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.