Scientific Report 2007

Molecular and Experimental Medicine

Division Of Biochemistry

NADH Dehydrogenases

T. Yagi, A. Matsuno-Yagi, B.B. Seo, E. Nakamaru-Ogiso, T. Yamashita, M. Marella, J. Barber-Singh, J. Torres-Bacete, P.K. Sinha

Structure and Function of NADH Dehydrogenases

The NADH-quinone (NADH-Q) oxidoreductases of the mitochondrial respiratory chain can be divided into 2 groups: the proton-translocating NADH-Q oxidoreductase (complex I) and the NADH-Q oxidoreductase lacking an energy coupling site (NDH-2). Mammalian complex I is composed of 45 unlike subunits, whereas NDH-2 is composed of a single polypeptide.

In one of our current projects, we are identifying the subunits that make up an inhibitor-binding pocket. We synthesized a photoreactive derivative of acetogenin, a complex I inhibitor, and found that the derivative specifically labeled subunit ND1, which is encoded by mitochondrial DNA.

In another project, to understand the biochemical basis for the function of yeast NDH-2 (Ndi1), we overexpressed Ndi1 in Escherichia coli. The Ndi1 purified from the membranes contained 1 FAD and had enzymatic activities comparable to those of original Ndi1. When extracted with detergent, isolated Ndi1 did not contain quinone. Reconstitution of the enzyme with quinone yielded a quinone-bound form. Quinone-bound Ndi1 had higher activity than did quinone-free Ndi1. Although both bound and free forms were inhibited by AC0-11, a quinone analog, the inhibitory mode for quinone-bound Ndi1 was distinct from that for quinone-free Ndi1.

The bound quinone was slowly released from Ndi1 by treatment with NADH or dithionite under anaerobic conditions. This release of quinone was prevented when Ndi1 was kept in the reduced state by NADH. When Ndi1 was incorporated into bovine submitochondrial particles, the quinone-bound form established the NADH-linked respiratory activity, which was insensitive to piericidin A but inhibited by potassium cyanate. Furthermore, Ndi1 produces hydrogen peroxide when isolated regardless of the presence of bound quinone, and this hydrogen peroxide was eliminated when the quinone-bound Ndi1 was incorporated into submitochondrial particles. The data suggest that Ndi1 bears at least 2 distinct quinone sites: one for bound quinone and the other for catalytic quinone (Fig. 1).

Fig. 1. A speculative mechanism of Ndi1. The reaction mechanism of Ndi1 is thought to follow ordered ping-pong. First, NADH binds to the enzyme, reduces FAD, and leaves as NAD+ (left). When the enzyme is in the reduced state (center), quinone (Q) can bind to the catalytic site, accepts electrons from FADH2, and is released as QH2 (right).

Molecular Remedy of Complex I Defects

Defects in complex I are involved in many human diseases. However, no remedies for the defects have been established. We have adopted a gene therapy approach that involves use of the gene NDI1, which encodes Ndi1, the yeast single polypeptide NADH dehydrogenase.

Recently, using rat dopaminergic cell lines, we investigated the protective effects of NDI1 against the generation of reactive oxygen species (ROS) by inhibitors of complex I. Incubation of nontransduced control cells with rotenone elicited oxidative damage to mitochondrial DNA as well as lipid peroxidation. In contrast, oxidative stress was significantly decreased when the cells were transduced with NDI1. Furthermore, mitochondria from the NDI1-transduced cells had a suppressed rate of formation of ROS by the complex I inhibitors (Fig. 2). We conclude that Ndi1 can suppress overproduction of ROS from defective complex I.

Fig. 2. Oxidative DNA damage by rotenone (Rot) and its prevention by Ndi1. In non–NDI1-transduced control cells, inhibition of complex I by rotenone or inhibition of complex III by antimycin (AntiA) triggers generation of ROS, resulting in oxidative modification of either mitochondrial or nuclear DNA or both as revealed by 8-oxo-deoxyguanine (8-oxo-dG) immunostaining. In NDI1-transduced cells, the DNA damage associated with inhibition of complex I, but not that associated with inhibition of complex III, was greatly reduced, indicating a protective effect of Ndi1. Mitochondria (Mito) were visualized by using MitoTracker.

Administration of 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) to mice and nonhuman primates causes a parkinsonian disorder. MPTP has been proposed to exert its neurotoxic effects through a variety of mechanisms, including inhibition of complex I, displacement of dopamine from vesicular stores, and formation of ROS from mitochondrial or cytosolic sources. However, the mechanism of MPTP-induced neurotoxic effects is still a matter of debate.

We used overexpression of Ndi1 in SK-N-MC cells and animals to determine the relative contribution of complex I inhibition in the toxic effects of MPTP. In cell culture, overexpression of Ndi1 abolished the toxic effects of 1-methyl-4-phenylpyridinium, the active metabolite of MPTP. Overexpression of Ndi1 through stereotactic administration of a viral vector harboring NDI1 into the substantia nigra protected mice against both neurochemical and behavioral deficits elicited by MPTP.

These data identify inhibition of complex I as a requirement for dopaminergic neurodegeneration and subsequent behavioral deficits induced by MPTP. Furthermore, combined with reports of a complex I deficit in patients with Parkinson's disease, our findings confirm the usefulness of MPTP in understanding the molecular mechanism that underlies neurodegeneration in Parkinson's disease.

Publications

Murai, M., Ishihara, A., Nishioka, T., Yagi, T., Miyoshi, H. The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I. Biochemistry 46:6409, 2007.

Richardson, J.R., Caudle, W.M., Guillot, T.S., Watson, J.L., Nakamaru-Ogiso, E., Seo, B.B., Sherer, T.B., Greenamyre, J.T., Yagi, T., Matsuno-Yagi, A., Miller, G.W. Obligatory role for complex I inhibition in the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Toxicol. Sci. 95:196, 2007.

Schnermann, M.J., Romero, F.A., Hwang, I., Nakamaru-Ogiso, E., Yagi, T., Boger, D.L. Total synthesis of piericidin A1 and B1 and key analogues. J. Am. Chem. Soc. 128:11799, 2006.

Seo, B.B., Marella, M., Yagi, T., Matsuno-Yagi, A. The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I. FEBS Lett. 580:6105, 2006.

Shere, T.B., Richardson, J.R., Testa, C.M., Seo, B.B., Panov, A.V., Yagi, T., Matsuno-Yagi, A., Miller, G.W., Greenamyre, J.T. Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson's disease.
J. Neurochem. 100:1469, 2007.

Yagi, T., Seo, B.B., Nakamaru-Ogiso, E., Marella, M., Barber-Singh, J., Yamashita, T., Matsuno-Yagi, A. Possibility of transkingdom gene therapy for complex I diseases. Biochim. Biophys. Acta 1757:708, 2006.

Yamashita, T., Nakamaru-Ogiso, E., Miyoshi, H., Matsuno-Yagi, A., Yagi, T. Roles of bound quinone in the single subunit NADH-quinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae. J. Biol. Chem. 282:6012, 2007.