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Mutagenesis of the L, M, and N subunits of Complex I from Escherichia coli indicates a common role in function.

Michel J, DeLeon-Rangel J, Zhu S, Van Ree K, Vik SB - PLoS ONE (2011)

Bottom Line: The 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.Corresponding mutations in the three subunits were found to have very similar effects on all activities measured.The 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, Texas, United States of America.

ABSTRACT

Background: The 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 findings: Nineteen 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/significance: The 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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Related in: MedlinePlus

Comparison of proton translocation rates with mutations at the first                        site: L_K169, M_K173, N_K158.The reactions were initiated with deamino-NADH (dNADH) to 250 µM final                        concentration. The fluorescence of ACMA (1 µM) was followed for                        several minutes. The uncoupler FCCP was added (1 µM) to collapse the                        generated proton gradient. In each panel the wild type strain is shown for                        comparison. The traces shown are representative of 2–3 experiments.                                (A) L subunit mutations K169C, E, R.                                (B) M subunit mutations K173C, E, R.                                (C) N subunit mutations K158C, E,                        R.
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pone-0017420-g005: Comparison of proton translocation rates with mutations at the first site: L_K169, M_K173, N_K158.The reactions were initiated with deamino-NADH (dNADH) to 250 µM final concentration. The fluorescence of ACMA (1 µM) was followed for several minutes. The uncoupler FCCP was added (1 µM) to collapse the generated proton gradient. In each panel the wild type strain is shown for comparison. The traces shown are representative of 2–3 experiments. (A) L subunit mutations K169C, E, R. (B) M subunit mutations K173C, E, R. (C) N subunit mutations K158C, E, R.

Mentions: Proton translocation assays were also carried out in preparations of membrane vesicles. The rates of deamino-NADH driven proton translocation were assessed by the quenching of fluorescence of the acridine dye ACMA (9-amino-6-chloro-2-methoxyacridine). For each of the 19 mutants the rate of proton translocation closely paralleled the rate of deamino-NADH oxidase. The rates for all three mutations at residues L_K169, M_K173, and N_K158, are shown in Figure 5, panels A, B, and C, respectively. Each assay is initiated by the addition of deamino-NADH, and completed with the addition of FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone), which collapses any proton gradient that has formed. In each panel, the rate of the wild type is rather more than that of the highest mutant, which is the K→R substitution. In each case, the other two substitutions, C and E, have lower rates than R. For comparison with deamino-NADH oxidase rates, see Figure 3.


Mutagenesis of the L, M, and N subunits of Complex I from Escherichia coli indicates a common role in function.

Michel J, DeLeon-Rangel J, Zhu S, Van Ree K, Vik SB - PLoS ONE (2011)

Comparison of proton translocation rates with mutations at the first                        site: L_K169, M_K173, N_K158.The reactions were initiated with deamino-NADH (dNADH) to 250 µM final                        concentration. The fluorescence of ACMA (1 µM) was followed for                        several minutes. The uncoupler FCCP was added (1 µM) to collapse the                        generated proton gradient. In each panel the wild type strain is shown for                        comparison. The traces shown are representative of 2–3 experiments.                                (A) L subunit mutations K169C, E, R.                                (B) M subunit mutations K173C, E, R.                                (C) N subunit mutations K158C, E,                        R.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046159&req=5

pone-0017420-g005: Comparison of proton translocation rates with mutations at the first site: L_K169, M_K173, N_K158.The reactions were initiated with deamino-NADH (dNADH) to 250 µM final concentration. The fluorescence of ACMA (1 µM) was followed for several minutes. The uncoupler FCCP was added (1 µM) to collapse the generated proton gradient. In each panel the wild type strain is shown for comparison. The traces shown are representative of 2–3 experiments. (A) L subunit mutations K169C, E, R. (B) M subunit mutations K173C, E, R. (C) N subunit mutations K158C, E, R.
Mentions: Proton translocation assays were also carried out in preparations of membrane vesicles. The rates of deamino-NADH driven proton translocation were assessed by the quenching of fluorescence of the acridine dye ACMA (9-amino-6-chloro-2-methoxyacridine). For each of the 19 mutants the rate of proton translocation closely paralleled the rate of deamino-NADH oxidase. The rates for all three mutations at residues L_K169, M_K173, and N_K158, are shown in Figure 5, panels A, B, and C, respectively. Each assay is initiated by the addition of deamino-NADH, and completed with the addition of FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone), which collapses any proton gradient that has formed. In each panel, the rate of the wild type is rather more than that of the highest mutant, which is the K→R substitution. In each case, the other two substitutions, C and E, have lower rates than R. For comparison with deamino-NADH oxidase rates, see Figure 3.

Bottom Line: The 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.Corresponding mutations in the three subunits were found to have very similar effects on all activities measured.The 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, Texas, United States of America.

ABSTRACT

Background: The 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 findings: Nineteen 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/significance: The 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.

Show MeSH
Related in: MedlinePlus