Abstract
To understand the biochemical basis for the function of the single subunit 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 to 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 the same concentration of AC0-11, the inhibitory mode of AC0-11 on Q-bound Ndi1 was distinct from that of Q-free Ndi1. When Ndi1 was incorporated into bovine heart submitochondrial particles (SMP), 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 SMP. The data suggest that the bound Q site is different from a catalytic site for Q and that Ndi1 in mitochondria bears the bound Q. Now, we are trying to identify the Q binding sites by site-directed mutagenesis technique. [J Physiol Sci. 2007;57 Suppl:S123]
