Exploring the catalytic core of complex I by Yarrowia lipolytica yeast genetics

We have developed Yarrowia lipolytica as a model system to study mitochondr ial complex I that combines the application of fast and convenient yeast ge netics with efficient structural and functional analysis of its very stable complex I isolated by his-tag affinity purification with high yield. Guide d by a structural model based on homologies between complex I and [NiFe] hy drogenases mutational analysis revealed that the 49 kDa subunit plays a cen tral functional role in complex I. We propose that critical parts of the ca talytic core of complex I have evolved from the hydrogen reactive site of [ NiFe] hydrogenases and that iron-sulfur cluster N2 resides at the interface between the 49 kDa and PSST subunits. These findings are in full agreement with the "semiquinone switch" mechanism according to which coupling of ele ctron and proton transfer in complex I is achieved by a single integrated p ump comprising cluster N2, the binding site for substrate ubiquinone, and a tightly bound quinone or quinoid group.