Locking loop movement in the ubiquinone pocket of complex I disengages the proton pumps

Abstract

Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the largest enzyme of the mitochondrial respiratory chain and a significant source of reactive oxygen species (ROS). We hypothesized that during energy conversion by complex I, electron transfer onto ubiquinone triggers the concerted rearrangement of three protein loops of subunits ND1, ND3, and 49-kDa thereby generating the power-stoke driving proton pumping. Here we show that fixing loop TMH1-2^ND3 to the nearby subunit PSST via a disulfide bridge introduced by site-directed mutagenesis reversibly disengages proton pumping without impairing ubiquinone reduction, inhibitor binding or the Active/Deactive transition. The X-ray structure of mutant complex I indicates that the disulfide bridge immobilizes but does not displace the tip of loop TMH1-2^ND3. We conclude that movement of loop TMH1-2^ND3 located at the ubiquinone-binding pocket is required to drive proton pumping corroborating one of the central predictions of our model for the mechanism of energy conversion by complex I proposed earlier.

Fig. 1

A disulfide cross-link is formed between subunits ND3 and PSST in mutant Q133C^PSST. Purified complex I from parental and mutant strains were incubated with either 5 mM dithiothreitol (DTT) or 0.1 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) for 5 min and then separated by non-reducing Tricine SDS-PAGE. a An additional band was observed in the sample from mutant Q133C^PSST treated with DTNB. b Mass spectrometric analysis of the corresponding gel slice confirmed the presence of cross-linked subunits PSST and ND3 and allowed label free quantification of their relative abundance as compared to the corresponding gel slice of the DTT treated sample (mean ± s.d.; n = 3 technical replicates; ***p < 0.001, ANOVA with Bonferroni correction). c Subunits PSST and ND3 were also identified by dSDS-PAGE, in which the cross-link was preserved in the first dimension (1D) and then reduced in the second dimension (2D) to separate both proteins at a shifted position in the mutant. Note that the spot containing subunit PSST does not disappear after cross-linking, because it also contains accessory subunits NUJM and NUPM

Fig. 2

Fixing loop TMH1-2^ND3 does not interfere with complex I activity and A/D transition. Mitochondrial membranes from parental and mutant strains were incubated with 5 mM dithiothreitol (DTT) or 0.1 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) for 5 min at room temperature. Complex I activities were measured in the absence and presence of 5 mM MgCl₂ (a) or 2 mM n-ethylmaleimide (NEM) (b). In all cases, 1 mM decylubiquinone (DBQ) was added to the samples before the reaction was started by the addition of DBQ activity buffer containing 110 µM dNADH and supplemented with either 5.5 mM MgCl₂ or 2.2 mM NEM. The final concentrations of DBQ and membranes were 100 µM and 50 µg ml^-1, respectively. n-Decyl-quinazoline-amine (DQA)-sensitive dNADH:DBQ oxidoreductase activities in Q133C^PSST normalized to their respective controls (parental) are indicated. Activities were measured at pH 8.5 since the inhibition of the D-form by divalent cations is stronger at alkaline pH values, as well as it favors the labeling of the exposed C40^ND3 by NEM. Data from three independent experiments (mean ± s.d.) are shown. *p < 0.05; ***p < 0.001; ****p < 0.0001, ANOVA with Bonferroni correction

Fig. 3

Blocking movement of loop TMH1-2^ND3 reversibly disengages the proton pumps. Proton pumping was monitored as 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quench in proteoliposomes containing either parental or mutant complex I treated with 5 mM dithiothreitol (DTT) (a, b) or 0.1 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) (c–f) for 5 min before starting the assay. As indicated, 0.5 µM ACMA, 70 µM decylubiquinone (DBQ). 100 µM NADH and 5 µM carbonylcyanide m-chlorophenylhydrazine (CCCP) were added during the assay. Addition of 5 mM DTT to DTNB treated proteoliposomes during the assay had no effect on parental complex I (e), but rapidly restored proton pumping of the mutant enzyme (f). Representative traces of one out of four datasets obtained with independent batches of proteoliposomes are shown

Fig. 4

The disulfide cross-link in mutant Q133C^PSST does not change proton permeability. Passive proton (H⁺) uptake was monitored as 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quench in proteoliposomes containing either parental (black lines) or mutant (gray lines) complex I treated with 5 mM dithiothreitol (DTT) (a, b) or 0.1 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) (c, d). In a, c 10 mM HCl were added to establish a transitory pH gradient. Proteoliposomes (5 µg ml⁻¹) were incubated for 5 min at room temperature in a reaction mixture containing 20 mM Mops pH 7.2 (Tris), 80 mM KCl and either 5 mM DTT or 0.1 mM DTNB. 0.5 µM ACMA and 0.5 µM valinomycin (VAL) were added immediately before recording the baseline. In b, d a K⁺ gradient was used to drive complex I independent H⁺ uptake. Proteoliposomes ([K⁺]_i = 80 mM) were incubated in 20 mM Mops pH 7.2 (Tris), 0.08 mM KCl, 160 mM sucrose and ΔΨ generation was induced by the addition of 0.5 µM valinomycin (VAL). A slow compensatory H⁺ uptake was observed. Finally, 0.05 mg ml⁻¹ alamethicin (ALA) was added to permeabilize the proteoliposomes and collapse the ion gradients. For the bottom traces shown in all panels, 5 µM carbonylcyanide m-chlorophenylhydrazine (CCCP) were added before the assay to dissipate the proton gradient. Note that neither complex I substrates nor inhibitors were used in these assays. Representative traces of one out of five technical replicates are shown

Fig. 5

Formation of the disulfide cross-link immobilizes loop TMH1-2^ND3 without displacing it. Section of the crystal structure of complex I from mutant Q133C^PSST showing the interface between membrane and peripheral arms in cartoon representation. The ND3 loop connecting TMH1 and TMH2 (light green) protrudes into the cavity formed by subunits PSST (blue), 49-kDa (blue-green), and ND1 (red). Residue C40^ND3 forms a disulfide bond with C133^PSST (stick representation). This was confirmed by a strong peak in the sulfur anomalous difference Fourier electron density map (yellow mesh) which was absent when analyzing wild-type complex I crystals. Loop TMH1-2^ND3 from the superimposed structure of wild-type complex I [2] is shown in orange