A Central Functional Role for the 49-kDa Subunit within the Catalytic Core of Mitochondrial Complex I

Abstract

We have analyzed a series of eleven mutations in the 49-kDa protein of mitochondrial complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica to identify functionally important domains in this central subunit. The mutations were selected based on sequence homology with the large subunit of [NiFe] hydrogenases. None of the mutations affected assembly of complex I, all decreased or abolished ubiquinone reductase activity. Several mutants exhibited decreased sensitivities toward ubiquinone-analogous inhibitors. Unexpectedly, seven mutations affected the properties of ironsulfur cluster N2, a prosthetic group not located in the 49-kDa subunit. In three of these mutants cluster N2 was not detectable by electron-paramagnetic resonance spectroscopy. The fact that the small subunit of hydrogenase is homologous to the PSST subunit of complex I proposed to host cluster N2 offers a straightforward explanation for the observed, unforeseen effects on this iron-sulfur cluster. We propose that the fold around the hydrogen reactive site of [NiFe] hydrogenase is conserved in the 49-kDa subunit of complex I and has become part of the inhibitor and ubiquinone binding region. We discuss that the fourth ligand of iron-sulfur cluster N2 missing in the PSST subunit may be provided by the 49-kDa subunit.Oxidative phosphorylation is a universal process that converts most of the energy provided by foodstuffs into the general energy source ATP (1). During this process electrons pass through a series of membrane-bound multiprotein complexes that translocate protons across the membrane. The resulting proton motive force is used by ATP synthase to make ATP (2). Complex I (NADH:ubiquinone oxidoreductase) is the first of these electron transfer complexes and accounts for up to 40% of the pumped protons. In human mitochondria, complex I is regarded as a major source of deleterious reactive oxygen species. Hereditary and acquired defects affecting this multiprotein complex have been implicated in numerous degenerative diseases and seem to promote aging (3). The physiological and medical importance of complex I contrasts sharply with our still rather limited knowledge about the molecular structure and the catalytic mechanism of this multisubunit membrane protein with a total mass close to 1000 kDa (4).The core of complex I is formed by 14 central subunits of which in animals and most fungi seven are encoded by the mitochondrial genome (5). The mitochondrial enzyme contains up to 29 additional "supernumerary" subunits. As bacterial enzymes comprise only the 14 central subunits, these must contain all functional modules required for electron and proton transfer. Except for the electron entry point that is formed by FMN and iron-sulfur center N3 in the 51-kDa subunit, the organization of these functional modules, the location and number of prosthetic groups, and the location of the ubiquinone substrate binding site are largely unknown. In particular, the function of nine of the 14 central subunits that carry none of the known prosthetic g...

The Redox-Bohr Group Associated with Iron-Sulfur Cluster N2 of Complex I

“…In earlier studies, we had changed this histidine to alanine, glutamine, or cysteine. These mutations also had only moderate effects on the catalytic activity of complex I, but the EPR signal of iron-sulfur cluster N2 was markedly reduced (16,20).…”

Functional Implications from an Unexpected Position of the 49-kDa Subunit of NADH:Ubiquinone Oxidoreductase

“…Specific mutations in the 49-kDa were identified, which cause severe cardiomyopathy and encephalopathy in humans (32). Several point mutations near the carboxyl terminus of the 49-kDa subunit were shown to cause resistance toward complex I inhibitors in Rhodobacter capsulatus (11,33) and Y. lipolytica (14). Other site-directed mutations in this subunit of Y. lipolytica complex I gave rise to specific alterations in the EPR spectra of the functionally critical iron-sulfur cluster N2 and virtually complete loss of ubiquinone reductase activity (14).…”

“…Evidence from different laboratories (10,11) and the identification of several pathogenic mutations (12) suggested a key mechanistic role for the 49-kDa, PSST, and TYKY subunits. Based on these indications and a well established homology (13) between the 49-kDa and PSST subunits of complex I and the large and small subunits of [NiFe] hydrogenases, we proposed that at least part of the ubiquinone binding pocket and possibly the proton translocation machinery of complex I have evolved from the domains surrounding the [NiFe] site of the hydrogenase (14). This catalytic core hypothesis was substantiated experimentally by a series of site-directed mutations in the 49-kDa subunit of complex I from Y. lipolytica (3).…”

Mammalian NADH:ubiquinone oxidoreductase (Complex I) and nicotinamide nucleotide transhydrogenase (Nnt) together regulate the mitochondrial production of H2O2—Implications for their role in disease, especially cancer

“…It may further explain e.g. why some point mutations in the 49 kDa subunit of the Y. lipolytica Complex I resulted in the disappearance of EPR signals from Fe-S clusters in other subunits (Kashani-Poor et al 2001;Grgic et al 2004;Zwicker et al 2006;Tocilescu et al 2010), whereas other mutations had no effect (Tocilescu et al 2007). Similar observations have been made with the E. coli enzyme (Belevich et al 2007).…”