Engineering the respiratory complex I to energy-converting NADPH:ubiquinone oxidoreductase

Abstract

The respiratory complex I couples the electron transfer from NADH to ubiquinone with a translocation of protons across the membrane. Its nucleotide-binding site is made up of a unique Rossmann fold to accommodate the binding of the substrate NADH and of the primary electron acceptor flavin mononucleotide. Binding of NADH includes interactions of the hydroxyl groups of the adenosine ribose with a conserved glutamic acid residue. Structural analysis revealed that due to steric hindrance and electrostatic repulsion, this residue most likely prevents the binding of NADPH, which is a poor substrate of the complex. We produced several variants with mutations at this position exhibiting up to 200-fold enhanced catalytic efficiency with NADPH. The reaction of the variants with NAD(P)H is coupled with proton translocation in an inhibitor-sensitive manner. Thus, we have created an energy-converting NADPH:ubiquinone oxidoreductase, an activity so far not found in nature. Remarkably, the oxidation of NAD(P)H by the variants leads to an enhanced production of reactive oxygen species.

FIGURE 1.

Structure of the nucleotide-binding site of complex I with bound NADH. The protein is shown in blue, iron in red, sulfur in yellow, the FMN in yellow, and the bound NADH in magenta. The radius of the sulfur ion was set to 1.7 Å and that of the iron ions to 0.7 Å (48). The length of the hydrogen bonds between Glu-183^F and the adenosine ribose hydroxyl groups are given in Å. The picture was drawn with Chimera (49) using the Protein Data Bank code 3IAM (13).

FIGURE 2.

Reduction of the Fe/S clusters of complex I and the E183H^F variant by nucleotides. Complex I was reduced with a 1,000-fold molar excess of both NADH (trace a) and NADPH (trace b). The E183H^F variant was reduced with a 1,000-fold molar excess NADPH (trace c). The spectra in A were recorded at 40 K and 2 milliwatts of microwave power, and the spectra in B were recorded at 13 K and 5 milliwatts of microwave power. Other EPR conditions were as follows: microwave frequency, 9.44 GHz; modulation amplitude, 0.6 mT; time constant: 0.124 s; scan rate: 17.9 mT/min.

FIGURE 3.

Generation of a proton gradient by complex I and the E183H^F variant. The proteins were reconstituted in proteoliposomes, and the ACMA fluorescence was monitored (28). The reaction of complex I (a) and the E183H^F variant (c) was started by adding either NADH (black curve) or NADPH (red curve) as indicated. The addition of NADPH to proteoliposomes containing complex I did not lead to the generation of a detectable proton gradient. As a control, either CCCP or piericidin A was added to the complex (b) before the reaction was started with the addition of NADH and to the variant (d) before the reaction was started with either NADH or NADPH.

FIGURE 4.

pH dependence of the NADH:decyl-ubiquinone (A) and NADPH:decyl-ubiquinone (B) oxidoreductase activity of complex I (squares) and the E183H^F variant (circles). The activity was measured between pH 5.0 and 8.0. All activities were more than 95% sensitive to the addition of 10 μm piericidin A. The maximum activity of the NADH and NADPH oxidation was 2.8 and 0.6 μm·min⁻¹·mg⁻¹ for complex I, respectively, and 4.0 and 1.3 μm·min⁻¹·mg⁻¹ for the E183H^F variant. The other variants showed a similar pH activity profile with a maximum NADH oxidation rate between pH 6.0. and 7.0 and a maximum NADPH oxidation rate between 6.0 and 6.5.

FIGURE 5.

NAD(P)H-induced superoxide production by complex I (A) and the E183H^F variant (B). The spectra were recorded with aliquots of the proteins reconstituted with phospholipids in the presence of 100 mm DEPMPO and 100 μm decyl-ubiquinone (trace a), plus 1 mm NADH (trace b), plus 1 mm NADPH (trace c), plus 1 mm NADPH and superoxide dismutase (100 units/ml) (trace d), plus 1 mm NADPH but without decyl-ubiquinone (trace e), and plus 1 mm NADPH and 20 μm piericidin A (trace f). Other EPR conditions were as follows: microwave frequency, 9.65 GHz; modulation amplitude, 0.1 mT; time constant: 0.164 s; scan rate: 5.4 mT/min.