S. Tsuyoshi Ohnishi’s research while affiliated with Treatment Research Institute, Philadelphia PA and other places

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Publications (140)


Malignant Hyperthermia: A Genetic Membrane Disease
  • Book

April 2022

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2 Reads

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2 Citations

S. Tsuyoshi Ohnishi

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Tomoko Ohnishi

Five Decades of Research on Mitochondrial NADH-quinone Oxidoreductase (complex I)

June 2018

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98 Reads

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48 Citations

NADH-quinone oxidoreductase (complex I) is the largest and most complicated enzyme complex of the mitochondrial respiratory chain. It is the entry site into the respiratory chain for most of the reducing equivalents generated during metabolism, coupling electron transfer from NADH to quinone to proton translocation, which in turn drives ATP synthesis. Dysfunction of complex I is associated with neurodegenerative diseases such as Parkinson’s and Alzheimer’s, and it is proposed to be involved in aging. Complex I has one noncovalently bound FMN, eight to ten iron-sulfur clusters, and protein-associated quinone molecules as electron transport components. Electron paramagnetic resonance (EPR) has previously been the most informative technique, especially in membrane in situ analysis. The structure of complex 1 has now been resolved from a number of species, but the mechanisms by which electron transfer is coupled to transmembrane proton pumping remains unresolved. Ubiquinone-10, the terminal electron acceptor of complex I, is detectable by EPR in its one electron reduced, semiquinone state. In the aerobic steady state of respiration the semiubiquinone anion has been observed and studied in detail. Two distinct protein-associated fast and slow relaxing, semiquinone signals have been resolved which were designated SQNf and SQNs. This review covers a five decade personal journey through the field leading to a focus on the unresolved questions of the role of the semiquinone radicals and their possible part in proton pumping. https://www.degruyter.com/view/j/bchm.2018.399.issue-11/hsz-2018-0164/hsz-2018-0164.xml




A new trend in the Complex I research field.

March 2013

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50 Reads

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7 Citations

Abstract To date, the 17th European Bioenergetics Conference (EBEC) in Freiburg, Germany (Sept. 15-20, 2012) was the most exciting conference for the complex I (NADH-Q oxidoreductase) research field. A whole day of oral presentations was dedicated to complex I, including 3 plenary lectures and 11 symposium talks. In addition, 43 posters were dedicated to complex I projects. This is a brief summary of an exciting paradigm shift from 'structure major' to 'structure + function', clearly observed in the complex I research field.


Functional Role of Two Protein-Associated Ubiquinone Molecules (Q-NF and Q-NS) for the Proton-Pumping Mechanism in Bovine Heart Complex I (NADH-Ubiquinone Oxidoreductase)

January 2013

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30 Reads

Biophysical Journal

We had characterized the function of two distinct protein-associated ubisemiquinone molecules in the proton-pumping mechanism in complex I (NADH-UQ oxidoreductase).We constructed most of the frame work of our proton-pumping hypothesis, utilizing EPR techniques before the X-ray structures of bacterial and mitochondrial complex I were reported by Sazanov's group and Brandt and his collaborators, respectively. The fast relaxing semiquinone (SQ-Nf) signal is extremely sensitive to the proton motive force (ΔP) imposed to the energy transducing membrane, strongly indicating its direct involvement in proton-pumping mechanism. Slow relaxing semiquinone (SQ-Ns) is not sensitive to ΔP. Although they show identical piericidin A sensitivity, they differ in rotenone sensitivity considerably as well as their SQ binding subunits. These differences were exploited using tightly coupled bovine heart submitochondrial particles with a high respiratory control ratio (>8).We determined the center-to-center distance of 12 A between SQ-Nf and its direct electron donor, iron-sulfur cluster N2 based on their spin-spin interaction analysis. We have extended this work using reconstituted bovine heart complex I proteoliposomes which shows a respiratory control ratio >5. Our recent Q-band (33.9 GHz) EPR analysis of SQ-Nf and SQ-Ns spectra in the reconstituted proteoliposome system also supports our two-semiquinone model. We will compare our EPR-based model with the X-ray structure based proton-pumping models by Sazanov's group and Brandt with his collaborators.



