31, 2703-2731). We have found that presence of the extra cysteine in B. subtilis SQR does not result in cluster conversion. It does, however, affect the EPR properties of the cluster S3, whereas those of the other two clusters remain normal. The results strongly support the view that residues in the most C-terminal cysteine motif in the iron-sulfur protein subunit of SQRs and QFRs ligate the trinuclear cluster. Compared to wild-type SQR, S3 in the B. subtilis mutant enzyme is not sensitive to methanol and the midpoint redox potential is close to normal. The quinone reductase activity of the mutant enzyme is only 35% of normal. Thus, the architecture around cluster S3 plays a role in electron transfer to quinone or in the binding of quinone to the enzyme.
Biochimica et Biophysica Acta 06/1995; 1229(3):356-62. · 4.66 Impact Factor
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ABSTRACT: Treatment of bovine heart ubiquinol-cytochrome c oxidoreductase (complex III, bc1 complex) with ethoxyformic anhydride (EFA) inhibits electron transfer between cytochromes b and c1 [Yagi et al., Biochemistry 21 (1982) 4777-4782]. This paper shows that EFA alters the EPR lineshape of the Rieske iron-sulfur cluster in complex III and in the isolated Rieske protein without a significant decrease of spin concentration. The effect of EFA on the Rieske iron-sulfur cluster is competitive with that of Qo site inhibitors, such as stigmatellin, and is completely reversed by hydroxylamine. These results are consistent with the possible ethoxyformylation by EFA of histidine ligands of the Rieske iron-sulfur cluster at the non-iron binding imidazole nitrogens.
FEBS Letters 11/1994; 353(1):103-7. · 3.54 Impact Factor
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Biochimica et Biophysica Acta 09/1994; 1187(2):121-4. · 4.66 Impact Factor
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ABSTRACT: This paper reports the first direct characterization of flavin (noncovalently bound FMN) in energy coupling site I of the mitochondrial respiratory chain. Thermodynamic parameters of its redox reactions were determined potentiometrically monitoring the g = 2.005 signal of its free radical form in isolated bovine heart NADH:ubiquinone oxidoreductase (complex I). The midpoint redox potentials of consecutive one-electron reduction steps are Em1/0 = -414 mV and Em2/1 = -336 mV at pH 7.5. This corresponds to a stability constant of the intermediate flavosemiquinone state of 4.5 x 10(-2). The pK values of the free radical (Fl.-<==>FlH.) and reduced flavin (FlH-<==>FlH2) were estimated as 7.7 and 7.1, respectively. The potentiometrically obtained g = 2.005 flavin free radical EPR signal revealed an unusually broad (2.4 mT) and pH-independent peak-to-peak line width. The spin relaxation of flavosemiquinone in complex I is much faster than that of flavodoxin due to strong dipole-dipole interaction with iron-sulfur cluster N3. Guanidine, an activator of NADH-ferricyanide reductase activity of complex I, was found to have a strong stabilizing effect on the flavin free radical generated both by equilibration with the NADH/NAD+ redox couple and by potentiometric redox titration. The addition of guanidine also leads to a slight modification of the EPR spectrum of iron-sulfur cluster N3. Anaerobic titration of flavosemiquinone free radical with the strictly n = 2 NADH/NAD+ and APADH/APAD+ redox couples revealed that nucleotide binding narrows the EPR signal line width of the flavin free radical to 1.7 mT and changes a shape of the titration curve.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 09/1994; 33(33):10069-75. · 3.42 Impact Factor
