Abstract
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.
Highlights
Proton-translocating NADH:ubiquinone oxidoreductase (EC 1.6.99.3, complex I) is the first complex of the mitochondrial respiratory chain
When Glu89, 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 gz region
To identify functionally important residues in the PSST homologue subunit of the Y. lipolytica complex I, we have introduced a series of single amino acid exchanges and replaced acidic residues that are strictly conserved among all known sequences
Summary
Proton-translocating NADH:ubiquinone oxidoreductase (EC 1.6.99.3, complex I) is the first complex of the mitochondrial respiratory chain. It couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the inner mitochondrial membrane [1, 2] In bovine heart, this enzyme is made up of 43 different subunits [3] with a molecular mass of nearly 1000 kDa. The homologous procaryotic complex I has a minimal number of 14 subunits with a total molecular mass of ϳ500 kDa [4]. The mechanism that couples proton translocation across the membrane to electron transfer from NADH through FMN and several iron-sulfur centers onto ubiquinone is unknown. Sitedirected mutagenesis in E. coli suggests the PSST homolog [14], and a similar study in Rhodobacter capsulatus [15] suggests the TYKY homolog as the most likely candidates to bind N-2
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