Tight binding of NADPH to the 39-kDa subunit of complex I is not required for catalytic activity but stabilizes the multiprotein complex.
In addition to the 14 central subunits, respiratory chain complex I from the aerobic yeast Yarrowia lipolytica contains at least 24 accessory subunits, most of which are poorly characterized. Here we investigated the role of the accessory 39-kDa subunit which belongs to the heterogeneous short-chain dehydrogenase/reductase (SDR) enzyme family and contains non-covalently bound NADPH. Deleting the chromosomal copy of the gene that codes for the 39-kDa subunit drastically impaired complex I assembly in Y. lipolytica. We introduced several site-directed mutations into the nucleotide binding motif that severely reduced NADPH binding. This effect was most pronounced when the arginine at the end of the second beta-strand of the NADPH binding Rossman fold was replaced by leucine or aspartate. Mutations affecting nucleotide binding had only minor or moderate effects on specific catalytic activity in mitochondrial membranes but clearly destabilized complex I. One mutant exhibited a temperature sensitive phenotype and significant amounts of three different subcomplexes were observed even at more permissive temperature. We concluded that the 39-kDa subunit of Y. lipolytica plays a critical role in complex I assembly and stability and that the bound NADPH serves to stabilize the subunit and complex I as a whole rather than serving a catalytic function.
Abdrakhmanova
Albina
A
Universität Frankfurt, Zentrum der Biologischen Chemie, Molekulare Bioenergetik, Theodor-Stern-Kai 7, Haus 26, D-60590 Frankfurt am Main, Germany.
Zwicker
Klaus
K
Kerscher
Stefan
S
Zickermann
Volker
V
Brandt
Ulrich
U
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004271:DNA, Fungal / Q000235:genetics; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025062:Yarrowia / Q000201:enzymology / Q000235:genetics
D004271:DNA, Fungal; D005656:Fungal Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D009249:NADP; D042967:Electron Transport Complex I