EPR analysis of two protein-associated ubiquinone (SQ(Nf) and SQ(Ns)) in the membrane in situ and in proteoliposomes of isolated bovine heart complex I

April 2012

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40 Reads

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29 Citations

Biochimica et Biophysica Acta

The success of Sazanov's group in determining the X-ray structure of the whole bacterial complex I is a great contribution to the progress of complex I research. In this mini-review of 35years' history of my laboratory and collaborators, we characterized the function of protein-associated semiquinone molecules in the proton-pumping mechanism in complex I (NADH-quinone oxidoreductase). We have constructed most of the frame work of our hypothesis, utilizing EPR techniques before the X-ray structures of complex I were reported by Sazanov's and Brandt's groups. One of the semiquinones (SQ(Nf)) is extremely sensitive to a proton motive force imposed on the energy-transducing membrane, while the other (SQ(Ns)) is insensitive. Their sensitivity to rotenone inhibition also differs. These differences were exploited using tightly coupled bovine heart submitochondrial particles with a high respiratory control ratio (>8). We determined the distance between SQ(Nf) and iron-sulfur cluster N2 on the basis of their direct spin-spin interaction. We are extending this line of work using reconstituted bovine heart complex I proteoliposomes which shows a respiratory control ratio >5. Two frontier research groups support our view point based on their mutagenesis studies. High frequency (33.9GHz; Q-band) EPR experiments appear to favor our two-semiquinone model. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).


Possible Roles of Two Quinone Molecules in Direct and Indirect Proton Pumps of Bovine Heart NADH-Quinone Oxidoreductase (Complex I)

December 2010

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50 Reads

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44 Citations

Biochimica et Biophysica Acta

In many energy transducing systems which couple electron and proton transport, for example, bacterial photosynthetic reaction center, cytochrome bc(1)-complex (complex III) and E. coli quinol oxidase (cytochrome bo(3) complex), two protein-associated quinone molecules are known to work together. T. Ohnishi and her collaborators reported that two distinct semiquinone species also play important roles in NADH-ubiquinone oxidoreductase (complex I). They were called SQ(Nf) (fast relaxing semiquinone) and SQ(Ns) (slow relaxing semiquinone). It was proposed that Q(Nf) serves as a "direct" proton carrier in the semiquinone-gated proton pump (Ohnishi and Salerno, FEBS Letters 579 (2005) 4555), while Q(Ns) works as a converter between one-electron and two-electron transport processes. This communication presents a revised hypothesis in which Q(Nf) plays a role in a "direct" redox-driven proton pump, while Q(Ns) triggers an "indirect" conformation-driven proton pump. Q(Nf) and Q(Ns) together serve as (1e(-)/2e(-)) converter, for the transfer of reducing equivalent to the Q-pool.


New insights into the superoxide generation sites in bovine heart NADH-ubiquinone oxidoreductase (Complex I): The significance of protein-associated ubiquinone and the dynamic shifting of generation sites between semiflavin and semiquinone radicals

December 2010

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139 Reads

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56 Citations

Biochimica et Biophysica Acta

Considerable disagreement still exists concerning the superoxide generation sites in the purified bovine heart NADH-ubiquinone oxidoreductase (complex I). Majority of investigators agree that superoxide is generated at the flavin site. Here we present a new hypothesis that the generation of superoxide reflects a dynamic balance between the flavosemiquinone (semiflavin or SF) and the semiquinone (SQ), like a "tug-of-war" through electrons. All preparations of bovine heart complex I, which have been isolated at Yoshikawa's laboratory, have one protein-bound endogenous ubiquinone per complex I (Shinzawa-Itoh et al., Biochemistry, 49 (2010) 487-492). Using these preparations, we measured (i) EPR signals of the SF, the SQ and iron-sulfur cluster N2 simultaneously with cryogenic EPR and (ii) superoxide production with both the room temperature spin-trapping technique and the partially acetylated cytochrome c method. Our experimental evidence was (1) without added decylubiquinone (DBQ), no catalytic oxidation of NADH occurs. The NADH addition produced mostly SF and it generated superoxide as reported by Kussmaul and Hirst (PNAS, 103 (2006) 7607-7612). (2) During catalytic electron transfer from NADH to DBQ, the superoxide generation site was mostly shifted to the SQ. (3) A quinone-pocket binding inhibitor (rotenone or piericidin A) inhibits the catalytic formation of the SQ, and it enhances the formation of SF and increases the overall superoxide generation. This suggests that if electron transfer was inhibited under pathological conditions, superoxide generation from the SF would be increased.


