NDUFA9 is a Q-module subunit of mitochondrial respiratory chain complex I that is required during a late assembly step to stabilize the junction between the membrane and matrix arms of the complex (PMID:23223238, PMID:28671271). Loss of NDUFA9 by gene knockout blocks complex I assembly and leaves an accumulating membrane-arm subcomplex lacking matrix-arm subunits, with re-expression restoring assembly (PMID:23223238); in patients, biallelic and homozygous NDUFA9 variants reduce steady-state protein, abolish fully assembled complex I, and abrogate complex I enzymatic activity, with the defect rescued by wild-type but not mutant complementation, establishing NDUFA9 as a functionally essential subunit (PMID:22114105, PMID:28671271, PMID:41069424). Structural modelling places mutational hotspots (Arg321, Arg360) at positions predicted to destabilize the protein or disrupt binding interfaces (PMID:22114105, PMID:41069424). Beyond its structural assembly role, NDUFA9 activity is tuned post-translationally by competing acyl modifications—crotonylation enhancing and acetylation inhibiting complex I activity, mitochondrial respiration, and ATP synthesis (PMID:38657899)—and its expression is driven transcriptionally by direct YY1 binding to the NDUFA9 promoter, with NDUFA9 levels in turn supporting oxidative phosphorylation and downstream Akt-mTOR signaling (PMID:42014681).