Tomoko Ohnishi, Ph.D. - US grants

We propose a multifaceted approach to the detailed structure-function analysis of electron and proton transfer processes which lead to energy transduction in the Site I and Site III segments of the respiratory chain. For Site I, we will: (1) Extend our efforts to identify and characterize essential redox components using resolved subunit polypeptides of both hydrophilic and hydrophobic fractions of mammalian Complex I (in collaboration with Hatefi's and Ragan's laboratories); (2) Critically compare proton pump and local chemiosmotic loop models for Site I energy coupling based on a detailed analysis of the response of all relevant redox components to the applied membrane potential (Delta X) and pH gradient (Delta pH) under various conditions; (3) Examine spin-spin and/or redox interactions of neighboring intrinsic redox components of Complex I and intact mitochondrial membrane preparations; (4) Conduct comparative studies of the Site I energy transduction mechanism using bacterial systems (Paracoccus denitrificans and Escherichia coli). The former contains redox components and energy coupling devices very similar to the mammalian system, but simpler in subunit structure, while E. coli seems to have simpler redox components with or without energy coupling at Site I. For Site III, we will: (1) Unravel the complexity of the molecular mechanism of cytochrome c oxidase utilizing functionally active but structurally perturbed double mutants (revertants) of subunit I, II, or III (in collaboration with Tzagoloff's and Slonimski's laboratories), and determine EPR, optical, thermodynamic, and kinetic parameters of individual redox centers of cytochrome c oxidase; (2) Examine spin-spin and/or redox interactions among the electron carriers and correlate them with their spatial organization relative to the neighboring redox components and to the inner mitochondrial membrane using continuous wave saturation and pulse EPR techniques.

Dr. Ohnishi is continuing to explore the relationship between the structure and function of the redox components in biological electron and proton transfer processes. Standard and advanced EPR techniques will be used in conjunction with state-of-the-art genetic engineering. These multidisciplinary studies are expected to enhanced the understanding of the molecular mechanism of the energy transduction in the Site II region of the respiratory chain. During the past few years a wealth of new information has been obtained from the study of mitochondria, photosynthetic bacteria and chloroplasts cytochrome bc type complexes leading to the consensus that all of these complexes function by some type of quinone cycle mechanism. However, the detailed molecular mechanism of proton and electron transfer is still not completely understood.***//

The ultimate goal of the project is to elucidate the structure- function relationship of the Site I and Site III energy coupling. We have three major long term objectives, namely, (A) To test the hypothesis that the mechanism of electron transfer in the Site I is of a cyclic type. This hypothesis enables us to explain (a) the extreme complexity of redox components and protein subunit structure, (b) a high (four to five) stoichiometry of H+/2e- (c) the extremely low midpotential of the cluster N-la. For this goal, we will do the following: (i) study the physico- chemical properties of the H+-carriers, (ii) investigate new inhibitors which are different from conventional rotenone-type compounds, (iii) measure the spatial organization of the redox components in the membrane using continuous wave and pulsed EPR techniques at cryogenic temperature, and (iv) study simpler bacterial systems, such as Paracoccus denitrificans and Thermus thermophilus HB-8. (B) To apply our experience in the study of Site I to clinical problems. Certain diseases are caused by a genetic defect in the iron sulfur proteins in the Site I. We have demonstrated that this defect can be clearly identified by EPR measurement on pathological specimens. We will continue this line of study. (C) To elucidate the structure function relationship of Site III respiration. Since we pioneered the measurement of spin-spin interaction between cyt.a3-NO and other paramagnetic species in bovine heart cytochrome oxidase, we will complete the study by conducting the following experiments: (i) definitive measurement of inter-cluster distance in bovine heart cytochrome oxidase, (ii) direct measurement of T1 and T2 by pulsed EPR with careful resolution of redox poise of cyt.a and CuA, (iii) use of yeast strain whose cyt. c oxidase protein and chromophore structure are perturbed by double mutation. Taking full advantage of studying microorganism, we will study spectral and thermodynamic characteristics of individual redox components and analysis of the redox and spin-spin interactions among redox centers. These studies are expected to open a new horizon in our understanding of the electron and proton transfer process in Site I and III on the molecular level.

