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 larg est and least understood enzyme of the respiratory chain. Complex I from bo vine mitochondria consists of more than forty different polypeptides. Subun it PSST has been suggested to carry iron-sulfur center N-2 and has more rec ently been shown to be involved in inhibitor binding. Due to its ps-depende nt midpoint; potential, N-2 has been proposed to play a central role both i n ubiquinone reduction and proton pumping, To obtain more insight into the functional role of PSST, we have analyzed site-directed mutants of conserve d acidic residues in the PSST homologous subunit of the obligate aerobic ye ast Yarrowia lipolytica, Mutations D136N and E140Q provided functional evid ence that conserved acidic residues in PSST play a central role in the prot on translocating mechanism of complex I and also in the interaction with th e substrate ubiquinone. When Glu(89), the residue that has been suggested t o 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 indica tes that Glu(89) is not a ligand of N-2, The results are discussed in the l ight of structural similarities to the homologous [NiFe] hydrogenases.