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J. Biol. Chem., Vol. 282, Issue 9, 6012-6020, March 2, 2007

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Roles of Bound Quinone in the Single Subunit NADH-Quinone Oxidoreductase (Ndi1) from Saccharomyces cerevisiae^*

Tetsuo Yamashita¹, Eiko Nakamaru-Ogiso, Hideto Miyoshi, Akemi Matsuno-Yagi, and Takao Yagi²

From the Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 and the Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606 8502, Japan

To understand the biochemical basis for the function of the rotenone-insensitive internal NADH-quinone (Q) oxidoreductase (Ndi1), we have overexpressed mature Ndi1 in Escherichia coli membranes. The Ndi1 purified from the membranes contained one FAD and showed enzymatic activities comparable with the original Ndi1 isolated from Saccharomyces cerevisiae. When extracted with Triton X-100, the isolated Ndi1 did not contain Q. The Q-bound form was easily reconstituted by incubation of the Q-free Ndi1 enzyme with ubiquinone-6. We compared the properties of Q-bound Ndi1 enzyme with those of Q-free Ndi1 enzyme, with higher activity found in the Q-bound enzyme. Although both are inhibited by low concentrations of AC0–11 (IC₅₀ = 0.2 µM), the inhibitory mode of AC0–11 on Q-bound Ndi1 was distinct from that of Q-free Ndi1. The bound Q was slowly released from Ndi1 by treatment with NADH or dithionite under anaerobic conditions. This release of Q was prevented when Ndi1 was kept in the reduced state by NADH. When Ndi1 was incorporated into bovine heart submitochondrial particles, the Q-bound form, but not the Q-free form, established the NADH-linked respiratory activity, which was insensitive to piericidin A but inhibited by KCN. Furthermore, Ndi1 produces H₂O₂ as isolated regardless of the presence of bound Q, and this H₂O₂ was eliminated when the Q-bound Ndi1, but not the Q-free Ndi1, was incorporated into submitochondrial particles. The data suggest that Ndi1 bears at least two distinct Q sites: one for bound Q and the other for catalytic Q.

Received for publication, November 16, 2006 , and in revised form, December 18, 2006.

^* This work was supported by National Institutes of Health Grant R01GM033712 (to A. M.-Y. and T. Y.) and supported in part by the Sam & Rose Stein Endowment Fund. This is publication 18349-MEM from The Scripps Research Institute (La Jolla, CA). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Kagawa University School of Medicine, Kagawa, Japan.

² To whom correspondence should be addressed: MEM-256, Division of Biochemistry, Dept. of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037. Fax: 858-784-2054; E-mail: yagi{at}scripps.edu.

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