Kinetic characterization of the rotenone-insensitive internal NADH: Ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae

Saccharomyces cerevisiae mitochondria contain an NADH:Q(6) oxidoreductase ( internal NADH dehydrogenase) encoded by NDI1 gene in chromosome XIII. This enzyme catalyzes the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. From a structural point o f view, the mature enzyme has a single subunit of 53 kDa with FAD as the on ly prosthetic group. Due to the fact that S. cerevisiae cells lack complex I, the expression of this protein is essential for cell growth under respir atory conditions. The results reported in this work show that the internal NADH dehydrogenase follows a ping-pong mechanism, with a K-m for NADH of 9. 4 muM and a K-m for oxidized 2,6-dichorophenolindophenol (DCPIP) of 6.2 muM . NAD(+), one of the products of the reaction, did not inhibit the enzyme w hile the other product, reduced DCPIP, inhibited the enzyme with a Ki of 11 .5 muM. Two dead-end inhibitors, AMP and flavone, were used to further char acterize the kinetic mechanism of the enzyme. AMP was a linear competitive inhibitor of NADH (K-i = 5.5 mM) and a linear uncompetitive inhibitor of ox idized DCPIP (K-i = 11.5 mM), in agreement with the ping-pong mechanism. On the other hand, flavone was a partial inhibitor displaying a hyperbolic un competitive inhibition regarding NADH, and a hyperbolic noncompetitive inhi bition with respect to oxidized DCPIP. The apparent intercept inhibition co nstant (K-ii = 5.4 muM) and the slope inhibition constant (K-is = 7.1 muM) were obtained by non linear regression analysis. The results indicate that the ternary complex F-DCPIPox-flavone catalyzes the reduction of DCPIP, alt hough with lower efficiency. The effect of pH on V-max was studied. The V-m ax profile shows two groups with pK(a) values of 5.3 and 7.2 involved in th e catalytic process. (C) 2001 Academic Press.