Quinone chemistry in respiratory complex I involves protonation of a conserved aspartic acid residue

Abstract

Respiratory complex I is a central metabolic enzyme coupling NADH oxidation and quinone reduction with proton translocation. Despite the knowledge of the structure of the complex, the coupling of both processes is not entirely understood. Here, we use a combination of site-directed mutagenesis, biochemical assays, and redox-induced FTIR spectroscopy to demonstrate that the quinone chemistry includes the protonation and deprotonation of a specific, conserved aspartic acid residue in the quinone binding site (D325 on subunit NuoCD in Escherichia coli). Our experimental data support a proposal derived from theoretical considerations that deprotonation of this residue is involved in triggering proton translocation in respiratory complex I.

Keywords: Escherichia coli; NADH dehydrogenase; NADH:quinone oxidoreductase; iron–sulfur cluster; proton‐coupled electron transfer; quinone reduction; redox‐induced FTIR spectroscopy; site‐directed mutagenesis.

Fig. 1

Structure of and electron transfer in complex I. (A) Structure of E. coli complex I (PDB: 7Z7S) with the subunits in gray [18]. The FMN and the Fe/S clusters are depicted in their standard atomic colors. The position of Q is in this structure is shown in red and green. (B) Scheme of the electron transfer in the peripheral arm of the complex from NADH (top) to the quinone (bottom). The amino acids mentioned in the text are depicted. (C) Arrangement of the amino acids in question in the Q binding site from T. thermophilus complex I (PDB: 4HEA). The Asp/His H‐bond is indicated by a dashed line and the distance is provided. The residues are shown in the T. thermophilus (purple) and in the E. coli (black) numbering. The position of the most distal Fe/S cluster N2 is shown at the top of the figure.

Fig. 2

Growth of the nuoCD mutant strains and stability of the variants. (A) Growth curves of the reference strain producing parental complex I (black) and the D325^CD mutant strains D325A^CD (red), D325E^CD (blue), D325N^CD (green), and D325Q^CD (violet) in minimal medium with acetate as the only carbon source. The arrow marks the induction of gene expression with 0.02% (w/v) L‐arabinose. (B) Sucrose gradients of detergent solubilized membranes from the parental strain (black) and the nuoCD mutant strains D325A^CD (red), D325E^CD (blue), D325N^CD (green) and D325Q^CD (violet). The NADH/ferricyanide oxidoreductase activity of each fraction is shown; the activities are normalized to 20 mg protein applied on each gradient.

Fig. 3

(A) Oxidized minus reduced FTIR difference spectra of the complex I and the D325A^CD variant at pH 6 in the −0.3/−0.1 V potential range. (B) Magnified view of the 1720–1670 cm⁻¹ spectral range.