Klaus Zwicker

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

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 iron-sulfur 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.

Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism?

The modular evolutionary origin of NADH:ubiquinone oxidoreductase (complex I) provides useful insights into its functional organization. Iron–sulfur cluster N2 and the PSST and 49 kDa subunits were identified as key players in ubiquinone reduction and proton pumping. Structural studies indicate that this ‘catalytic core’ region of complex I is clearly separated from the membrane. Complex I from Escherichia coli and Klebsiella pneumoniae was shown to pump sodium ions rather than protons. These new insights into structure and function of complex I strongly suggest that proton or sodium pumping in complex I is achieved by conformational energy transfer rather than by a directly linked redox pump.

The iron-sulphur protein Ind1 is required for effective complex I assembly.

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi‐subunit protein complex containing eight iron–sulphur (Fe–S) clusters. Little is known about the assembly of complex I and its Fe–S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide‐binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe–S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two‐dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe–S clusters showed that only a minor fraction (∼20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe–4S] cluster that was readily transferred to an acceptor Fe–S protein. Our data suggest that Ind1 facilitates the assembly of Fe–S cofactors and subunits of complex I.

Biophysical and structural characterization of proton-translocating NADH-dehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica

Mitochondrial proton-translocating NADH-dehydrogenase (complex I) is one of the largest and most complicated membrane bound protein complexes. Despite its central role in eukaryotic oxidative phosphorylation and its involvement in a broad range of human disorders, little is known about its structure and function. Therefore, we have started to use the powerful genetic tools available for the strictly aerobic yeast Yarrowia lipolytica to study this respiratory chain enzyme. To establish Y. lipolytica as a model system for complex I, we purified and characterized the multisubunit enzyme from Y lipolytica and sequenced the nuclear genes coding for the seven central subunits of its peripheral part. Complex I from Y lipolytica is quite stable and could be isolated in a highly pure and monodisperse state. One binuclear and four tetranuclear iron-sulfur clusters, including N5, which was previously known only from mammalian mitochondria, were detected by EPR spectroscopy. Initial structural analysis by single particle electron microscopy in negative stain and ice shows complex I from Y. lipolytica as an L-shaped particle that does not exhibit a thin stalk between the peripheral and the membrane parts that has been observed in other systems.

Architecture of complex I and its implications for electron transfer and proton pumping.

Proton pumping NADH:ubiquinone oxidoreductase (complex I) is the largest and remains by far the least understood enzyme complex of the respiratory chain. It consists of a peripheral arm harbouring all known redox active prosthetic groups and a membrane arm with a yet unknown number of proton translocation sites. The ubiquinone reduction site close to iron-sulfur cluster N2 at the interface of the 49-kDa and PSST subunits has been mapped by extensive site directed mutagenesis. Independent lines of evidence identified electron transfer events during reduction of ubiquinone to be associated with the potential drop that generates the full driving force for proton translocation with a 4H(+)/2e(-) stoichiometry. Electron microscopic analysis of immuno-labelled native enzyme and of a subcomplex lacking the electron input module indicated a distance of 35-60 A of cluster N2 to the membrane surface. Resolution of the membrane arm into subcomplexes showed that even the distal part harbours subunits that are prime candidates to participate in proton translocation because they are homologous to sodium/proton antiporters and contain conserved charged residues in predicted transmembrane helices. The mechanism of redox linked proton translocation by complex I is largely unknown but has to include steps where energy is transmitted over extremely long distances. In this review we compile the available structural information on complex I and discuss implications for complex I function.

Exploring the Ubiquinone Binding Cavity of Respiratory Complex I

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 site-directed 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.

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.

Function of Conserved Acidic Residues in the PSST Homologue of Complex I (NADH:Ubiquinone Oxidoreductase) from Yarrowia lipolytica

Proton-translocating NADH:ubiquinone oxidoreductase (complex I) is the largest and least understood enzyme of the respiratory chain. Complex I from bovine mitochondria consists of more than forty different polypeptides. Subunit PSST has been suggested to carry iron-sulfur center N-2 and has more recently been shown to be involved in inhibitor binding. Due to its pH-dependent midpoint potential, N-2 has been proposed to play a central role both in ubiquinone reduction and proton pumping. To obtain more insight into the functional role of PSST, we have analyzed site-directed mutants of conserved acidic residues in the PSST homologous subunit of the obligate aerobic yeast Yarrowia lipolytica. Mutations D136N and E140Q provided functional evidence that conserved acidic residues in PSST play a central role in the proton translocating mechanism of complex I and also in the interaction with the substrate ubiquinone. When Glu89, the residue that has been suggested to be the fourth ligand of iron-sulfur center N-2 was changed to glutamine, alanine, or cysteine, the EPR spectrum revealed an unchanged amount of this redox center but was shifted and broadened in the gzregion. This indicates that Glu89 is not a ligand of N-2. The results are discussedin the light of structural similarities to the homologous [NiFe] hydrogenases.

Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids

NADH:ubiquinone oxidoreductase (complex I) is the largest multiprotein complex of the mitochondrial respiratory chain. His-tagged complex I purified from the strictly aerobic yeast Yarrowia lipolytica exhibited electron transfer rates from NADH to n-decylubiquinone of less than 2% when compared to turnover numbers calculated for native mitochondrial membranes from this organism. Reactivation was observed upon addition of asolectin, purified phospholipids and different phospholipid mixtures. Maximal activities of 6-7 micromol NADH min(-1) mg(-1) were observed following incubation with a mixture of 76% phosphatidylcholine, 19% phosphatidylethanolamine and 5% cardiolipin. For full reactivation, 400-500 phospholipid molecules per complex I were needed. This demonstrated that the inactivation of complex I from Y. lipolytica by general delipidation could be fully reversed simply by returning the phospholipids that had been removed during the purification procedure. Thus, our homogeneous and highly pure complex I preparation had retained its full catalytic potential and no specific, functionally essential component had been lost. As the purified enzyme was also found to contain only substoichiometric amounts of ubiquinone-9 (0.2-0.4 mol/mol), a functional requirement of this endogeneous ubiquinone could also be excluded.

The Redox-Bohr group associated with iron-sulfur cluster N2 of complex I.

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 iron-sulfur 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 redox-Bohr 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.