Mutagenesis of the L, M, and N subunits of Complex I from Escherichia coli indicates a common role in function |
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Authors: | Michel Jose DeLeon-Rangel Jessica Zhu Shaotong Van Ree Kalie Vik Steven B |
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Institution: | Department of Biological Sciences, Southern Methodist University, Dallas, Texas, United States of America. |
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Abstract: | BackgroundThe membrane arm of Complex I (NADH:ubiquinone oxidoreductase) contains three
large, and closely related subunits, which are called L, M, and N in
E. coli. These subunits are homologous to components of
multi-subunit Na+/H+ antiporters, and so are
implicated in proton translocation.Methodology/Principal FindingsNineteen site-specific mutations were constructed at two corresponding
positions in each of the three subunits. Two positions were selected in each
subunit: L_K169, M_K173, N_K158 and L_Q236, M_H241, N_H224. Membrane
vesicles were prepared from all of the resulting mutant strains, and were
assayed for deamino-NADH oxidase activity, proton translocation,
ferricyanide reductase activity, and sensitivity to capsaicin. Corresponding
mutations in the three subunits were found to have very similar effects on
all activities measured. In addition, the effect of adding exogenous
decylubiquinone on these activities was tested. 50 µM decylubiquinone
stimulated both deamino-NADH oxidase activity and proton translocation by
wild type membrane vesicles, but was inhibitory towards the same activities
by membrane vesicles bearing the lysine substitution at the L236/M241/N224
positions.Conclusions/SignificanceThe results show a close correlation with reduced activity among the
corresponding mutations, and provide evidence that the L, M, and N subunits
have a common role in Complex I. |
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