Lucie Sokolova

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.

Functional Dissection of the Proton Pumping Modules of Mitochondrial Complex I

A catalytically active subcomplex of respiratory chain complex I lacks 14 of its 42 subunits yet retains half of its proton-pumping capacity, indicating that its membrane arm has two pump modules.

Tryptophan-accelerated electron flow across a protein-protein interface.

We report a new metallolabeled blue copper protein, Re126W122Cu(I) Pseudomonas aeruginosa azurin, which has three redox sites at well-defined distances in the protein fold: Re(I)(CO)3(4,7-dimethyl-1,10-phenanthroline) covalently bound at H126, a Cu center, and an indole side chain W122 situated between the Re and Cu sites (Re-W122(indole) = 13.1 Å, dmp-W122(indole) = 10.0 Å, Re-Cu = 25.6 Å). Near-UV excitation of the Re chromophore leads to prompt Cu(I) oxidation (<50 ns), followed by slow back ET to regenerate Cu(I) and ground-state Re(I) with biexponential kinetics, 220 ns and 6 μs. From spectroscopic measurements of kinetics and relative ET yields at different concentrations, it is likely that the photoinduced ET reactions occur in protein dimers, (Re126W122Cu(I))2 and that the forward ET is accelerated by intermolecular electron hopping through the interfacial tryptophan: *Re//←W122←Cu(I), where // denotes a protein-protein interface. Solution mass spectrometry confirms a broad oligomer distribution with prevalent monomers and dimers, and the crystal structure of the Cu(II) form shows two Re126W122Cu(II) molecules oriented such that redox cofactors Re(dmp) and W122-indole on different protein molecules are located at the interface at much shorter intermolecular distances (Re-W122(indole) = 6.9 Å, dmp-W122(indole) = 3.5 Å, and Re-Cu = 14.0 Å) than within single protein folds. Whereas forward ET is accelerated by hopping through W122, BET is retarded by a space jump at the interface that lacks specific interactions or water molecules. These findings on interfacial electron hopping in (Re126W122Cu(I))2 shed new light on optimal redox-unit placements required for functional long-range charge separation in protein complexes.

Laser-induced liquid bead ion desorption-MS of protein complexes from blue-native gels, a sensitive top-down proteomic approach

We have developed an experimental approach that combines two powerful methods for proteomic analysis of large membrane protein complexes: blue native electrophoresis (BNE or BN‐PAGE) and laser‐induced liquid bead ion desorption (LILBID) MS. Protein complexes were separated by BNE and eluted from the gel. The masses of the constituents of the multiprotein complexes were obtained by LILBID MS, a detergent‐tolerant method that is especially suitable for the characterisation of membrane proteins. High sensitivity and small sample volumes required for LILBID MS resulted in low demands on sample quantity. Eluate from a single band allowed assessing the mass of an entire multiprotein complex and its subunits. The method was validated with mitochondrial NADH:ubiquinone reductase from Yarrowia lipolytica. For this complex of 947 kDa, typically 30 μg or 32 pmol were sufficient to obtain spectra from which the subunit composition could be analysed. The resolution of this electrophoretic small‐scale approach to the purification of native complexes was improved markedly by further separation on a second dimension of BNE. Starting from a subcellular fraction obtained by differential centrifugation, this allowed the purification and analysis of the constituents of a large multiprotein complex in a single LILBID spectrum.

ATP synthases: cellular nanomotors characterized by LILBID mass spectrometry.

Mass spectrometry of membrane protein complexes is still a methodological challenge due to hydrophobic and hydrophilic parts of the species and the fact that all subunits are bound non-covalently together. The present study with the novel laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) reports on the determination of the subunit composition of the F(1)F(o)-ATP synthase from Bacillus pseudofirmus OF4, that of both bovine heart and, for the first time, of human heart mitochondrial F(1)F(o)-ATP synthases. Under selected buffer conditions the mass of the intact F(1)F(o)-ATP synthase of B. pseudofirmus OF4 could be measured, allowing the analysis of complex subunit stoichiometry. The agreement with theoretical masses derived from sequence databases is very good. A comparison of the ATP synthase subunit composition of 5 different ATPases reveals differences in the complexity of eukaryotic and bacterial ATP synthases. However, whereas the overall construction of eukaryotic enzymes is more complex than the bacterial ones, functionally important subunits are conserved among all ATPases.

Heme-copper terminal oxidase using both cytochrome c and ubiquinol as electron donors

The cytochrome c oxidase Cox2 has been purified from native membranes of the hyperthermophilic eubacterium Aquifex aeolicus. It is a cytochrome ba3 oxidase belonging to the family B of the heme-copper containing terminal oxidases. It consists of three subunits, subunit I (CoxA2, 63.9 kDa), subunit II (CoxB2, 16.8 kDa), and an additional subunit IIa of 5.2 kDa. Surprisingly it is able to oxidize both reduced cytochrome c and ubiquinol in a cyanide sensitive manner. Cox2 is part of a respiratory chain supercomplex. This supercomplex contains the fully assembled cytochrome bc1 complex and Cox2. Although direct ubiquinol oxidation by Cox2 conserves less energy than ubiquinol oxidation by the cytochrome bc1 complex followed by cytochrome c oxidation by a cytochrome c oxidase, ubiquinol oxidation by Cox2 is of advantage when all ubiquinone would be completely reduced to ubiquinol, e.g., by the sulfide∶quinone oxidoreductase, because the cytochrome bc1 complex requires the presence of ubiquinone to function according to the Q-cycle mechanism. In the case that all ubiquinone has been reduced to ubiquinol its reoxidation by Cox2 will enable the cytochrome bc1 complex to resume working.

Analysis of AcrB and AcrB/DARPin ligand complexes by LILBID MS.

