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Probing the mechanistic role of the long α‐helix in subunit L of respiratory Complex I from Escherichia coli by site‐directed mutagenesis
Author(s) -
Belevich Galina,
Knuuti Juho,
Verkhovsky Michael I.,
Wikström Mårten,
Verkhovskaya Marina
Publication year - 2011
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2011.07883.x
Subject(s) - biology , helix (gastropod) , mutagenesis , protein subunit , amphiphile , biophysics , amino acid , escherichia coli , biochemistry , mutation , stereochemistry , gene , chemistry , ecology , organic chemistry , snail , copolymer , polymer
Summary 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.