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Mitochondial NADH:ubiquinone oxidoreductase (complex I) the largest multiprotein enzyme of the respiratory chain, catalyses the transfer of two electrons from NADH to ubiquinone, coupled to the translocation of four protons across the membrane. In addition to the 14 strictly conserved central subunits it contains a variable number of accessory subunits. At present, the best characterized enzyme is complex I from bovine heart with a molecular mass of about 980 kDa and 32 accessory proteins. In this study, the subunit composition of mitochondrial complex I from the aerobic yeast Y. lipolytica has been analysed by a combination of proteomic and genomic approaches. The sequences of 37 complex I subunits were identified. The sum of their individual molecular masses (about 930 kDa) was consistent with the native molecular weight of approximately 900 kDa for Y. lipolytica complex I obtained by BN-PAGE. A genomic analysis with Y. lipolytica and other eukaryotic databases to search for homologues of complex I subunits revealed 31 conserved proteins among the examined species. A novel protein named “X” was found in purified Y. lipolytica complex I by MALDI-MS. This protein exhibits homology to the thiosulfate sulfurtransferase enzyme referred to as rhodanese. The finding of a rhodanese-like protein in isolated complex I of Y. lipolytica allows to assume a special regulatory mechanism of complex I activity through control of the status of its iron-sulfur clusters. The second part of this study was aimed at investigating the possible role of one of these extra subunits, 39 kDa (NUEM) subunit which is related to the SDRs-enzyme family. The members of this family function in different redox and isomerization reactions and contain a conserved NAD(P)H-binding site. It was proposed that the 39 kDa subunit may be involved in a biosynthetic pathway, but the role of this subunit in complex I is unknown. In contrast to the situation in N. crassa, deletion of the 39 kDa encoding gene in Y. lipolytica led to the absence of fully assembled complex I. This result might indicate a different pathway of complex I assembly in both organisms. Several site-directed mutations were generated in the nucleotide binding motif. These had either no effect on enzyme activity and NADPH binding, or prevented complex I assembly. Mutations of arginine-65 that is located at the end of the second b-strand and responsible for selective interaction with the 2’-phosphate group of NADPH retained complex I activity in mitochondrial membranes but the affinity for the cofactor was markedly decreased. Purification of complex I from mutants resulted in decrease or loss of ubiquinone reductase activity. It is very likely that replacement of R65 not only led to a decrease in affinity for NADPH but also caused instability of the enzyme due to steric changes in the 39 kDa subunit. These data indicate that NADPH bound to the 39 kDa subunit (NUEM) is not essential for complex I activity, but probably involved in complex I assembly in Y. lipolytica.
Mitochondrial complex I has a key role in cellular energy metabolism, generating a major portion of the proton motive force that drives aerobic ATP synthesis. The hydrophilic arm of the L-shaped ~1 MDa membrane protein complex transfers electrons from NADH to ubiquinone, providing the energy to drive proton pumping at distant sites in the membrane arm. The critical steps of energy conversion are associated with the redox chemistry of ubiquinone. We report the cryo-EM structure of complete mitochondrial complex I from the aerobic yeast Yarrowia lipolytica both in the deactive form and after capturing the enzyme during steady-state activity. The site of ubiquinone binding observed during turnover supports a two-state stabilization change mechanism for complex I.