We propose to study the mechanism of electron and proton transfer pathways in mitochondrial NADH-quinone oxidoreductase (often referred to as Complex I). this complex plays a central role in the oxidation of NADH, the reducing product of cellular metabolism, by the respiratory chain. Complex I is the most complicated and least understood energy transducing proton-motive enzyme of the respiratory chain. the proposed research takes new directions in the investigation of the mechanism, kinetics, and regulatory properties of this complex in both the isolated and membrane-bound states. The specific studies will include: [I] determination of the minimal structure of Complex I capable of catalyzing NADH oxidation which is coupled with vectorial transfer of protons across the membrane; [II] determination of the internal electron transfer sequence within intrinsic redox components of NADH-quinone oxidoreductase by utilizing artificial electron acceptors (transition metal complexes) with proper redox potentials and appropriate other parameters (hydrophilicity, charge, size); [III] physico-chemical studies on the ubisemiquinones associated with the specific binding sites in Complex I which was recently discovered in Russian Co-P.I.'s laboratory; [IV] studies of molecular events involved in the slow active/inactive transition of Complex I, rediscovered recently by the Russian Co-P.I., which will be extended to searches of physiologically relevant factors which are involved in the control of hysteretic behavior of this complex. The P.I. has extensive experience with EPR studies and with thermodynamic analysis of the intrinsic paramagnetic redox centers of mitochondrial and microbial systems. Specifically, the P.I. has made significant contribution in the characterization of iron-sulfur clusters, flavin and ubiquinone free radicals. The Russian Co-P.I. is an established biochemist and has recently developed unique submitochondrial particle preparations having tightly coupled Site I energy transduction. The collaborative program between these investigators, therefore, is expected to greatly enhance the understanding of the mechanism of energy coupling and its regulation.

9418694 Ohnishi This research involves studies on the molecular mechanism of electron and proton transfer processes in two segments of the respiratory chain, (1) the quinol-cytochrome c oxidoreductase, and (2) succinate-quinone oxidoreductase, with a particular emphasis on their structure-function relationships. 1 Quinol-cytochrome c oxidoreductase: The Rieske iron-sulfur protein plays an essential role in quinol oxidation at the Qo site of quinol-cyt. c oxidoreductase. We plan to delineate amino acid residues that are involved in quinone binding at the Rieske domain of the Qo pocket. We also plan to investigate quinone interactions with its surroundings via hydrogen bonds and the weak interaction with the aqueous medium by 17O-quinone EPR and 1H- and 2H-ENDOR techniques (with Dr. G. Babcock) combined with site directed mutagenesis. 2 Succinate-quinone oxidoreductase: We will investigate the molecular mechanism of quinone binding and protein architecture of the Qs domain utilizing three different bacterial systems, Escherichia coli and Bacillus subtilis succinate-quinone reductase (with Drs R. Gennis and L. Hederstedt, respectively) and E. coli quinol-fumarate reductase (with Dr. G. Cecchini). We will use advanced EPR, ENDOR and genetic engineering techniques. The structural implication of these findings will be studied using site-directed mutagenesis in combination with various spectroscopic methods including EPR and 57Fe-ENDOR (in collaboration with Dr. B. Hoffman's group). A soluble 7 kDa Streptomyces griseolus ferredoxin bearing a single trinuclear iron-sulfur cluster will be analyzed as a model system for a cluster which is not readily converted to a 4Fe-4S cluster. %%% This research covers studies on the molecular mechanism of electron and proton transfer in two segments of the respiratory chain, the quinol-cytochrome c oxidoreductase, and succinate-quinone oxidoreductase, with particular emphasis on their structure-function relationships. The strength of this research is the com bination of molecular biological approaches with biochemical and biophysical techniques. The experimental outcome will provide detailed insight on the energy coupling mechanism at two crucial proton and electron interaction sites and on a non-energy coupling site. ***

