Kinetics of the mitochondrial three-subunit NADH dehydrogenase interaction with hexammineruthenium(III)

Abstract

The steady-state kinetics of the NADH dehydrogenase activity of the three-subunit flavo-iron-sulfur protein (FP, Type II NADH dehydrogenase) in the presence of the one-electron acceptor hexammineruthenium(III) (HAR) were studied. The maximal catalytic activities of FP with HAR as electron acceptor calculated on the basis of FMN content were found to be approximately the same for the submitochondrial particles, Complex I and purified FP. This result shows that the protein structure responsible for the primary NADH oxidation by FP is not altered during the isolation procedure and the lower (compared with Complex I) catalytic capacity of the enzyme previously reported was due to the use of inefficient electron acceptors. Simple assay procedures for NADH dehydrogenase activity with HAR as the electron acceptor are described. The maximal activity at saturating concentrations of HAR was insensitive to added guanidine, whereas at fixed concentration of the electron acceptor, guanidine stimulated oxidation of low concentrations of NADH and inhibited the reaction at saturating NADH. The inhibitory effect of guanidine was competitive with HAR. The double-reciprocal plots 1/v vs. 1/[NADH] at various HAR concentrations gave a series of straight lines intercepting on the ordinate. The plots 1/v vs. 1/[HAR] at various NADH concentrations gave a series of straight lines intercepting in the fourth quadrant. The kinetics support the mechanism of the overall reaction where NADH is oxidized by the protein-Ru(NH3)3+(6) complex in which positively charged electron acceptor is bound at the specific site close to FMN, thus stabilizing the flavosemiquinone intermediate.

Redox-Dependent Change of Nucleotide Affinity to the Active Site of the Mammalian Complex I

“…It has been shown previously that NADH-OH rapidly reacts with the oxidants ferricyanide and 2,6-dichlorophenolindophenol (26). Figure 2 shows that this is also true for HAR, the most efficient electron acceptor for complex I (21,22). Brief preincubation of NADH-OH with 0.5 mM HAR (the concentration used for the assay of the NADH dehydrogenase activity of complex I) completely abolished the inhibitory effect ( Figure 2, curves 1 and 2).…”

“…For mechanistic models of the nucleotide substrate (product) interaction with the enzyme active site(s), only limited steadystate kinetic (18)(19)(20)(21)(22)(23)(24) and recent electrochemical (25) data are available. A very potent and specific inhibitor of the mitochondrial complex I, a derivative of NADH (tentatively identified as β-2,6-dihydroxydihydronicotinamide adenine dinucleotide), has been recently discovered (26).…”

Function of Conserved Acidic Residues in the PSST Homologue of Complex I (NADH:Ubiquinone Oxidoreductase) from Yarrowia lipolytica

“…Among the representative membrane preparations listed in Table I, only the membranes from mutants E89C and E89A that were prepared in parallel batches exhibited a 45% higher activity. As the nonphysiological dNADH:HAR activity only depends on a functional 51-kDa subunit (31), this demonstrated that the complex I content of the membranes was not dramatically altered by the mutations. The observed differences in HAR activity were obviously because of variations in growth conditions and quality of the membranes.…”

Assembly defects induce oxidative stress in inherited mitochondrial complex I deficiency

“…4C). Indeed, ROS production at the Q site should generate the formation of semiquinones (Ohnishi et al, 2010), while the FMN site involves a reduced, free Q4 flavin, a state favoured by high NADH to NAD+ ratio (Birrell and Hirst, 2013;Gavrikova et al, 1995;Kussmaul and Hirst, 2006). As DQ addition both increased the ratio of reduced (QH2) to oxidized quinone (Q) and reduces the ratio of NADH to NAD+, this should indicate the site involved in ROS formation between the FMN and quinone sites.…”

“…NADH Ubiquinone Reductase (NUR) activity was measured as described Medja et al, 2009). NADH dehydrogenase activity was measured by following the NADH oxidation at 340 nm with hexammineruthenium (HAR) as an electron acceptor (NADH:HAR activity) (Gavrikova et al, 1995). Assays were realized in a KH 2 PO 4 buffer (20 mM, pH 7.5) with 50 M NADH, 3.5 mM HAR, 4 g of solubilized OXPHOS proteins.…”