title: Probing the mechanistic role of the long α-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis.
abstract: The C-terminus of the NuoL subunit of Complex I includes a long amphipathic α-helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston-like function. First, NuoL was truncated at its C- and N-termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the amphipathic α-helix: Asp and Lys residues and a Pro were substituted for acidic, basic or neutral residues. All these variants exhibited almost a wild-type phenotype. Third, several substitutions and insertions were made to reduce rigidity of the amphipathic α-helix, and/or to change its geometry. Most insertions/substitutions resulted in a normal growth phenotype, albeit often with reduced stability of Complex I. In contrast, insertion of six to seven amino acids at a site of the long α-helix between NuoL and M resulted in substantial loss of proton pumping efficiency. The implications of these results for the proton pumping mechanism of Complex I are discussed.
authors: Helsinki Bioenergetics Group, Institute of Biotechnology, FIN-00014, University of Helsinki, Helsinki, Finland.
authors: Galina
authors: G
authors: Belevich
authors: Juho
authors: J
authors: Knuuti
authors: Michael I
authors: MI
authors: Verkhovsky
authors: Mårten
authors: M
authors: Wikström
authors: Marina
authors: M
authors: Verkhovskaya
mesh_terms: D019943:Amino Acid Substitution; D042967:Electron Transport Complex I / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D021122:Protein Subunits; D009245:NADH Dehydrogenase; C479743:NuoL protein, E coli; D042967:Electron Transport Complex I