Respiratory complex I with charge symmetry in the membrane arm pumps protons

Abstract

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H + /e − stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.

Membrane-domain mutations in respiratory complex I impede catalysis but do not uncouple proton pumping from ubiquinone reduction

“…First, the D648N Nqo12 variant was reported to pump three protons per NADH oxidized ( 32 ). However, the NADH:UQ oxidoreductase activities of both the wild-type and variant enzymes following reconstitution into proteoliposomes were extremely low (∼1.6 μmol min –1 mg –1 , compared to 35.0 ± 0.2 μmol min –1 mg –1 for P. denitrificans complex I (Figure S3), and 32.8 μmol min –1 mg –1 reported recently for wild-type E. coli complex I in detergent ( 46 )). Furthermore, the decreased stoichiometry was determined by single-point comparisons of ACMA (9-amino-6-chloro-2-methoxyacridine) fluorescence quenching (which provides an indirect and semiquantitative measure of ΔpH across a liposomal membrane).…”

“…Here, we show unambiguously that mutations of all three residues (D648N Nqo12 , H320L, and H346Q) pump four protons per NADH oxidized, and we therefore challenge the conclusions of decreased stoichiometries for the matching E. coli variants ( 32 , 27 ). Recently, a decreased proton stoichiometry for the E405K variant in E. coli ND4/Nqo13 has also been proposed on the basis of ACMA measurements on enzymes catalyzing at extremely low rates (∼0.2 μmol min –1 mg –1 ) in proteoliposomes ( 46 ). Although we have not studied the E405K variant here, we further question the validity of this conclusion, based on such low rates of turnover.…”

“…Our results confirm, like those of earlier studies ( 49 , 50 ), that the Glu-Lys ion pair and terminal protonatable residue (E141, K232, and E405 in Nqo13/ND4) are essential for catalysis, consistent with both mechanisms. Recent data showing that the E. coli E405K variant retains ∼80% NADH:UQ oxidoreductase activity suggests flexibility in the identity and charge of the terminal residue, with both the ND4-Glu and ND2/ND5-Lys catalyzing effectively in this position in ND4/Nqo13 ( 46 ). A substantial decrease in NADH:UQ oxidoreductase activity was also observed here for the K263Q variant (Figure 2 ), supporting its central role, although the remaining activity (18% of wild type) was not sufficient for stoichiometry measurements.…”

Horizontal proton transfer across the antiporter-like subunits in mitochondrial respiratory complex I

“…The putative proton transfer pathways in antiporter-like subunits have been investigated by structural biology techniques, site-directed mutagenesis and computational approaches. [3][4][5][6][7][8][9][10][11][12][13][14][15][16] It has been proposed that each of the three antiporter-like subunits pick protons from the N side and release to the P side as a response to redox activity in the quinone binding tunnel. Proton transfer from the N side is assumed to rst occur to a conserved Lys residue in transmembrane helix (TMH) 8 (Lys241 in ND2), located in the central part of the subunit (all amino acid numbering corresponds to complex I from Yarrowia lipolytica, unless otherwise stated).…”

“…Proton transfer from the N side is assumed to rst occur to a conserved Lys residue in transmembrane helix (TMH) 8 (Lys241 in ND2), located in the central part of the subunit (all amino acid numbering corresponds to complex I from Yarrowia lipolytica, unless otherwise stated). Subsequent proton transfer from this to another conserved lysine in ND2 (Lys383 or a glutamate in ND4 but with function shared 13 ), and putatively to the P side is driven by the dynamics and/or protonation reactions of conserved Lys/Glu pair from TMH 7/5. 1,15,16 However, how this is achieved at a molecular level remains elusive and debated.…”