Citations (74)


... MH is an inherited pharmacogenetic disorder triggered by volatile anesthetics, such as halothane, first reported by Denborough and Lovell (38) (for review and books, see Refs. 16,85,175,200). The main manifestations of MH are a rapid and sustained rise in body temperature that can exceed 43°C, generalized muscular contracture, and a severe metabolic acidosis, all of which might result from an increased Ca 2ϩ concentration in muscle cells. If not properly treated, MH can rapidly lead to severe tissue damage and, eventually, death. ...

Reference:

Calcium-Induced Calcium Release in Skeletal Muscle
Malignant Hyperthermia: A Genetic Membrane Disease
  • Citing Book
  • April 2022

... and complex II, also known as succinate dehydrogenase (EC 1.3.5.1), play a crucial role in energy generation in the mitochondrial respiratory chain. They are found in the inner mitochondrial membrane in eukaryotes and translocate electrons across it, generating the electrochemical gradient required for ATP synthesis [59]. Complex I requires FMN, whereas complex II requires FAD; both cofactors can accept and give one or two electrons through the tricyclic heteroaromatic isoalloxazine ring, being able to participate in a wide range of oxidation and reduction reactions. ...

Five Decades of Research on Mitochondrial NADH-quinone Oxidoreductase (complex I)
  • Citing Article
  • June 2018

... The method was fully described and generalized with other spin-traps [116,117]. Similar experiments, using Fe-MGD and a microwave frequency of 1.2 GHz, were simultaneously described by Zweier's group in rat brains subjected to ischemia-reperfusion or in ischemic rat hearts [40,86,118]. Finally a third group attempted to quantitate NO by ex vivo and in vivo experiments in several regions of mice brain making use of X and L-bands [119,120]. ...

Electron Paramagnetic Resonance Imaging of Nitric Oxide in Tissues
  • Citing Chapter
  • January 1998

... These include NO, [133] anti-adhesion molecules, [47] the surfactant poloxamer-188, [134] levocarnitine, [180] arginine, [181] zileuton, a 5-lipoxygenase inhibitor, [182] green tea, [183,184] aged garlic, [183,184] and herbal extracts. [185] Some of these agents are being used on an investigational basis. ...

Green tea extract and aged garlic extract inhibit potassium-chloride cotrantport in sickle cells in vitro, but vitamine C and vitamin E do not.
  • Citing Article
  • November 2000

Blood

... Examination of the solvent accessible surface within the expected lipid dielectric allows observation of a 30 Å long narrow curved cavity, extending up to the proposed Q-head binding residues (see below) in the vicinity of N2. From assigned EPR spin-spin interactions between N2 and fast relaxing semiquinone (SQNf ; see section 2.2.1) a distance of 12 Å was calculated (Ohnishi, Ohnishi et al. 2012), agreeing with the structural information and electron tunneling theory. The Q-head extends almost 20 Å above the cytoplasmic membrane plane and is detected within the hydrogen bonding distance from the highly conserved Nqo4 residues H38, Y87 and D139. ...

EPR analysis of two protein-associated ubiquinone (SQNf and SQNs) in the membrane in situ and in proteoliposomes of isolated bovine heart complex I
  • Citing Article
  • October 2012

Biochimica et Biophysica Acta (BBA) - Bioenergetics

... In this case the tail of the exchangeable UQ will always remain in the hydrophobic membrane surrounding. The existence of two UQ-binding sites has been proposed by Ohnishi and co-authors16171819 based on their EPR study of Complex I in the natural membrane fragments, submitochondrial vesicles. Under turnover, Complex I showed two semiquinone EPR signals originating from two different semiquinone species with different spinrelaxation behavior (Q fast and Q slow ) and distances from N2; only one of which being sensitive to transmembrane electric potential. ...

