Figure 3.

Enzymatic activities of the respiratory complexes are not temperature-sensitive in the absence of DsbA. The rates of oxygen consumption were determined polarographically at 25°C using a Clark-type oxygen electrode, in DDM-solubilized chromatophore membranes from cells grown in liquid enriched medium at 25°C under Res conditions. All assays were carried out using 2 ml of 50 mM MOPS, 5 mM MgCl₂, pH 7.2 assay buffer, containing 50–300 μg of DDM-dispersed membrane proteins [protein: detergent (w/w) of 1:1] from wild type and ΔdsbA strains. Depending on the segment of the respiratory chain to be assayed, the O₂ consumption reaction was initiated by addition of 1 mM NADH (for NADH dehydrogenase) (A), 200 μM DBH₂ (for bd-Qox) (B) or 250 μM N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) together with 10 mM sodium ascorbate (for cbb₃-Cox) (C). The same measurements at 25°C were also repeated after incubation of the membranes at 35°C for 2 h (gray bars) to assess the temperature response of the activities. The rates were determined as μmoles of O₂ reduced/min/mg total protein, and are shown as % of oxygen consumed taking as 100% the rate of the wild type strain. Average values of at least 3 independent measurements are shown. Error bars represent the range of values (maximum and minimum) obtained for each condition.