Roles of bound quinone in the single subunit NADH-quinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae

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