The AcrA/AcrB/TolC complex is responsible for intrinsic multidrug resistance (MDR) in Escherichia coli. Together with the periplasmic adaptor protein AcrA and the outer membrane channel TolC, the inner membrane component AcrB forms an efflux complex that spans both the inner and outer membrane and bridges the periplasm of the Gram-negative cell. Within the entire tripartite complex, homotrimeric AcrB plays a central role in energy transduction and substrate selection. In vitro selected designed ankyrin repeat proteins (DARPin) that specifically bind to the periplasmic domain of AcrB were shown to ameliorate diffraction resolution of AcrB/DARPin protein co-crystals (G. Sennhauser, P. Amstutz, C. Briand, O. Storchenegger, M.G. Grutter, Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors, PLoS Biol 5 (2007) e7). Structural analysis by X-ray crystallography revealed that 2 DARPin molecules were bound to the trimeric AcrB wildtype protein in the crystal, whereas the V612F and G616N AcrB variant crystal structures show 3 DARPin molecules bound to the trimer. These specific stoichiometric differences were analyzed in solution via densitometry after microchannel electrophoresis, analytical ultracentrifugation and via laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). Using the latter technology, we investigated the gradual disassembly of the AcrB trimer and bound DARPin ligands in dependence on laser intensity in solution. At low laser intensity, the release of the detergent molecule micelle from the AcrB/DARPin complex was observed. By increasing laser intensity, dimeric and monomeric AcrB species with bound DARPin molecules were detected showing the high affinity binding of DARPin to monomeric AcrB species. High laser intensity LILBID MS experiments indicated a spectral shift of the monomeric AcrB peak of 3.1kDa, representing a low molecular weight ligand in all detergent-solubilized AcrB samples and in the AcrB crystal. The identity of this ligand was further investigated using phospholipid analysis of purified AcrB and AcrB variant samples, and indicated the presence of phosphatidylethanolamine and possibly cardiolipin, both constituents of the Escherichia coli membrane.

LILBID-mass spectrometry of the mitochondrial preprotein translocase TOM

In the present work we applied a novel mass spectrometry method termed laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS) to the outer mitochondrial membrane protein translocon TOM to analyze its subunit composition and stoichiometry. With TOM core complex, purified at high pH, we demonstrate that a TOM core complex of Neurospora crassa is composed of at least two Tom40 and Tom22 molecules, respectively, and more than five small Tom subunits between 5.5 and 6.4 kDa. We show that the multiprotein complex has a total molecular mass higher than 170 depending on the number of Tom5, Tom6 and Tom7 molecules bound.

Mass Spectrometric Characterization of Oligomers in Pseudomonas aeruginosa Azurin Solutions

We have employed laser-induced liquid bead ion desorption mass spectroscopy (LILBID MS) to study the solution behavior of Pseudomonas aeruginosa azurin as well as two mutants and corresponding Re-labeled derivatives containing a Re(CO)(3)(4,7-dimethyl-1,10-phenanthroline)(+) chromophore appended to a surface histidine. LILBID spectra show broad oligomer distributions whose particular patterns depend on the solution composition (pure H(2)O, 20-30 mM NaCl, 20 and 50 mM NaP(i) or NH(4)P(i) at pH = 7). The distribution maximum shifts to smaller oligomers upon decreasing the azurin concentration and increasing the buffer concentration. Oligomerization is less extensive for native azurin than its mutants. The oligomerization propensities of unlabeled and Re-labeled proteins are generally comparable, and only Re126 shows some preference for the dimer that persists even in highly diluted solutions. Peak shifts to higher masses and broadening in 20-50 mM NaP(i) confirm strong azurin association with buffer ions and solvation. We have found that LILBID MS reveals the solution behavior of weakly bound nonspecific protein oligomers, clearly distinguishing individual components of the oligomer distribution. Independently, average data on oligomerization and the dependence on solution composition were obtained by time-resolved anisotropy of the Re-label photoluminescence that confirmed relatively long rotation correlation times, 6-30 ns, depending on Re-azurin and solution composition. Labeling proteins with Re-chromophores that have long-lived phosphorescence extends the time scale of anisotropy measurements to hundreds of nanoseconds, thereby opening the way for investigations of large oligomers with long rotation times.

Identification and characterization two isoforms of NADH:ubiquinone oxidoreductase from the hyperthermophilic eubacterium Aquifex aeolicus

The NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the respiratory chain and the entry point for most electrons. Generally, the bacterial complex I consists of 14 core subunits, homologues of which are also found in complex I of mitochondria. In complex I preparations from the hyperthermophilic bacterium Aquifex aeolicus we have identified 20 partially homologous subunits by combining MALDI-TOF and LILBID mass spectrometry methods. The subunits could be assigned to two different complex I isoforms, named NQOR1 and NQOR2. NQOR1 consists of subunits NuoA2, NuoB, NuoD2, NuoE, NuoF, NuoG, NuoI1, NuoH1, NuoJ1, NuoK1, NuoL1, NuoM1 and NuoN1, with an entire mass of 504.17 kDa. NQOR2 comprises subunits NuoA1, NuoB, NuoD1, NuoE, NuoF, NuoG, NuoH2, NuoI2, NuoJ1, NuoK1, NuoL2, NuoM2 and NuoN2, with a total mass of 523.99 kDa. Three Fe-S clusters could be identified by EPR spectroscopy in a preparation containing predominantly NQOR1. These were tentatively assigned to a binuclear center N1, and two tetranuclear centers, N2 and N4. The redox midpoint potentials of N1 and N2 are -273 mV and -184 mV, respectively. Specific activity assays indicated that NQOR1 from cells grown under low concentrations of oxygen was the more active form. Increasing the concentration of oxygen in the bacterial cultures induced formation of NQOR2 showing the lower specific activity.