The NADH:ubiquinone oxidoreductase (complex I) is a large membrane bound protein complex coupling the redox reaction of NADH oxidation and quinone reduction to vectorial proton translocation across bioenergetic membranes. The mechanism of proton pumping is still unknown; it seems however that the reduction of quinone induces conformational changes which drive proton uptake from one side and release at the other side of the membrane. In this study the proposed quinone and inhibitor binding pocket located at the interface of the 49-kDa and PSST subunits was explored by a large number of point mutations introduced into complex I from the strictly aerobic yeast Yarrowia lipolytica. Point mutations were systematically chosen based on the crystal structure of the hydrophilic domain of complex I from Thermus thermophilus. In total, the properties of 94 mutants at 39 positions which completely cover the lining of the large putative quinone and inhibitor binding cavity are described and discussed here. A structure/function analysis allowed the identification of functional domains within the large putative quinone binding cavity. A possible quinone access path ranging from the N-terminal beta-sheet of the 49-kDa subunit into the pocket to tyrosine 144 could be defined, since all exchanges introduced here, caused an almost complete loss of complex I activity. A region located deeper in the proposed quinone binding pocket is apparently not important for complex I activity. In contrast, all exchanges of tyrosine 144, even the very conservative mutant Y144F, essentially abolished dNADH:DBQ oxidoreductase activity of complex I. However, with higher concentrations of Q1 or Q2 the dNADH:Q oxidoreductase activity was largely restored in the mutants with the more conservative exchanges. Proton pumping experiments showed that this activity was also coupled to proton translocation, indicating that these quinones were reduced at the physiological site. However, the apparent Km values for Q1 or Q2 were drastically increased, clearly demonstrating that tyrosine 144 is central for quinone binding and reduction. These results further prove that the enzymatically relevant quinone binding site of complex I is located at the interface of the 49-kDa and PSST subunits. The quinone binding pocket is thought to comprise the binding sites for a plethora of specific complex I inhibitors that are usually grouped into three classes. The large array of mutants targeting the quinone binding cavity was examined with a representative of each inhibitor class. Many mutants conferring resistance were identified which, depending on the inhibitor tested, clustered in well defined and partially overlapping regions of the large putative quinone and inhibitor binding cavity. Mutants with effects on type A (DQA) and type B (rotenone) inhibitors were found in a subdomain corresponding to the former [NiFe] site in homologous hydrogenases, whereby the type A inhibitor DQA seems to bind deeper in this domain. Mutants with effects on the type C inhibitor (C12E8) were found in a narrow crevice. Exchanging more exposed residues at the border of these well defined domains affected all three inhibitor types. Therefore, the results as a whole provide further support for the concept that different inhibitor classes bind to different but partially overlapping binding sites within a single large quinone binding pocket. In addition, they also indicate the approximate location of the binding sites within the structure of the large quinone and inhibitor binding cavity at the interface of the 49 kDa and the PSST subunit. It has been proposed earlier that the highly conserved HRGXE-motif in the 49-kDa subunit forms a part of the quinone binding site of complex I. Mutagenesis of the HRGXE-motif, revealed that these residues are rather critical for complex I assembly and seem to have an important structural role. The question why iron-sulfur cluster N1a is not detectable by EPR in many models organisms is not solved yet. Introducing polar and positively charged amino acid residues close to this cluster in order to increase its midpoint potential did not result in the appearance of the cluster N1a EPR signal in mitochondrial membranes from the mutants. Clearly, further research will be necessary to gain insights to the function of this iron-sulfur cluster in complex I. In an additional project, a new and simple in vivo screen for complex I deficiency in Y. lipolytica was developed and optimized. This assay probes for defects in complex I assembly and stability, oxidoreductase activity and also proton pumping activity by complex I. Most importantly, this assay is applicable to all Y. lipolytica strains and could be used to identify loss-of-function mutants, gain-of-functions mutants (i.e. resistance towards complex I inhibitors) and revertants due to mutations in both nuclear and mitochondrially encoded genes of complex I subunits.
1. Fab co-complexes of proton pumping NADH:ubiquinone oxidoreductase (complex I) Fab fragments suitable for co-crystallization with complex I were generated using an immobilized papainbased protocol. The binding of the antibody fragments to complex I was verified using Surface Plasmon Resonance and size exclusion chromatography. The binding constants of the antibodies and their respective Fab fragments were found to be in the nanomolar range. This work presents the first report on successful crystallization of complex I (proton pumping NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica with proteolytic Fab fragments. The quality of the crystals was significantly improved when compared to the initial experiments and the best crystals diffracted X-rays to a resolution of ~7 Å. The activity of complex I remained uninfluenced by antibody fragment binding. The initial diffraction data suggest that the complex I/Fab co-complex crystals represent a space group different to the one observed for the native protein. Ongoing experiments are aimed at further enhancements of the diffraction quality of the crystals. Providing a different space group the CI/Fab co-complexes may become a very useful approach for structure determination of the enzyme. Moreover, the bound Fab offers an additional possibility to generate phase information. The antibody-mediated crystallization represents a valuable tool in structural characterization of the NADH:oxidoreductase subcomplexes or even single subunits. 2. UDP-glucose pyrophosphorylase UDP-glucose pyrophosphorylase from Yarrowia lipolytica displays affinity towards Ni2+ NTA and was first detected in a contaminated sample of complex I. Following, separation from complex I, Ugp1p was purified using anion exchange chromatography. Sequence similarity studies revealed high identity to other known pyrophosphorylases. As indicated by laser-based mass spectrometry method (LILBID) Ugp1p from Y. lipolytica builds octamers similarly to the enzyme from Saccharomyces cerevisiae. The initial crystals grew as thin needles favorably in sitting drop setups. The size of the crystals was increased by employment of a micro batch technique. The improved crystals diffracted X-rays to a resolution of 3.2 Å at the synchrotron beamline. Structural characterization is under way using a molecular replacement approach based on the published structure of baker’s yeast UGPase.