Membrane vesicles were prepared from cultures of all mutants and wild type grown
in rich media at 30°C. Cultures were shaken at ∼230 rpm and harvested at
A600 = 1.4. The cells were harvested, as
previously described [32] (link), [45] (link). For proton translocation assays membranes underwent
a third centrifugation, 1 hour at 355,000×g. The membranes were tested for
deamino-NADH driven proton translocation by measuring the fluorescence quenching
of ACMA over the course of several minutes, using excitation and emission
wavelengths of 410 and 490 nm respectively. Deamino-NADH oxidase activity assays
and proton translocation assays were performed after the second centrifugation
in 50 mM MOPS, 10 mM MgCl2, pH 7.3 at room temperature, using 150
µg/ml membrane protein. Deamino-NADH oxidase activity was assayed using
oxygen as a terminal electron acceptor. The oxidase assays were started with
0.25 mM deamino-NADH (extinction coefficient 6.22 mM−1cm−1) and the absorbance monitored at 340 nm for 2 minutes.
Decylubiquinone was added from an ethanol stock to the reaction cuvette
containing membranes, and the samples were incubated for several minutes at room
temperature before addition of deamino-NADH. Complex I inhibitor capsaicin was
added at 0.3 mM from a 100 mM ethanol stock. The uncoupler FCCP was added to a
final concentration of 1 µM from a 1 mM ethanol stock. For proton
translocation assays, ACMA was added to 1 µM, while other concentrations
were the same as for oxidase assays. Ferricyanide reductase assays were
conducted at room temperature and the absorbance monitored at 410 nm for 2
minutes in buffer containing 10 mM potassium phosphate (pH 7.0), 1 mM EDTA, 1 mM
K3FeCN6, and 10 mM KCN [46] (link). 40–100 µg/ml of
membrane protein was used in each assay. Ferricyanide was used as the terminal
electron acceptor (extinction coefficient of 1.0 mM−1cm−1). The assays were started with 0.15 mM
deamino-NADH.
in rich media at 30°C. Cultures were shaken at ∼230 rpm and harvested at
A600 = 1.4. The cells were harvested, as
previously described [32] (link), [45] (link). For proton translocation assays membranes underwent
a third centrifugation, 1 hour at 355,000×g. The membranes were tested for
deamino-NADH driven proton translocation by measuring the fluorescence quenching
of ACMA over the course of several minutes, using excitation and emission
wavelengths of 410 and 490 nm respectively. Deamino-NADH oxidase activity assays
and proton translocation assays were performed after the second centrifugation
in 50 mM MOPS, 10 mM MgCl2, pH 7.3 at room temperature, using 150
µg/ml membrane protein. Deamino-NADH oxidase activity was assayed using
oxygen as a terminal electron acceptor. The oxidase assays were started with
0.25 mM deamino-NADH (extinction coefficient 6.22 mM−1cm−1) and the absorbance monitored at 340 nm for 2 minutes.
Decylubiquinone was added from an ethanol stock to the reaction cuvette
containing membranes, and the samples were incubated for several minutes at room
temperature before addition of deamino-NADH. Complex I inhibitor capsaicin was
added at 0.3 mM from a 100 mM ethanol stock. The uncoupler FCCP was added to a
final concentration of 1 µM from a 1 mM ethanol stock. For proton
translocation assays, ACMA was added to 1 µM, while other concentrations
were the same as for oxidase assays. Ferricyanide reductase assays were
conducted at room temperature and the absorbance monitored at 410 nm for 2
minutes in buffer containing 10 mM potassium phosphate (pH 7.0), 1 mM EDTA, 1 mM
K3FeCN6, and 10 mM KCN [46] (link). 40–100 µg/ml of
membrane protein was used in each assay. Ferricyanide was used as the terminal
electron acceptor (extinction coefficient of 1.0 mM−1cm−1). The assays were started with 0.15 mM
deamino-NADH.
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