Roles of Subunit ND2/NuoN in the Proton Pumping Coupling Mechanism of Complex I

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Respiration

Background:

• Complex I (NADH:quinone oxidoreductase, CI) is a vital enzyme in aerobic energy metabolism, featuring a conserved L-shaped structure with distinct electron transfer and proton pumping arms.
• The precise mechanism by which these two functional sites are coupled, particularly the role of the membrane subunit ND2/NuoN, remains incompletely understood.

Purpose of the Study:

• To elucidate the mechanistic role of conserved residues within the membrane subunit ND2/NuoN in the proton pumping and electron transfer coupling of Complex I.
• To investigate the functional consequences of specific mutations in the *E. coli* NuoN subunit on Complex I activity.

Main Methods:

• Site-directed mutagenesis of highly conserved residues in the *E. coli* NuoN subunit.
• Purification of mutant Complex I enzymes.
• Assessment of electron transfer and proton pumping activities using instant membrane reconstitution with *E. coli* double knockout (DKO) membrane vesicles and conventional proteoliposome reconstitution.
• Analysis of a NuoN knockout (ΔNuoN) mutant using blue native PAGE.

Main Results:

• Mutations at Lys247 and Lys395 in NuoN abolished both electron transfer and proton pumping activities, highlighting their essentiality.
• Mutation at Lys217 reduced NADH oxidase activity by ~50% without affecting proton pumping, while Glu133 showed no significant decrease in activity.
• A purified ΔNuoN mutant exhibited residual Complex I activity when reconstituted into DKO membranes, suggesting partial assembly facilitated by endogenous membrane subunits.

Conclusions:

• The membrane subunit ND2/NuoN plays a critical role in the coupling mechanism of Complex I.
• Specific highly conserved lysine residues (Lys247 and Lys395) within ND2/NuoN are indispensable for the functional coupling of electron transfer and proton translocation.
• The assembly and function of Complex I can be influenced by the availability of membrane subunits, as demonstrated by the ΔNuoN mutant reconstitution.