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Abstract
The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is a multisubunit enzymatic complex. It has a characteristic L-shaped form with two domains, a hydrophilic peripheral domain and a hydrophobic membrane domain. The membrane domain contains three antiporter-like subunits (NuoL, NuoM, and NuoN, Escherichia coli naming) that are considered to be involved in the proton translocation. Deletion of either NuoL or NuoM resulted in an incomplete assembly of NDH-1 and a total loss of the NADH-quinone oxidoreductase activity. We have truncated the C terminus segments of NuoM and NuoL by introducing STOP codons at different locations using site-directed mutagenesis of chromosomal DNA. Our results suggest an important structural role for the C-terminal segments of both subunits. The data further advocate that the elimination of the last transmembrane helix (TM14) of NuoM and the TM16 (at least C-terminal seven residues) or together with the HL helix and the TM15 of the NuoL subunit lead to reduced stability of the membrane arm and therefore of the whole NDH-1 complex. A region of NuoL critical for stability of NDH-1 architecture has been discussed.
Highlights
Enzyme is the principal source of reactive oxygen species in mitochondria and that its deficiencies have been shown to be the origin of many human neurodegenerative diseases [3,4,5]
All subunits of NDH-1 are homologous to the 14 subunits that constitute the core of the mitochondrial complex I [8, 9]. Both eukaryotic complex I and prokaryotic NDH-1 have a characteristic L-shaped form with two clearly defined domains, a hydrophilic peripheral arm projected into the mitochondrial matrix/bacterial cytoplasm, and a hydrophobic arm embedded in the inner mitochondrial/ cytoplasmic membrane [10]
In the present work, using E. coli NDH-1, we attempted to clarify the structural role of the C-terminal segments of NuoL and NuoM that are most distantly located from the peripheral domain
Summary
Enzyme is the principal source of reactive oxygen species in mitochondria and that its deficiencies have been shown to be the origin of many human neurodegenerative diseases [3,4,5]. We measured the electron transfer activities of NDH-1 in membrane vesicles obtained from E. coli MC4100 wild type and the ctNuoL and ctNuoM mutants (Table 1).
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