D1-arginine257 mutants (R257E, K, and Q) of Chlamydomonas reinhardtii have a lowered QB redox potential: analysis of thermoluminescence and fluorescence measurements

Abstract

Arginine257 (R257), in the de-helix that caps the Q(B) site of the D1 protein, has been shown by mutational studies to play a key role in the sensitivity of Photosystem II (PS II) to bicarbonate-reversible binding of the formate anion. In this article, the role of this residue has been further investigated through D1 mutations (R257E, R257Q, and R257K) in Chlamydomonas reinhardtii. We have investigated the activity of the Q(B) site by studying differences from wild type on the steady-state turnover of PS II, as assayed through chlorophyll (Chl) a fluorescence yield decay after flash excitation. The effects of p-benzoquinone (BQ, which oxidizes reduced Q(B), Q(B)(-) ) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, which blocks electron flow from Q(A)(-) to Q(B)) were measured. The equilibrium constants of the two-electron gate were obtained through thermoluminescence measurements. The thermoluminescence properties were changed in the mutants, especially when observed after pretreatment with 100 microM BQ. A theoretical analysis of the thermoluminescence data, based mainly on the recombination pathways model of Rappaport et al. (2005), led to the conclusion that the free-energy difference for the recombination of Q(B)(-) with S(2) was reduced by 20-40 mV in the three mutants (D1-R257K, D1-R257Q, and D1-R257E); this was interpreted to be due to a lowering of the redox potential of Q(B)/Q(B)(-). Further, since the recombination of Q(A)(-) with S(2) was unaffected, we suggest that no significant change in redox potential of Q(A)/Q(A)(-) occurred in these three mutants. The maximum variable Chl a fluorescence yield is lowered in the mutants, in the order R257K > R257Q > R257E, compared to wild type. Our analysis of the binary oscillations in Chl a fluorescence following pretreatment of cells with BQ showed that turnover of the Q(B) site was relatively unaffected in the three mutants. The mutant D1-R257E had the lowest growth rate and steady-state activity and showed the weakest binary oscillations. We conclude that the size and the charge of the amino acid at the position D1-257 play a role in PS II function by modulating the effective redox potential of the Q(B)/Q(B)(-) pair. We discuss an indirect mechanism mediated through electrostatic and/or surface charge effects and the possibility of more pleiotropic effects arising from decreased stability of the D1/D2 and D1/CP47 interfaces.

Fig. 1

Structure of the acceptor side of Photosystem II. A Location of the de-helix on the D1 protein. The D1 (red) and D2 (blue) proteins are shown in cartoon representation of the protein backbone. The loop of the D1 protein between transmembrane helices D and E of PS II is shown. D1-R257 mutated in this study is shown as ball-and-stick, located at the C-terminal end of the de-helix. The location of plastoquinones bound at the Q_A and Q_B sites are shown in yellow. The bicarbonate anion and the Fe atom to which it is ligated are shown as ball-and-stick and a van der Waals sphere, respectively. The orientation is parallel to the membrane plane. B Detailed view of the Q_B site and residues near D1-R257. Protein side chains are shown as ball-and-stick attached to a cartoon representation of the protein backbone. The D1 is red, D2 is blue, and CP47 is green. Q_A (yellow), the Fe atom and its ligands (the bicarbonate ion, and four histidines) are shown to the left of the image. Q_B is bound below the de-helix shown in the center of the image. To the right are D1-R257 and the nearby residues D1-Q261, D2-D25, and CP47-E489. The orientation is perpendicular to the membrane plane. Both A and B were created using VMD (Humphrey et al. 1996) from coordinates from PDB file 2AXT (Loll et al. 2005)

Fig. 2

Photoautotrophic growth. C. reinhardtii cells were grown in high salt minimal medium (Sueoka 1960) at 25 °C, bubbled with 3% CO₂ (v/v), and illuminated with 100 μE m⁻² s⁻¹ white light. The growth curve was determined by measuring the optical density (OD) of the cell suspension at 750 nm. Wild type control, pBA157 (open circles); R257K (upside down triangles); R257Q (closed circles); R257E (triangles)

Fig. 3

Variable Chl a fluorescence decay curves, after flashes 1 or 2 (at 1 Hz). Samples from 6 to 7 days old C. reinhardti cultures were treated with 100 μM BQ for 10 min in the dark. Panel A is for pBA157, previously shown to act as wild type; panel B is for R257K; panel C for R257Q, and panel D for R257E. The first flash is designated by squares (black curve), the second flash by circles (red curve). Insets in each of the panels show the sub-ms data of the corresponding curves in that panel. The standard error was approximately the size of the symbols used. F_t is fluorescence at time t, and F₀ is the initial minimal fluorescence. [Chl], 7 μg/ml

Fig. 4

Variable Chl a fluorescence decay curves in the presence of DCMU. Cells of three mutants (R257K, R257Q, and R257E) and the wild type (pBA157) C. reinhardtii were treated as in Fig. 3. DCMU was added to a final concentration of 10 μM. A single actinic flash was delivered after 10 min dark adaptation with the DCMU present. A train of measuring pulses was used up to 10 s. F_t is fluorescence at time t, and F_o is the initial minimal fluorescence. [Chl], 7 μg/ml

Fig. 5

Variable Chl a fluorescence decay curves in the R257E mutant of C. reinhardtii cells, pretreated with different BQ concentrations. Panel A is in the absence of BQ; panel B is with 10 μM BQ; panel C is with 25 μM BQ, and panel D is with 100 μM BQ. The first flash is designated by squares (black curve), the second flash by circles (red curve). Seven-day-old C. reinhardtii cultures of the R257E strain were used, dark adapted for 10 min. F_t is fluorescence at time t, and F_o is the initial minimal fluorescence. [Chl], 7 μg/ml

Fig. 6

Binary oscillations in Chl a fluorescence: the two-electron gate on the acceptor side of PS II. Panel A shows variable Chl a fluorescence data at 195 μs after a series of flashes, given at 1 Hz, for the wild type C. reinhardtii cells; panel B is for R257K; panel C is for R257Q; and panel D is for R257E mutant cells. Cells were treated with 100 μM (squares), 50 μM (circles), 25 μM (triangles), and 10 μM (inverted triangles) of p-benzoquinone. F_t is fluorescence at time t, and F_o is the initial minimal fluorescence. [Chl], 7 μg/ml

Fig. 7

Thermoluminescence curves from DCMU-treated cells. Data show the Q-band (due to ${\text{S}}_2{\text{Q}}_{\text{A}}^{-}$ recombination) in C. reinhardtii cells from wild type (pBA157) and three D1-R257 mutant strains (R257K, R257Q, and R257E). Note that in all cases the Q-band is at ∼ 10°C suggesting that the mutation did not change the ${\text{Q}}_{\text{A}}/{\text{Q}}_{\text{A}}^{-}$ redox potential. Thermoluminescence curves were staggered vertically for clarity. For details, see text

Fig. 8

Thermoluminescence curves of C. reinhardtii cells. Panel A is for the untreated cells of wild type (pBA157) and three D1-R257 mutant strains (R257K, R257Q, and R257E). Panel B is for 25 μM BQ, and panel C is for 100 μM BQ-treated cells. Thermoluminescence curves were staggered vertically for clarity. The ordinate is plotted in arbitrary units (AU)

Scheme 1

The equilibria in the acceptor complex determining K_app