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NADH Dehydrogenases

E.K.L. Chan, S. Di Bernardo, T. Kitajima-Ihara, A. Matsuno-Yagi, Y. Miyoshi,* T. Ohnishi,** I.E. Scheffler,*** B.B. Seo, T. Yagi, T. Yano

* Kyoto University, Kyoto, Japan
** University of Pennsylvania, Philadelphia, PA
*** University of California, San Diego, CA

STRUCTURE AND FUNCTION OF PROTON-TRANSLOCATING NADH-QUINONE OXIDOREDUCTASE

The proton-translocating NADH-quinone oxidoreductase (complex I) of mammalian mitochondria is composed of at least 43 different subunits and probably has the most intricate structure of any known membrane-bound enzyme complex. Complex I contains 1 FMN and at least 5 iron-sulfur clusters, detectable by electron paramagnetic resonance, as cofactors.

Aerobically grown Paracoccus denitrificans, which is called "a free-living mitochondrion," expresses a mammalian mitochondrial-type respiratory chain. The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus membranes is quite similar to mammalian complex I in terms of electron carriers and amino acid sequences. However, in contrast to the mitochondrial enzyme, NDH-1 is composed of 14 unlike subunits, suggesting that the structure of Paracoccus NDH-1 is relatively simpler than that of its mitochondrial counterpart. Therefore, Paracoccus NDH-1 is a useful model system for studying the structure and function of complex I.

In previous studies, a gene cluster encoding the Paracoccus NDH-1 was cloned and sequenced. This cluster is composed of 14 structural genes (designated NQO1 to NQO14) and 6 unidentified reading frames. On the basis of the deduced primary structures of the Paracoccus NDH-1 subunits, subunits NQO1, 2, 3, 6, and 9 are expected to contain iron-sulfur clusters. To verify these assumptions, we expressed single Paracoccus NDH-1 subunits in Escherichia coli and characterized the iron-sulfur clusters.

Properties of the Paracoccus NQO9 subunit that is predicted to contain 2 [4Fe-4S] clusters were investigated by using heterologous and homologous expression and immunologic techniques. When the full-length NQO9 subunit was coexpressed with thioredoxin in E coli at ambient temperature, the subunit was found only in the membrane fraction. When relatively hydrophobic and less conserved N-terminal stretches (30 or 40 amino acid residues long) of the subunit were genetically deleted, the truncated NQO9 subunits were expressed as soluble forms in the cytoplasm. Although deletion of the C-terminal regions (13 or 23 amino acid residues long) was less effective, the NQO9 subunits in which both the N- and the C-terminal regions had been removed were more soluble. When the truncated forms of the subunit were expressed in Paracoccus with a broad-host-range plasmid, the products were expressed predominantly in the cytoplasm, similar to the expression in E coli. When His-tagged truncated forms of NQO9 subunits were purified from E coli by column chromatography, the products contained reducible iron-sulfur clusters. These results indicate that the Paracoccus NQO9 subunit contains the iron-sulfur clusters in its hydrophilic part.

Localization of the NQO9 subunit in the Paracoccus NDH-1 complex was investigated by using Paracoccus membranes and immunologic techniques. The subunit could be extracted from membranes by treatment with chaotropic reagents or with alkaline buffer. These results suggest that the subunit is anchored to the intrinsic membrane part of the enzyme complex similar to anchoring of the Rieske iron-sulfur subunit of the cytochrome c₁ complex. Stoichiometry of the NQO9 subunit relative to other subunits (NQO1 to NQO6) in the NDH-1 enzyme complex was determined by using immunologic techniques. Each of the NQO subunits 1--6 and 9 is present as a single molecule in the NDH-1 enzyme complex. These results provide an estimate of the total number of FMN and iron-sulfur clusters. Namely, the Paracoccus NDH-1 enzyme complex contains 1 molecule of FMN and up to 8 iron-sulfur clusters.

MOLECULAR REMEDY OF COMPLEX I DEFECTS

Research in recent years has shown that structural and functional defects of complex I are involved in many human diseases. These diseases include Leber's hereditary optic neuropathy, Parkinson's disease, dystonia, severe lactic acidosis, various encephalomyopathies, and possibly Huntington's disease. Dysfunction of complex I causes 3 problems: (1) impairment of the ability of the respiratory chain to oxidize NADH back to NAD; (2) impairment of the ability of complex I to pump protons, resulting in a decrease in ATP synthesis; and (3) production of superoxide radicals. Our overall goal is to find a remedy for the diseases caused by dysfunction of complex I.

Of the 3 problems, impairment of proton pumping by 1 of the 3 proton-translocation sites does not appear to be as severe a health hazard as the inability of mitochondria to oxidize NADH and damage by superoxide production. Yeast (Saccharomyces cerevisiae) mitochondria lack complex I and contain instead an NADH dehydrogenase composed of a single subunit: Ndi1. We intend to use yeast Ndi1 enzyme to transmit electrons from NADH to ubiquinone-10 in mammalian mitochondria that lack a functional complex I.

NDI1, the gene that encodes rotenone-insensitive internal NADH-quinone oxidoreductase of yeast mitochondria, was transfected to complex I--deficient Chinese hamster CCL16-B2 cells. Stable transfected cells were obtained by screening with antibiotic G418. NDI1 was expressed in the transfected cells. Immunoblotting and confocal immunofluorescence microscopic analyses indicated that the expressed Ndi1 enzyme was localized to mitochondria. Studies with digitonin-permeabilized cells indicated that the transfected cells, but not nontransfected control cells, had electron-transfer activities, with glutamate/malate as the respiratory substrates. The activities were inhibited by flavone, antimycin A, and potassium cyanate but not by rotenone. Added NADH did not serve as the substrate, suggesting that the expressed Ndi1 enzyme was located on the matrix side of the inner mitochondrial membranes.

Furthermore, whereas nontransfected cells could not survive in a medium low in glucose, which is a substrate of glycolysis, the NDI1-transfected cells grew in medium containing 0.6 mM glucose. When glycolysis is slow, either at low glucose concentrations or in the presence of galactose, respiration is required for cells to survive. The mutant cells did not survive at low glucose concentrations or in galactose, but they could be rescued by Ndi1. These results indicate that the yeast Ndi1 was functionally expressed in CCL16-B2 cells and that it catalyzed electron transfer from NADH in the matrix to ubiquinone-10 in the inner mitochondrial membranes. We conclude that NDI1 provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.

PUBLICATIONS

Kitajima-Ihara, T., Yagi, T. Rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria: The enzyme expressed in Escherichia coli acts as a member of the respiratory chain in the host cells. FEBS Lett. 421:37, 1998.

Ohnishi, T., Sled, V.D., Yano, T., Yagi, T., Burbaev, D.S., Vinogradov, A.D. Structure-function studies of iron-sulfur clusters and semiquinones in the NADH-Q oxidoreductase segment of the respiratory chain. Biochim. Biophys. Acta, in press.

Ohshima, M., Miyoshi, H., Sakamoto, K., Takegami, K., Iwata, J., Kuwabara, K., Iwamura, H., Yagi, T. Characterization of the ubiquinone reduction site of mitochondrial complex I using bulky synthetic ubiquinone. Biochemistry, in press.

Yagi, T., Yano, T., De Bernardo, S., Matsuno-Yagi, A. Procaryotic complex I (NDH-1). Biochim. Biophys. Acta, in press.