Functional Significance of Conserved Histidines and Arginines in the 49-kDa Subunit of Mitochondrial Complex I

Grgic, Ljuban; Zwicker, Klaus; Kashani‐Poor, Noushin; Kerscher, Stefan; Brandt, Ulrich

doi:10.1074/jbc.m313180200

Cited by 75 publications

(70 citation statements)

References 25 publications

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“…The low activity of the mutant does not, however, allow us to establish the pumping stoichiometry, which may be lower than in the wild-type enzyme. Substantial inhibition of the oxidoreduction activity has also been observed by mutation of His-38 into alanine (26,27). All these findings suggest that the His-Asp motif is central for the protoncoupled electron transfer function of complex I.…”

Section: Resultsmentioning

confidence: 48%

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

Sharma

Belevich

Gámiz‐Hernández

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

109

170

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Complex I functions as a redox-linked proton pump in the respiratory chains of mitochondria and bacteria, driven by the reduction of quinone (Q) by NADH. Remarkably, the distance between the Q reduction site and the most distant proton channels extends nearly 200 Å. To elucidate the molecular origin of this long-range coupling, we apply a combination of large-scale molecular simulations and a site-directed mutagenesis experiment of a key residue. In hybrid quantum mechanics/molecular mechanics simulations, we observe that reduction of Q is coupled to its local protonation by the His-38/Asp-139 ion pair and Tyr-87 of subunit Nqo4. Atomistic classical molecular dynamics simulations further suggest that formation of quinol (QH 2 ) triggers rapid dissociation of the anionic Asp-139 toward the membrane domain that couples to conformational changes in a network of conserved charged residues. Site-directed mutagenesis data confirm the importance of Asp-139; upon mutation to asparagine the Q reductase activity is inhibited by 75%. The current results, together with earlier biochemical data, suggest that the proton pumping in complex I is activated by a unique combination of electrostatic and conformational transitions.NADH-quinone oxidoreductase | electron transfer | molecular dynamics simulations | QM/MM simulations | cell respiration C omplex I (NADH-quinone oxidoreductase) is the largest (550-980 kDa) and one of the most enigmatic enzymes of the electron transport chains of mitochondria and bacteria. It catalyzes electron transfer (eT) from reduced nicotinamide adenine dinucleotide (NADH) to quinone (Q) and couples the reaction to translocation of three to four protons across the membrane (1, 2). The established electrochemical proton gradient is further used to synthesize adenosine triphosphate (ATP) for active transport (3). Due to its central role in cellular respiration, elucidating the catalytic mechanism of complex I is crucial for understanding the molecular principles of biological energy transduction and for unveiling the origins of many mitochondrial disorders (4).The electrons donated by NADH to complex I are transferred via flavin mononucleotide (FMN) to Q, bound at the lower edge of the hydrophilic domain at a distance of ∼80 Å from the FMN (Fig. 1). The eT process is mediated by seven to eight iron-sulfur (FeS) clusters, depending on the organism, and takes place in ∼100 μs (5). It is believed that the eT process does not couple to proton translocation, which is likely to occur on millisecond timescales (5, 6), but it is rather the oxidoreduction chemistry of the bound Q molecule that drives the proton pump (2, 5-9; cf. ref. 10).The proton-pumping machinery of complex I is located in the membrane domain of the enzyme (9) and is responsible for pumping three to four protons across the membrane (Fig. 1) (8, 11). Biochemical and structural studies suggest that the reduction of Q activates the proton pump via a conformationaldriven coupling mechanism, accompanied by electrostatic gating (2, 6-8, 12-14). A ...

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Section: Resultsmentioning

confidence: 48%

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

Sharma

Belevich

Gámiz‐Hernández

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

109

170

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“…We have shown previously that the mutation of histidine 226 in the 49-kDa subunit to methionine has little effect on steady-state activity and inhibitor sensitivity of complex I (20). However, when we determined the redox midpoint potential of complex I from mutant H226M at pH 7, we found that it was shifted to the negative from Ϫ140 mV to Ϫ220 mV ( Fig.…”

Section: His 226 Is the Redox-bohr Group Associated With Iron-sulfur mentioning

confidence: 93%

“…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).…”

