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. 2011 Mar 18;286(11):9287-97.
doi: 10.1074/jbc.M110.175547. Epub 2011 Jan 10.

Reaction mechanism of single subunit NADH-ubiquinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae: evidence for a ternary complex mechanism

Yu Yang  1 , Tetsuo Yamashita, Eiko Nakamaru-Ogiso, Takeshi Hashimoto, Masatoshi Murai, Junsuke Igarashi, Hideto Miyoshi, Nozomu Mori, Akemi Matsuno-Yagi, Takao Yagi, Hiroaki Kosaka
Affiliations

Reaction mechanism of single subunit NADH-ubiquinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae: evidence for a ternary complex mechanism

Yu Yang et al. J Biol Chem. .

Abstract

The flavoprotein rotenone-insensitive internal NADH-ubiquinone (UQ) oxidoreductase (Ndi1) is a member of the respiratory chain in Saccharomyces cerevisiae. We reported previously that bound UQ in Ndi1 plays a key role in preventing the generation of reactive oxygen species. Here, to elucidate this mechanism, we investigated biochemical properties of Ndi1 and its mutants in which highly conserved amino acid residues (presumably involved in NADH and/or UQ binding sites) were replaced. We found that wild-type Ndi1 formed a stable charge transfer (CT) complex (around 740 nm) with NADH, but not with NADPH, under anaerobic conditions. The intensity of the CT absorption band was significantly increased by the presence of bound UQ or externally added n-decylbenzoquinone. Interestingly, however, when Ndi1 was exposed to air, the CT band transiently reached the same maximum level regardless of the presence of UQ. This suggests that Ndi1 forms a ternary complex with NADH and UQ, but the role of UQ in withdrawing an electron can be substitutable with oxygen. Proteinase K digestion analysis showed that NADH (but not NADPH) binding induces conformational changes in Ndi1. The kinetic study of wild-type and mutant Ndi1 indicated that there is no overlap between NADH and UQ binding sites. Moreover, we found that the bound UQ can reversibly dissociate from Ndi1 and is thus replaceable with other quinones in the membrane. Taken together, unlike other NAD(P)H-UQ oxidoreductases, the Ndi1 reaction proceeds through a ternary complex (not a ping-pong) mechanism. The bound UQ keeps oxygen away from the reduced flavin.

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Figures

FIGURE 1.
FIGURE 1.
Effect of detergents on purity and content of bound UQ of Ndi1. A, the absorption spectra of Ndi1, which was purified using TX (5 μm; solid line), TX/DM (5 μm; dotted line), and DM (5 μm; dashed line). The spectra were measured on a Hitachi U-3310 spectrophotometer at 25 °C in a buffer containing 50 mm Mops-KOH (pH 7.0), 0.1 mm EDTA, 10% glycerol, 1 m NaCl, and 0.02% TX (for TX enzyme) or 0.02% DM (for TX/DM and DM enzymes). B, each sample (10 μg) was subjected to SDS-polyacrylamide gel (9%) electrophoresis. C, UQ8 content of each sample was calculated from the results of reverse-phase HPLC. * denotes an undetectable level of UQ8. Each value and vertical bar represent the mean ± S.E.
FIGURE 2.
FIGURE 2.
Effect of bound UQ on formation of CT complex in Ndi1. The absorption spectra of UQ-free (100 μm; A) and UQ6-reconstituted Ndi1 (about 1 mol of UQ6/mol of enzyme; 100 μm; B) were measured on an Olis DW2000 spectrophotometer at 25 °C in a buffer containing 50 mm Mops-KOH (pH 7.0), 0.1 mm EDTA, 10% glycerol, and 0.02% DM. The experiments were carried out using an anaerobic chamber (Coy Laboratory Products Inc.) The method of incorporation of UQ6 into the purified Ndi1 was described in Ref. . UQ-free Ndi1 (A) and UQ6-reconstituted Ndi1 (B) were reduced by the addition of 1 mm NADH (dotted line), and then 200 μm DBQ was added (dashed-dotted line). After the addition of DBQ, the cuvette was opened under aerobic conditions. The dashed line indicates the maximum height of the broad band. Inset, change of broad band over time after the addition of NADH under aerobic condition (in the absence of DBQ).
FIGURE 3.
FIGURE 3.
Effect of NADH binding on Ndi1 structure. A, Ndi1 (1 mg of protein/ml) was incubated with 1 mm NADH, 1 mm NAD+, 1 mm dithionite, 100 μm UQ1, or 5% EtOH in a buffer containing 50 mm Tris-HCl (pH 8.0), 1 mm EDTA, 1 mm CaCl2, 200 mm NaCl, and 10% glycerol and digested by proteinase K for 1–3 h under strict anaerobic conditions. The reaction was stopped by the addition of 2 mm PMSF. Then the samples were subjected to SDS-polyacrylamide gel (9%) electrophoresis. B, the intensities of each digested peptide band of non-treated (non) protein were compared with those of proteins that were treated under different conditions. The intensities of each peptide band were determined by NIH Image. The band numbers correspond to the number of digested peptides in A. *, p < 0.05; **, p < 0.01 compared with untreated protein. The p value was calculated by unpaired Student's t test. Each value and vertical bar represent the mean ± S.E. (n = 3).
FIGURE 4.
FIGURE 4.
Predicted UQ binding site of Ndi1. A, FAD (yellow and green) and nicotinamide-ribose (NADP+, blue; NAD+, white) of DmTR and PpLipDH are displayed as a stick model. B, the amino acid residues of DmTR and PpLipDH (in parentheses) surrounding FAD are displayed as a stick model colored in blue and white, respectively. C, amino acid sequence alignment from the group A family of NDH-2, DmTR, and PpLipDH. The GXGXXG consensus motifs are shown with a gray background. Residues marked with an asterisk indicate conserved residues among the NDH-2 enzymes, and those in bold are mutated residues in this study. ScNDI1, S. cerevisiae Ndi1 (NCBI accession number CAA89160); Ec, E. coli (NCBI accession number CAA23586.1); Av, Azotobacter vinelandii (NCBI accession number AAK19737.1); Tb, Trypanosoma brucei (NCBI accession number AAM95239).
FIGURE 5.
FIGURE 5.
Inhibitory effects of Reactive Blue-2 and ubiquinol on NADH-UQ1 reductase activity of Ndi1. A, structures of UQ1, UQ1H2, and several inhibitors for NQO1. B, NADH-UQ1 reductase activities were measured in the presence of increasing concentrations of RB-2 (closed circle). The NADH-UQ1 reductase activity was corrected for nonspecific inhibition using the value in the presence of 1 nm RB-2 as 100% activity (open circle). 100% activity was 900 μmol of NADH oxidized/min/mg of Ndi1. Each value and vertical bar represent the mean ± S.E.
FIGURE 6.
FIGURE 6.
Inhibition mode of RB-2 and UQ1HinfMACROS BELOW ARE FOR THE VISUAL2. Inhibition kinetics for NADH (A and C; 60 μm UQ1) and UQ1 (B and D; 100 μm NADH) were measured in the presence of RB-2 (A and B) and UQ1H2 (C and D). The concentrations of RB-2 and UQ1H2 are as indicated in the figure.
FIGURE 7.
FIGURE 7.
Elution profiles of quinones extracted from Ndi1 purified using TX/DM from control membrane (TX/DM-1) and UQ10-fused membrane (TX/DM-2) on reverse-phase HPLC. Both samples contained UQ6 as an internal standard. Authentic UQ6, UQ8, and UQ10 were used for the standard (STD). mAU, milliabsorbance units.
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