DESCRIPTION: NADH-quinone oxidoreductase (Complex I) is the largest, most elaborate and least understood of the energy transducing enzymes. It has a uniquely high proton/electron stoichiometry (4-5 H+/2e-). Since Complex I encoding genes comprise more than half of the protein-encoding genes in mitochondrial DNA (7 out of 13), a majority of mitochondrial genetic diseases are associated with dysfunction of Complex I. Thus, the study of Complex I is of great medical importance. This research project places particular emphasis on the study of (I) the structure-function relationship in Complex I, combining state-of-the-art molecular genetic technology and cryogenic EPR spectroscopy, and (II) Complex I-related mitochondrial diseases. The P.I.'s group has shown that, contrary to a long-held view, Complex I consists of a large hydrophilic arm protruding out from the membrane (electron injector containing one flavin and several iron-sulfur clusters) and a hydrophobic part within the membrane (proton-pumping reactor containing three distinct quinone-binding sites). The P.I.'s group proposes a new quinone-gated proton-pump model for the energy coupling mechanism in Complex I. The following experimental strategies will be employed: (1) Determination of the spatial locations of cluster N2 and of quinone-binding sites; (2) further characterization of semiquinone-involving reactions using specific inhibitors, ionophores, and uncouplers; (3) identification of three different quinone-binding sites by isolation and characterization of inhibitor-resistant mutants: (4) targeting of the sequence motifs, which are in common with proton channel containing polypeptides by site-directed mutagenesis; (5) studies of Complex I-related diseases, such as Leber's Hereditary Optic Neuropathy and Parkinson's disease, using patient derived mitochondria propagated in cultured cells. The P.I. will collaborate on this project with a multi-disciplinary research team, consisting of renowned experts of molecular biology, mitochondrial disease, biochemistry and biophysics.

Discussion of how one can clearly differentiate 2 and 4 iron centers in expressed

The NADH-quinone oxidoreductase (complex I) is the largest (M.W. = approximately 1 mega-daltons) and most complicated (43 subunits) energy-transducing system in mitochondria. Many mitochondria-linked genetic diseases have been discovered, and the majority of them originate from a complex I defect(s). The elucidation of the structure-function relationship of complex I is vital not only for the study of bioenergetics, but also for the understanding of the nature of these diseases, in order to develop therapies. Based upon our previous findings, we will extend our studies in the following directions: (1) The NADH-binding site, one FMN molecule, and a majority of iron-sulfur clusters with low midpoint potential are localized in the hydrophilic promontory domain of complex I. In contrast, the iron-sulfur cluster N2 (which has the highest midpoint redox potential) and three distinct ubisemiquinone species are located within the membrane domain. We hypothesized that cluster N2 and these semiquinones play key roles in the proton and electron transfer in complex I. We found that cluster N2 resides in either of TYKY or PSST subunits. Both subunits are at least partially buried within the membrane. Determining the subunit location and ligand structure of cluster N2 has been one of the most important yet difficult tasks in complex I study. Recently, we have developed systems with much simpler bacterial complex I counterparts in which these two candidate subunits can be separated. Using these systems, we will identify which subunit harbors cluster N2. Furthermore, we will study the unique functions of cluster N2 employing site-directed mutagenesis techniques. (2) The subunit PSST (not TYKY) contains a specific and tight binding site for various complex I inhibitors. We have discovered that the distinct ubisemiquinone species respond differently to these inhibitors. Using vari9us inhibitors with different specificity, we will study the functional roles of both cluster N2 and the three quinone species in the energy-coupling mechanism in complex I. (3) We have found that the complex I counterpart in Thermus thermophilus has extreme thermo-stability and that its purified subunits are very stable. We will use this bacterium for crystallization and X-ray crystallographic studies. (4) We will determine physicochemical properties and spatial organization of all important redox components by combining state-of-the-art molecular genetic technology with sophisticated physical techniques such as EPR, ENDOR, ESEEM, and cyclic voltammetry as collaborative efforts. (5) We have developed an exciting bacterial model system, which allows us to study mechanisms of mitochondria-linked diseases by making clinically significant point mutations.