Photosynthetic electron transport involved in PxcA-dependent proton extrusion in Synechocystis sp. Strain PCC6803: effect of pxcA inactivation on CO2, HCO3-, and NO3- uptake

Abstract

The product of pxcA (formerly known as cotA) is involved in light-induced Na+-dependent proton extrusion. In the presence of 2, 5-dimethyl-p-benzoquinone, net proton extrusion by Synechocystis sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO2 fixation by glyceraldehyde in a cytochrome c oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO2 fixation is inhibited. At pH 8.0, NO3- uptake activity was very low in the pxcA mutant at low [Na+] (approximately 100 microM). At pH 6.5, the pxcA strain did not take up CO2 or NO3- at low [Na+] and showed very low CO2 uptake activity even at 15 mM Na+. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO2, HCO3-, and NO3- is discussed.

FIG. 1

Net proton movements in suspensions of WT (A, B, D, and E) and pxcA (C and F) cells upon switching the light on (arrow down) and off (arrow up). The cells were suspended in 0.2 mM TES-KOH buffer (pH 8.0) containing 15 mM KCl (A and D) or NaCl (B, C, E, and F) in the absence (A to C) and presence (D to F) of 1 mM DMBQ. The chlorophyll concentration in the cell suspension was 14 μg/ml.

FIG. 2

Net proton movement in the suspensions of psaAB (A to C), psbDIC/psbDII (D and E), and coxAB (F and G) cells upon switching the light on (arrow down) and off (arrow up). The cells were suspended in 0.2 mM TES-KOH buffer containing 15 mM KCl (A) and NaCl (B to G). DMBQ was added prior to illumination in panels C, E, and F. The chlorophyll concentration in the cell suspension was 1.4 μg/ml for the psaAB mutant and 14 μg/ml for the psbDIC/psbDII and coxAB mutants.

FIG. 3

Effect of DMBQ, PNDA, DCMU, and DBMIB on net proton movements in WT Synechocystis cells. The light was switched on (arrow down) and off (arrow up) as indicated. The cells were suspended in 0.2 mM TES-KOH buffer (pH 8.0) containing 15 mM NaCl. DMBQ (final concentration, 1 mM) (D to E), PNDA (3 mM) (G to I), DCMU (20 μM) (B, E, and H), and DBMIB (10 μM) (C, F, and I) were added as indicated. All additions were done prior to illumination.

FIG. 4

Effect of GA, KCN, and DMBQ on net proton movements involving WT (A to C and G to I) and coxAB (D to F) cells of Synechocystis. The light was switched on (arrow down) and off (arrow up). The cells were suspended in 0.2 mM TES-KOH buffer (pH 8.0) containing 15 mM NaCl at a chlorophyll concentration of 14 μg/ml. GA (final concentration, 20 mM) (B, C, E, and F) and KCN (5 mM) (H and I) were added prior to illumination and DMBQ (1 mM) (C, F, and H) was added in the dark to the cell suspensions after the profiles in the presence of the inhibitors were obtained.

FIG. 5

Rates of CO₂, HCO₃⁻, and NO₃⁻ uptake in WT and pxcA cells of Synechocystis at pH 8.0 and pH 6.5 in the presence of 15 mM NaCl (N-Na⁺) or KCl (L-Na⁺).