Two aspartic acid residues in the PSST-homologous NUKM subunit of complex I from Yarrowia lipolytica are essential for catalytic activity.
Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of water-soluble [NiFe] hydrogenases.
Garofano
Aurelio
A
Johann Wolfgang Goethe-Universität Frankfurt am Main, Fachbereich Medizin, Gustav Embden Zentrum der Biologischen Chemie, Institut für Biochemie I, Theodor-Stern-Kai 7, Haus 25B, D-60590 Frankfurt am Main, Germany.
Zwicker
Klaus
K
Kerscher
Stefan
S
Okun
Pamela
P
Brandt
Ulrich
U
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry*; D002384:Catalysis; D017124:Conserved Sequence; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D025062:Yarrowia / Q000201:enzymology*
D005656:Fungal Proteins; D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; D001224:Aspartic Acid; D042967:Electron Transport Complex I