Exploring the Ubiquinone Binding Cavity of Respiratory Complex I

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 sitedirected 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.Respiratory chain NADH:ubiquinone oxidoreductase (complex I) is a large membrane protein complex that catalyzes electron transfer from NADH to ubiquinone and thereby pumps protons across the inner mitochondrial or bacterial plasma membrane (1). Electron microscopy revealed that complex I is L-shaped (2-9) and is composed of a hydrophobic arm embedded in the membrane and a peripheral arm protruding into the mitochondrial matrix or the bacterial cytosol. The peripheral arm contains all known redox centers, one FMN and eight or nine iron-sulfur clusters. Based on sequence comparisons (10, 11), mutational analysis (12-15), and photoaffinity labeling studies (16), we have proposed previously (14, 17) that the PSST and the 49-kDa subunit that are homologous to the small and large subunit of [NiFe] hydrogenases form part of the quinone reducing catalytic core of complex I (note that the bovine nomenclature will be used for the central subunits of complex I throughout). Recently the crystal structure of the peripheral domain of complex I from Thermus thermophilus has been solved at 3.3 Å resolution (18). This structure (Fig. 1) shows a wire of iron-sulfur clusters connecting the NADH-binding site near FMN with a broad cavity formed by the PSST and the 49-kDa subunit that should comprise the active site for ubiquinone reduction and the binding region for the large number of inhibitors that have been found for complex I (19).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...

Redox-induced activation of the proton pump in the respiratory complex I

“…This finding is consistent with earlier biochemical data, suggesting that the Q headgroup is in hydrogen-bonding contact with Tyr-87 of subunit Nqo4 (19). Moreover, the structural data showed that the Q headgroup is also in hydrogen-bonding contact with His-38 of Nqo4, which in turn is further stabilized by an ion pair with Asp-139 (Fig.…”

Amyloid-β and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer's disease mice

“…NUCM likely has a central role within the catalytic core of mitochondrial complex I (26). Interestingly, NUCM and NIDM were up-regulated in triple AD brain, probably as a compensatory response to the functional failure of OX-PHOS.…”

Mechanistic insight from the crystal structure of mitochondrial complex I

“…Supporting the notion that the position of the substrate analogs also reflects a with a role in ubiquinone or inhibitor binding (27) are located nearby (Fig 5). (29,30). Indeed, binding of ubiquinone and piericidin A next to these residues and closer to cluster N2 was modelled into the structure of the bacterial enzyme (10) revealing a marked difference (Fig.…”