Energy Transducing Roles of Antiporter-like Subunits in Escherichia coli NDH-1 with Main Focus on Subunit NuoN (ND2)

摘要

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) contains a peripheral and a membrane domain. Three antiporter-like subunits in the membrane domain, NuoL, NuoM, and NuoN (ND5, ND4 and ND2, respectively), are structurally similar. We analyzed the role of NuoN in Escherichia coli NDH-1. The lysine residue at position 395 in NuoN (NLys³⁹⁵) is conserved in NuoL (LLys³⁹⁹) but is replaced by glutamic acid (MGlu⁴⁰⁷) in NuoM. Our mutation study on NLys³⁹⁵ suggests that this residue participates in the proton translocation. Furthermore, we found that MGlu⁴⁰⁷ is also essential and most likely interacts with conserved LArg¹⁷⁵. Glutamic acids, NGlu¹³³, MGlu¹⁴⁴, and LGlu¹⁴⁴, are corresponding residues. Unlike mutants of MGlu¹⁴⁴ and LGlu¹⁴⁴, mutation of NGlu¹³³ scarcely affected the energy-transducing activities. However, a double mutant of NGlu¹³³ and nearby KGlu⁷² showed significant inhibition of these activities. This suggests that NGlu¹³³ bears a functional role similar to LGlu¹⁴⁴ and _MGlu¹⁴⁴ but its mutation can be partially compensated by the nearby carboxyl residue. Conserved prolines located at loops of discontinuous transmembrane helices of NuoL, NuoM, and NuoN were shown to play a similar role in the energy-transducing activity. It seems likely that NuoL, NuoM, and NuoN pump protons by a similar mechanism. Our data also revealed that _NLys¹⁵⁸ is one of the key interaction points with helix HL in NuoL. A truncation study indicated that the C-terminal amphipathic segments of _NTM14 interacts with the _Mβ sheet located on the opposite side of helix HL. Taken together, the mechanism of H⁺ translocation in NDH-1 is discussed.