Kinetics and regulation of mammalian NADH-ubiquinone oxidoreductase (Complex I)

Abstract

NADH-ubiquinone oxidoreductase (Complex I, European Commission No. 1.6.5.3) is one of the respiratory complexes that generate the proton-motive force required for the synthesis of ATP in mitochondria. The catalytic mechanism of Complex I has not been well understood, due to the complicated structure of this enzyme. Here, we develop a kinetic model for Complex I that accounts for electron transfer from NADH to ubiquinone through protein-bound prosthetic groups, which is coupled to the translocation of protons across the inner mitochondrial membrane. The model is derived based on the tri-bi enzyme mechanism combined with a simple model of the conformational changes associated with proton transport. To study the catalytic mechanism, parameter values are estimated by analyzing kinetic data. The model is further validated by independent data sets from additional experiments, effectively explaining the effect of pH on enzyme activity. Results imply that matrix pH significantly affects the enzyme turnover processes. The overall kinetic analysis demonstrates a hybrid ping-pong rapid-equilibrium random bi-bi mechanism, consolidating the characteristics from previously reported kinetic mechanisms and data.

Figure 1

(A) Schematic representation of the mechanism of Complex I. The NADH²⁻ dehydrogenase module (I) accepts electrons from NADH²⁻ and transfers them to the hydrogenase module (II) via the flavin (FMN) and the iron-sulfur clusters (N1–N5). Further transfer to N2 and finally to ubiquinone (Q) is linked with a redox-driven proton translocation (black arrows). The conformational changes of the proton transporter module (III), which transfer protons from the matrix to the cytosol, are mediated by the overall electron transfer by a yet unknown coupling mechanism. (B) Kinetic scheme of the tri-bi enzyme mechanism in combination with the mechanism of conformational changes. The forward reaction is read in the clockwise direction. The enzyme has three binding sides: site 1 binds to the proton (triangles), site 2 binds to substrate A or the corresponding product P (circles), and site 3 binds to substrate B or the corresponding product Q (squares). It is assumed that the mechanism involves conformational changes that are accompanied by phenomenological transfer of the H₂ moiety from sites 1 and 2 to site 3.

Figure 2

(A) Complex I activity as a function of NADH²⁻ concentration at different Q₁ concentrations in the absence of products. Kinetic data were obtained from Fig. 4 of Nakashima et al. (8). The Q₁concentrations in the assay were 50 μM (♦), 25 μM (▪), and 10 μM (•). (B) Complex I activity as a function of NADH²⁻ concentration at different NAD⁻ concentrations. Kinetic data were obtained from Fig. 5 A of Nakashima et al. (8). The NAD⁻ concentrations were 0μM(▴), 20 μM (♦), 80 μM (▪), and 200 μM (•) in the assay. Q₁ concentration was fixed to at 25 μM in these assays. Solid lines are results of model fitting to the data points represented by symbols based on the optimization of kinetic parameters (Table 1). Dashed lines are results of model fitting based on the optimization of rate constants and dissociation constants (Table 1).

Figure 3

(A) Complex I activity as a function of Q₁ concentration at different NAD⁻ concentrations. Kinetic data were obtained from Fig. 5 B of Nakashima et al. (8). The NAD⁻ concentrations in the assay were 0 μM (▴), 100 μM (♦), 200 μM (▪), and 400 μM (•). NADH²⁻ concentration was fixed at 6 μM in these assays. (B) Complex I activity as a function of Q₁ at different Q₁H₂ concentrations. Kinetic data were obtained from Fig. 5 C of Nakashima et al. (8). Q₁H₂ concentrations in the assay were 0 μM (♦), 25 μM (▪), and 75 μM (•). NADH²⁻ concentration was fixed at 6 μM in these assays. Solid lines are results of model fitting to the data points represented by symbols based on the optimization of kinetic parameters (Table 1). Dashed lines are results of model fitting based on the optimization of rate constants and dissociation constants (Table 1).

Figure 4

(A) Complex I activity as a function of NAD⁻ concentration at pH 9.0. Kinetic data were obtained from Fig. 1 (upper left) of Hano et al. (9). Symbols •, ▪, ♦, and ▴ represent data obtained in the presence of 25 μM, 50 μM, 100 μM, and 150 μM DQ in the assay. No products were present in the assay. The solid, dash-dotted, dashed, and dotted lines are results of model fitting for the data points represented by symbols •, ▪, ♦, and ▴, respectively. (B) Complex I activity as a function of NAD⁻ concentration at pH 6.5. Kinetic data were obtained from Fig. 1 (upper right) of Hano et al. (9). Symbols •, ▪, ♦, and ▴ represent data obtained in the presence of 25 μM, 50 μM, 100 μM, and 150 μM DQ in the assay. No products were present in the assay. Solid, dash-dotted, dashed, and dotted lines are results of model fitting for the data points represented by symbols •, ▪, ♦, and ▴, respectively.

Figure 5

Effect of pH on the activity of Complex I from rat hearts. The measured flux as a function of pH was obtained from Fig. 2 A of Sadek et al. (10). The reaction medium contains 40 μM Q₁ and 40 μM NADH²⁻. The solid line is the result of model fitting to the data point represented by the solid circle.

Figure 6

Model prediction of Complex I activity as a function of Q₁ concentration at different NADH²⁻ concentrations. Symbols ▪, ♦, ▴, and • represent Complex I activity measured in the presence of 4.27 μM, 7.13 μM, 14.19 μM, and 75 μM NADH²⁻ in the assay obtained from Fig. 2 of Fato et al. (7). The solid, dashed, dash-dotted, and dotted lines are model predictions to the data points represented by the symbols •, ▪, ♦, and ▴, respectively.