Function of conserved acidic residues in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica.
Proton-translocating NADH:ubiquinone oxidoreductase (complex I) is the largest and least understood enzyme of the respiratory chain. Complex I from bovine mitochondria consists of more than forty different polypeptides. Subunit PSST has been suggested to carry iron-sulfur center N-2 and has more recently been shown to be involved in inhibitor binding. Due to its pH-dependent midpoint potential, N-2 has been proposed to play a central role both in ubiquinone reduction and proton pumping. To obtain more insight into the functional role of PSST, we have analyzed site-directed mutants of conserved acidic residues in the PSST homologous subunit of the obligate aerobic yeast Yarrowia lipolytica. Mutations D136N and E140Q provided functional evidence that conserved acidic residues in PSST play a central role in the proton translocating mechanism of complex I and also in the interaction with the substrate ubiquinone. When Glu(89), the residue that has been suggested to be the fourth ligand of iron-sulfur center N-2 was changed to glutamine, alanine, or cysteine, the EPR spectrum revealed an unchanged amount of this redox center but was shifted and broadened in the g(z) region. This indicates that Glu(89) is not a ligand of N-2. The results are discussedin the light of structural similarities to the homologous [NiFe] hydrogenases.
Ahlers
P M
PM
Universitätsklinikum Frankfurt, Institut für Biochemie I, D-60590 Frankfurt am Main, Federal Republic of Germany.
Zwicker
K
K
Kerscher
S
S
Brandt
U
U
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D017124:Conserved Sequence; D004578:Electron Spin Resonance Spectroscopy; D018698:Glutamic Acid / Q000235:genetics; D006864:Hydrogenase / Q000737:chemistry; D007425:Intracellular Membranes / Q000201:enzymology; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009245:NADH Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017494:Proton Pumps / Q000235:genetics / Q000378:metabolism*; D004718:Saccharomycetales / Q000201:enzymology*; D020539:Sequence Analysis, Protein; D017384:Sequence Deletion; D014451:Ubiquinone / Q000378:metabolism*
D007506:Iron-Sulfur Proteins; D008024:Ligands; D017494:Proton Pumps; D014451:Ubiquinone; D001224:Aspartic Acid; D018698:Glutamic Acid; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D009245:NADH Dehydrogenase