Abstract
Proton pumping respiratory complex I is a major player in mitochondrial energy conversion. Yet little is known about the molecular mechanism of this large membrane protein complex. Understanding the details of ubiquinone reduction will be prerequisite for elucidating this mechanism. Based on a recently published partial structure of the bacterial enzyme, we scanned the proposed ubiquinone binding cavity of complex I by site-directed mutagenesis in the strictly aerobic yeast Yarrowia lipolytica. The observed changes in catalytic activity and inhibitor sensitivity followed a consistent pattern and allowed us to define three functionally important regions near the ubiquinone-reducing iron-sulfur cluster N2. We identified a likely entry path for the substrate ubiquinone and defined a region involved in inhibitor binding within the cavity. Finally, we were able to highlight a functionally critical structural motif in the active site that consisted of Tyr-144 in the 49-kDa subunit, surrounded by three conserved hydrophobic residues.
Highlights
Because reduction of ubiquinone is likely to be a key event in the energy-coupling mechanism of complex I [1, 20], the quinone-binding site in the PSST and the 49-kDa subunit next to iron sulfur-cluster N2 is of particular interest
To identify the domains essential for catalytic activity and inhibitor binding, we introduced a set of point mutations in the PSST and the 49-kDa subunits of complex I from our model organism, the strictly aerobic yeast Yarrowia lipolytica
Positions for point mutations were chosen by analyzing the T. thermophilus structure so that they would probe all parts of the proposed quinone binding cavity and some surrounding residues
Summary
Strains and Site-directed Mutagenesis—The Y. lipolytica nucm⌬ and nukm⌬ deletion strains described earlier [21, 22] were transformed with the replicative plasmids pUB26 containing a genomic fragment of the NUCM gene or pUB4 containing a genomic fragment of the NUKM gene. After transformation [23] into Y. lipolytica strain nucm⌬ or nukm⌬, plasmids were recovered, and the entire open reading frames were sequenced to verify the introduced point mutations and exclude other sequence changes. Small Scale Preparation of Mitochondrial Membranes—Mitochondrial membranes were isolated essentially according to published protocols [24]. After phenylmethylsulfonyl fluoride was added to a final concentration of 2 mM, mitochondrial membranes were homogenized, shock-frozen, and stored in liquid nitrogen. Aliquots of preparations were used for activity measurements and gel electrophoresis. The activity was measured at 30 °C in 20
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