EPR detection of protein-associated ubiquinone molecules in purified bovine heart NADH–ubiquinone oxidoreductase (complex I)
  • Citing Article
  • July 2010

Biochimica et Biophysica Acta (BBA) - Bioenergetics

... With the recovery of brain perfusion, bringing oxygen and glucose to basal levels at the end of the HI procedure, a second loss of high-energy phosphate levels takes place (Folbergrov a et al., 1992(Folbergrov a et al., , 1995, bringing a burst of reactive oxygen species (ROS) production (Hagberg et al., 1987;Lafemina et al., 2006) associated with downregulation of antioxidant enzymes (Andersen et al., 1996;Numagami et al., 1997;Blomgren and Hagberg, 2006;Guglielmotto et al., 2009); these events initiate the secondary neuronal damage. Both depletion of ATP and high levels of ROS are proved to inhibit the activity of Na 1 ,K 1 -ATPase, both in the adult (Wyse et al., 2000) and in the neonatal brain (Weis et al., 2011).The subsequent neuronal membrane depolarization leads to excessive glutamate release and activation of NMDA, AMPA, and kainite receptors, coupled to calcium influx and nitric oxide (NO) release by nitric oxide synthase activity (McLean and Ferriero, 2004;Buonocore and Groenendaal, 2007), resulting in mitochondrial dysfunction and cell death by apoptosis. ...

EFFECT OF ALLOPURINOL ON FREE RADICAL FORMATION DURING CEREBRAL HYPOXIA IN NEWBORN PIGLETS. ??? 247
  • Citing Article
  • April 1996

Pediatric Research

... The most frequently used method for reproducing GCI is four-vessel occlusion (4VO), which was developed by W.A. Pulsinelli (Pulsinelli and Brierley, 1979). However, 4VO is associated with a number of serious technical problems, including difficulty of electrocoagulation of the vertebral arteries through the alar foramina of the first cervical vertebra, risk of spinal cord damage, and risk of excess bleeding (Chen, 2009;Ohnishi and Ohnishi, 1995;Pulsinelli and Buchan, 1988), a preconditioning effect (Yamaguchi et al., 2005), incomplete ischemia of the brain stem (Pulsinelli and Brierley, 1979). As an alternative to the 4VO model, Shcherbak et al. (2012) proposed a new way of performing GCI modeling in rats by reversible occlusion of the three main vessels that branch off from the aorta and supply the brain (brachiocephalic trunk, left subclavian artery, and left common carotid artery). ...

Central nervous system trauma : research techniques
  • Citing Book
  • January 1995

... The electron transfer from NADH to the last Fe-S cluster in the chain takes ∼100 μs, and turnover is ∼3 ms (Verkhovskaya et al. 2008); therefore, it is plausible that the energy-converting step is time limiting, and thus the lifetime of the semiquinone state could be long enough to detect it upon Complex I turnover. The electron paramagnetic resonance (EPR) signal assigned to semiquinone was detected in the isolated Complex I (Suzuki and King 1983;Ohnishi et al. 2005;Narayanan et al. 2015), tightly coupled submitochondrial particles (Kotlyar et al. 1990;Magnitsky et al. 2002;Yano, Dunham and Ohnishi 2005;Ohnishi and Ohnishi 2013) and Complex I reconstituted into proteoliposomes (Ohnishi et al. 2012;Narayanan et al. 2015). However, further EPR studies of the semiquinone radical are complicated due to its low amplitude and strong signal deriving from the cluster N1a (g z = 2.00) overlapping the radical area and difficulties in freezing Complex I in functioning state. ...

A new trend in the Complex I research field.
  • Citing Article
  • March 2013

... Many observations provide convincing evidence for the effective role of UA in MS. Hooper and colleagues found that treatment of EAE with high doses of UA completely protected mice from the disease [16]. In addition, serum UA is reported to be significantly lower in people with MS [17][18][19][20][21]. Furthermore, it has been shown that during treatment with MS immunotherapies, an increased serum UA level was observed. ...

Prevention of Experimental Allergic Encephalomyelitis by Targeting Nitric Oxide and Peroxynitrite: Implications for the Treatment of Multiple Sclerosis
  • Citing Article
  • March 1997

Proceedings of the National Academy of Sciences