Assembly of E. coli complex I and its interaction with other proteins
Final Report Abstract
Respiratory chains convert the free energy derived from cell metabolism into an ion gradient across the membrane. This gradient is used to drive energy-consuming processes. One major component of aerobic respiratory chains is the NADH:ubiquinone oxidoreductase, respiratory complex I. Complex I couples the electron transfer from NADH to ubiquinone with a proton-translocation across the membrane. In humans, a dysfunction of complex I is associated with several neurodegenerative diseases and aging. The bacterial homologue is often used as a less complicated model to study possible causes for dysfunctions. The bacterial complex I from Escherichia coli is composed of 13 different subunits called NuoA to NuoN and contains one flavin mononucleotide and 9 iron-sulfur (Fe/S) clusters as cofactors. The assembly of such a complicated molecular machine, its detailed function and its interaction with other components of the aerobic respiratory chains are still unclear. We have shown that all 13 subunits of the complex are essential for the assembly of a stable and active enzyme. Inactivation of the gene nuoL resulted in the accumulation of an intermediate in the mutant membrane. Purification of the intermediate led to two populations. One population showed full electron transfer activity but only half of the proton translocation activity most likely due to the lack of an unusual 110 Å long amphipathic helix that comprises the C-terminal part of NuoL. Furthermore, we showed that a conserved amino acid on that helix is essential for the translocation of one proton and that this helix does not significantly move during catalytic activity. The other population was inactive due to the lack of the most distal Fe/S-cluster. Surprisingly, this population was equipped with a non-cognate protein, the inducible lysine decarboxylase. We propose that this protein keeps the population in an assembly competent state contributing to the formation of the missing Fe/S-cluster. To determine the distribution and dynamics of enzyme complexes of aerobic respiratory chains in E. coli we fused fluorescent proteins to NADH- and succinatedehydrogenase, to cytochromes bd-I and bo terminal oxidases and to ATP-synthase by chromosomal recombination. With different pairs of labeled enzyme complexes and using real-time multicolor single-molecule fluorescence imaging and superresolution particle tracking on live cells of we found that the enzyme complexes are compartmentalized as assemblages of distinct, dynamic islands indicating an organization that is unexpectedly different to that found in mitochondria where socalled supercomplexes are formed.
Publications
- (2011) Engineering the respiratory complex I to an energyconverting NADPH:ubiquinone oxidoreductase. J. Biol. Chem. 286, 34267-34634
Morina, K., Schulte, M., Hubrich, F., Dörner, K., Steimle, S., Stolpe, S., and Friedrich, T.
(See online at https://doi.org/10.1074/jbc.M111.274571) - (2012) Asp563 of the horizontal helix of subunit NuoL is involved in proton translocation by the respiratory complex I. FEBS Lett. 586, 699-704
Steimle, S., Willistein, M., Hegger, P., Janoschke, M., Erhardt, H. and Friedrich, T.
(See online at https://doi.org/10.1016/j.febslet.2012.01.056) - (2012) Disruption of individual nuo-genes leads to the formation of partially assembled NADH:ubiquinone oxidoreductase (complex I) in Escherichia coli. Biochim. Biophys. Acta 1817, 863-871
Erhardt, H., Steimle, S., Muders, V., Pohl, T., Walter, J., and Friedrich, T.
(See online at https://doi.org/10.1016/j.bbabio.2011.10.008) - (2014) Organization of the Escherichia coli aerobic enzyme complexes of oxidative phosphorylation in dynamic domains within the cytoplasmic membrane. MicrobiologyOpen 33, 316-326
Erhardt, H., Dempwolff, F., Pfreundschuh, M., Riehle, M., Schäfer, C., Pohl, T., Graumann, P. and Friedrich, T.
(See online at https://doi.org/10.1002/mbo3.163) - (2014): Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation. In: Biochimica et biophysica acta 1837 (6), S. 811–824
Llorente-Garcia, I., Lenn, T., Erhardt, H., Harriman, O.L., Liud, L., Robson, A., Chiua, S-W., Matthews, S., Willis, N.J., Brayd, C.D., Liphardt, J., Friedrich, T., Mullineaux, C.W. and Leake, M.C.
(See online at https://doi.org/10.1016/j.bbabio.2014.01.020) - (2015) Cysteine scanning reveals minor local rearrangements of the horizontal helix of respiratory complex I. Mol. Microbiol. 98, 151-161
Steimle, S., Schnick, C., Burger, E-M, Nuber, F., Krämer, D., Dawitz, H., Brander, S., Matlosz, B., Schäfer, J., Maurer, K., Glessner, U. and Friedrich, T.
(See online at https://doi.org/10.1111/mmi.13112) - (2016) Assembly of the Escherichia coli NADH:ubiquinone oxidoreductase (respiratory complex I). Biochim. Biophys. Acta 1857, 214-223
Friedrich, T., Kreuzer-Dekovic, D. and Burschel, S.
(See online at https://doi.org/10.1016/j.bbabio.2015.12.004)