Section: Discussionmentioning

confidence: 99%

“…In water-soluble [NiFe] hydrogenases a conserved histidine of the large subunit forms a hydrogen bond to the iron-sulfur cluster ligated to the small subunit (19). Remarkably, a fully conserved histidine is also found at the corresponding position in the 49-kDa subunit of complex I. Mutating this histidine (His 226 ) to methionine in the 49-kDa subunit of complex I from the strictly aerobic yeast Yarrowia lipolytica shifts the EPR spectrum of reduced iron sulfur cluster N2 to slightly lower field values but does not significantly affect the catalytic activity of complex I (20). Here we show that residue His 226 in the 49-kDa subunit of complex I from Y. lipolytica is the redox-Bohr group associated with iron-sulfur cluster N2 but is not involved in the proton pumping mechanism of complex I.…”

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confidence: 99%

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

Zwicker¹,

Galkin²,

Dröse

et al. 2006

Journal of Biological Chemistry

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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...

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“…Complex I from both mutant and wild-type was affinitypurified from isolated mitochondrial membranes that were solubilized with n-dodecyl-␤-D-maltoside essentially as described previously (29). Construction of the mutant and its characterization have been reported elsewhere (30). Protein concentrations were determined according to a modified Lowry protocol (32).…”

Section: Methodsmentioning

confidence: 99%

Superoxide Radical Formation by Pure Complex I (NADH:Ubiquinone Oxidoreductase) from Yarrowia lipolytica

Galkin¹,

Brandt

2005

Journal of Biological Chemistry

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149

110

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Generation of reactive oxygen species (ROS) is increasingly recognized as an important cellular process involved in numerous physiological and pathophysiological processes. Complex I (NADH:ubiquinone oxidoreductase) is considered as one of the major sources of ROS within mitochondria. Yet, the exact site and mechanism of superoxide production by this large membrane-bound multiprotein complex has remained controversial. Here we show that isolated complex I from Yarrowia lipolytica forms superoxide at a rate of 0.15% of the rate measured for catalytic turnover. Superoxide production is not inhibited by ubiquinone analogous inhibitors. Because mutant complex I lacking a detectable iron-sulfur cluster N2 exhibited the same rate of ROS production, this terminal redox center could be excluded as a source of electrons. From the effect of different ubiquinone derivatives and pH on this side reaction of complex I we concluded that oxygen accepts electrons from FMNH 2 or FMN semiquinone either directly or via more hydrophilic ubiquinone derivatives.Over the last decade the processes leading to the production of superoxide and other reactive oxygen species (ROS) 1 have gained much attention. ROS seem to be involved in apoptosis, the development of various pathological states, aging, and the regulation of cell metabolism. It is generally accepted that production of reactive oxygen species is an inherent property of the mitochondrial respiratory chain of eucaryotic cells. Oxidation of certain redox centers in complex I and III by molecular oxygen results in the production of superoxide anion radical O 2. (see Ref. 1 for a review). Superoxide can then convert into hydrogen peroxide, the highly active hydroxyl radical (OH ⅐ ), and other ROS. It has been shown that O 2 . production by complex I occurs in the mitochondrial matrix, whereas the cytochrome bc 1 complex reduces oxygen primarily on the intermembrane side (2, 3) (see, however, Ref. 4). It has been demonstrated for the cytochrome bc 1 complex that oxygen reduction occurs at the Q P site and is increased markedly under conditions of "oxidant-induced reduction" (5, 6). However, much less is known about the site and mechanism of O 2 . generation in complex I. Thermodynamically, any of the complex I redox centers in the reduced state is capable of donating an electron to molecular oxygen to form a superoxide anion. Eucaryotic NADH:ubiquinone oxidoreductase (complex I or type I NADH dehydrogenase) is the largest and most complex enzyme of the respiratory chain, residing in the inner membrane of mitochondria. In mammals, the enzyme is composed of 46 different subunits (7) and contains non-covalently bound FMN and up to eight iron-sulfur clusters as redox cofactors. Two complex I-associated, electron paramagnetic resonancedetectable semiquinone species with different spin relaxation times have been characterized (8, 9). Complex I catalyzes the transfer of electrons from matrix NADH to membrane ubiquinone coupled to the translocation of four protons across the membrane (10, 11...

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Functional Significance of Conserved Histidines and Arginines in the 49-kDa Subunit of Mitochondrial Complex I

Cited by 75 publications

References 25 publications

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

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

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

Superoxide Radical Formation by Pure Complex I (NADH:Ubiquinone Oxidoreductase) from Yarrowia lipolytica

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