Probing the mechanistic role of the long α-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis.

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.

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