Clarke type oxygen electrode

TbQSOX activity was measured at 25 °C by monitoring oxygen consumption in a Clarke-type oxygen electrode (Hansatech Instruments Ltd). TbQSOX at 50 nM (or 100 nM when measuring activity of certain TbQSOX mutants) was assayed with various DTT concentrations. Reactions were initiated by injection of DTT into the reaction chamber and were repeated three times for each DTT concentration. Turnover numbers were calculated from slopes of oxygen depletion progress curves. Double-injection assays were performed and analysed as follows. According to the Michaelis–Menten model, an enzyme with a K_M of 65 μM is expected to function at 94% of V_max on 1 mM substrate. At increasing substrate concentrations, the ratio of the reaction rate to the rate at 1 mM substrate should plateau at 1.065. Oxygen consumption rates at various DTT concentrations were measured relative to the rate on 1 mM DTT by first supplying TbQSOX (50 nM) with 1 mM DTT, recording the oxygen consumption rate for 1 min, making a second injection of DTT (or only buffer) to bring the final DTT concentration to between 1 and 80 mM, and recording the new oxygen consumption rate. The ratio of the two rates for each sample, after correction for small volume changes, was then taken, and ratios were averaged for multiple measurements at the same final DTT concentration (n=3–6). Error bars represent s.d. of the ratios.

In vivo affinity for bicarbonate of HC- or LC-acclimated cultures of the Synechocystis strains used in this study was determined by the measurement of C_i-dependent photosynthetic activity through a Clarke-type oxygen electrode (Hansatech). Cultures were re-suspended in fresh BG11 pH 8.0 medium at an OD₇₅₀ = 3 (approximately 10 µg chlorophyll a per ml). Measurements were performed on 3 mL of the culture, illuminated with 300 µmol photons m^-2 s^-1 and increasing amounts of HCO₃^-. Oxygen evolution rates, normalized to the chlorophyll a content, were used to calculate the affinities to C_i of each strain as function of K_m values. K_m values were obtained from the linear regression of the bicarbonate/oxygen evolution curve, where V_max = 1/m and K_m = V_max * Y intercept.