Asp563 of the horizontal helix of subunit NuoL is involved in proton translocation by the respiratory complex I

► The respiratory complex I contains an unusual, 'horizontal' helix on subunit NuoL. ► We mutated residues of the helix to investigate their role in proton translocation. ► The variants D563XL (X = E, N, Q and A) showed a reduced H+/e− stoichiometry. ► D563L is involved in proton transfer to the membranous translocation site.