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

Abstract

Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near ironsulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redoxBohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H ؉ /2e ؊ . This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.In mammalian cells mitochondrial complex I (NADH: ubiquinone oxidoreductase) provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme (1) cause numerous human disorders (2). Very recently, the x-ray structure of the peripheral arm of complex I from Thermus thermophilus has been solved (3). However, it is still unclear how complex I couples electron transfer from NADH to ubiquinone with the vectorial translocation of four protons across the bioenergetic membrane (4, 5). Numerous hypothetical mechanisms have been proposed for this energy conversion over the years (6 -9). None of the known redox groups of complex I (FMN and 8 -9 ironsulfur clusters) resides in the membrane domain (3, 10). The Fe 4 S 4 cluster N2 is the last component in a "wire" of seven iron-sulfur clusters connecting FMN, the immediate oxidant for NADH, and substrate ubiquinone (3,11). While all other clusters in this chain have a redox midpoint potential of around Ϫ250 mV, iron-sulfur cluster N2 has a significantly more positive E m7 of typically Ϫ150 mV (12, 13). Moreover, the redox midpoint potential of cluster N2 exhibits pronounced pH dependence between pH 6 and 8 (12). Based on this "redoxBohr" effect (14) and the comparably more positive potential, cluster N2 has been implicated as a key component of the proton pump (6, 15).The structural model for the ubiquinone reducing catalytic core of complex I (1, 16) that had been deduced from distinct homologies between [NiFe] hydrogenase and complex I (17, 18) was now confirmed by the structure of the peripheral arm of complex I (3). Like the proximal iron-sulfur cluster of hydrogenase, iron-sulfur cluster N2 is at the interface between two subunits, the PSST subunit and the 49-kDa subunit (Fig. 1). In water-soluble [NiFe] hydrogenases a conserved histidine of the l...

Redox-Dependent Change of Nucleotide Affinity to the Active Site of the Mammalian Complex I

“…Since the FMN is located almost 90 Å from the membrane plane (16), it is virtually impossible to construct any FMN-catalyzed proton-translocating loop mechanism. The alternative might be that redox-dependent conformational coupling (13,(45)(46)(47)(48) is operating together with a direct ubiquinone reduction-associated loop mechanism which may result in the observed overall H + /e stoichiometry of 4. The redox-dependent nucleotide binding change as reported here may be a part of such a conformational coupling.…”

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

“…The assembly of the complex was usually quite normal (Kashani-Poor et al 2001b;Grgic et al 2004). One mutation (H226M) led to a 80 mV decrease of the midpoint potential of the N2 signal and an apparent loss of the pH dependence of its midpoint potential (Zwicker et al 2006). Other mutations in the 49-kDa subunit and a series mutations in the PSST subunit, some very close to its Fe-S cluster assumed by these authors to be cluster N2 (Zickermann et al 2009), did not induce any qualitative changes in the EPR signal of N2 (or any of the other signals) of the mutant enzyme ).…”

Assessing mitochondrial dysfunction in cells

“…Several techniques have been described for monitoring cell respiration in a multiwell format using oxygen-dependent fluorescence quenching to monitor the oxygen concentration in the medium. These techniques either operate at steady-state, where oxygen consumption is balanced by inward diffusion of oxygen [63], or measure oxygen depletion in a sealed chamber [64,65]. Although these can be used to monitor or compare basal cell respiration rates, their low time resolution and the limited ability to make additions during the run preclude them from being used for sophisticated respiration assays.…”