Tomoko Ohnishi

IRON-SULFUR CLUSTERS/SEMIQUINONES IN COMPLEX I

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.

Relationship between free radical production and lipid peroxidation during ischemia-reperfusion injury in the rat brain

Forebrain ischemia was produced in the rat by bilateral occlusion of the common carotid arteries combined with hemorrhagic hypotension (30 mmHg). The whole cerebral cortex was homogenized in the presence of the spin trap agent N-tert-butyl-alpha-phenyl-nitrone, followed by a Folch extract. Spin-adducts were detected using electron spin resonance spectroscopy. The lipid peroxidation was estimated from both the amount of thiobarbituric acid reactive substance and the formation of conjugated diene. After 10 or 20 min of ischemia, reperfusion was initiated which induced an abrupt burst of free radical formation. The formation peaked at 5 min, and the peak value increased with the ischemia time. The degree of lipid peroxidation, which was measured after 20 min of reperfusion, also increased with the ischemia time. The results suggest that the lipid peroxidation may be the direct consequence of the action of free radicals formed during ischemia and reperfusion periods.

The chromone inhibitor stigmatellin - binding to the ubiquinol oxidation center at the C-side of the mitochondrial membrane

Cytochrome bc2 complex Inhibitor Fe2S2 protein EPR Cytochrome b Stigmatellin

A reductant-induced oxidation mechanism for Complex I

A model for energy conversion in Complex I is proposed that is a conservative expansion of Mitchells 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.

Dre2, a Conserved Eukaryotic Fe/S Cluster Protein, Functions in Cytosolic Fe/S Protein Biogenesis

ABSTRACT In a forward genetic screen for interaction with mitochondrial iron carrier proteins in Saccharomyces cerevisiae, a hypomorphic mutation of the essential DRE2 gene was found to confer lethality when combined with Δmrs3 and Δmrs4. The dre2 mutant or Dre2-depleted cells were deficient in cytosolic Fe/S cluster protein activities while maintaining mitochondrial Fe/S clusters. The Dre2 amino acid sequence was evolutionarily conserved, and cysteine motifs (CX2CXC and twin CX2C) in human and yeast proteins were perfectly aligned. The human Dre2 homolog (implicated in blocking apoptosis and called CIAPIN1 or anamorsin) was able to complement the nonviability of a Δdre2 deletion strain. The Dre2 protein with triple hemagglutinin tag was located in the cytoplasm and in the mitochondrial intermembrane space. Yeast Dre2 overexpressed and purified from bacteria was brown and exhibited signature absorption and electron paramagnetic resonance spectra, indicating the presence of both [2Fe-2S] and [4Fe-4S] clusters. Thus, Dre2 is an essential conserved Fe/S cluster protein implicated in extramitochondrial Fe/S cluster assembly, similar to other components of the so-called CIA (cytoplasmic Fe/S cluster assembly) pathway although partially localized to the mitochondrial intermembrane space.

Potentiation of nitric oxide formation following bilateral carotid occlusion and focal cerebral ischemia in the rat: in vivo detection of the nitric oxide radical by electron paramagnetic resonance spin trapping

We have directly demonstrated in vivo that nitric oxide (NO) is produced in the ischemic rat brain. Using diethyldithiocarbamate and Fe as spin-trapping agents, NO spin adducts were detected by cryogenic electron paramagnetic resonance. The cerebral cortex which was exposed to focal ischemia or bilateral carotid artery occlusion generated an increased amount of spin-adducts of NO radicals (g = 2.039, a hyperfine coupling constant aN = 13 gauss). This signal disappeared by the preischemic administration of NG-nitro-L-arginine methylester, a NO synthase inhibitor.

Energy-dependent Complex I-associated ubisemiquinones in submitochondrial particles

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 at 40 K) and slow‐relaxing ) 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 ( ). The spin‐spin interaction between SQNf and the iron‐sulfur cluster N2 was detected as split peaks of the 2.05 signal with a coupling constant of 1.65 mT, revealing their mutual distance of 8–11 Å. 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.

Conformation-driven and semiquinone-gated proton-pump mechanism in the NADH-ubiquinone oxidoreductase (complex I)

We propose that the proton pump is operated by redox‐driven conformational changes of the quinone binding protein. In the input state, semiquinone is reduced to quinol, acquiring two protons from the N (matrix) side of the mitochondrial inner membrane and an electron from the low potential (NADH) side of the respiratory chain. A conformational change brings the protons into position for release at the P (inter‐membrane space) side of the membrane via a proton‐well. Concomitantly, an electron is donated to the quinone pool at the high potential side of the coupling site. The system then returns to the original state to repeat the cycle. This hypothesis provides a useful frame work for further investigation of the mechanism of proton translocation in complex I.

EPR Characterization of Ubisemiquinones and Iron-Sulfur Cluster N2, Central Components of the Energy Coupling in the NADH-Ubiquinone Oxidoreductase (Complex I) In Situ

AbstractThe proton-translocating NADH-ubiquinone oxidoreductase (complex I) is the largest and least understood respiratory complex. The intrinsic redox components (FMN and iron–sulfur clusters) reside in the promontory part of the complex. Ubiquinone is the most possible key player in proton-pumping reactions in the membrane part. Here we report the presence of three distinct semiquinone species in complex I in situ, showing widely different spin relaxation profiles. As our first approach, the semiquinone forms were trapped during the steady state NADH-ubiquinone-1 (Q1) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQNf (fast-relaxing component), SQNs (slow-relaxing), and SQNx (very slow relaxing). This indicates the presence of at least three different quinone-binding sites in complex I. In the current study, special attention was placed on the SQNf, because of its high sensitivities to