Site Specific Mutagenesis Reveals a Critical Role of Histidine 252 of the D1 subunit in the Two-Electron Gate of Photosystem IISean Padden1, Jun Minagawa2, Atsuko Kanazawa3, Govindjee4, Antony Crofts5

1 Program in Physiological and Molecular Plant Biology , School of Integrative Biology, University of Illinois at Urbana-Champaign, 286 Morrill Hall, 505 South Goodwin Ave., Urbana, IL 61801, USA; 2Division of Environmental Photobiology, National Institute for Basic Biology, Okazaki 444-8585, Japan 3: Michigan State University, East Lansing, MI 48824-1312 USA; 4 Department of Plant Biology, University of Illinois at Urbana-Champaign 265 Morrill Hall, 505 S. Goodwin Ave., Urbana IL 61801, USA5: Department of Biochemistry, University of Illinois at Urbana-Champaign,

419 Roger Adams Lab, 600 S. Morrill Ave., Urbana IL 61801, USA

Abstract:

Site-directed mutagenesis of histidine 252 in the QB site of the D1 protein in Photosystem II of the green alga Chlamydomonas reinhardtii has revealed a plausible route for the first proton transfer to the plastosemiquinone at the QB site. When D1H252 was mutated, only one mutation retained photosynthetic capacity (H252D) while three others were rendered non-photosynthetic (H252K, N, Q). In all mutants, the first electron transfer from QA

- to QB was altered to various degrees, the timing of the back-reaction from the acceptor side to the oxygen evolving complex was modified in the presence of diuron, and only the H252D mutant could produce oxygen under constant light. Thermoluminescence data from the H252 mutants showed changes in the thermodynamic profile of the QB-site and an effect on the QA

-/S2 equilibrium constant. The results suggest that H252 facilitates protonic stabilization of the semiquinone intermediate, and is required for H+ transfer associated with the reduction of QB

- on the second electron transfer. When compared to the QB site of the bacterial reaction center, a semi-conserved route of proton transfer is revealed.

Fig. 5: TL analysis of the WT and D1H252 mutant strains of C. reinhardtii WT and D1H252 mutant cells. Fig 5A and 5B: black: H252D; red: H252K; blue: H252Q; teal: pBA158 (WT): A: TAP pH 7.0 100 µM BQ wash, 10 µM 18crown6; B: as A plus 1 µM DCMU C: Fig 5C: mutant – WT from Fig 5A; Fig 5D: mutant – WT from Fig 5B where black: H252D; red: H252K; blue: H252Q.

Introduction:

The two – electron gate is the current paradigm for the electron transfer on the acceptor side of Photosystem II (PS II) (Bouges-Bocquet, 1973; Velthyus, 1974, 1981). In this model, the tightly bound quinone QA passes electrons in two sequential transfers to the weakly bound quinone at the QB site. Electron transfer from QA

- to QB produces a semiquinone anion (QB-) which is stabilized at the QB site.

On the basis of a model to account for the pH dependence of the QAQB reaction (Robinson et. al., 1984), Crofts et al., (1987) suggested that D1H252 in the D1 protein of PSII might be the ‘putative ligand for the proton which stabilizes the semiquinone’. This proposal was based on a model of the PSII acceptor side using the B. viridis structure (Michel et. al., 1986) as a template for the D1 protein from Anacystis nidulans (Golden et. al. 1985).

In the bacterial reaction center (BRC) the route for the first protonation event has been termed the ‘serine hydrogen bond switch’ (Wraight, 2004). In this mechanism when in the dark LS223 is hydrogen bonded to LD213, and the quinone is in the distal position. When exposed to light LD213 protonates allowing LS223 to hydrogen bond to the quinone, forming a network from LD213 – LS223 –QB for proton transfer as the quinone moves into the proximal position (Fig. 1A). In PSII the crystal structures (Loll, et. al. 2005, Guskov et. al. 2009) are in agreement with the model of Crofts et al. 1987 where D1H252 corresponds to LD213 and D1S264 corresponds to LD223. Thus a similar system exists in PSII (Fig. 1B) but with one caveat. In the BRC LE212 protonates on electron transfer stabilizing the anionic semiquinone (Paddock et. al. 1989), but the QB site of PSII does not have an equilivalent residue. Instead D1H252 must serve as both the stabilizing influence for the anionic semiquinone and the initial proton.

In order to test if D1H252 was the ligand used to stabilize the semiquinone as proposed we mutated the residue in the unicellular green alga, Chlamydomonas reinhardtii The mutations constructed were D1H252D, D1H252K, D1H252N and D1H252Q. In each of these mutants the following parameters were measured: QA

- fluorescence decay out to 10 ms (Fig. 2), the binary oscillation of the two-electron gate (Fig. 3), production of oxygen under continuous light (Table 1), the kinetics of back reaction from DCMU inhibited cells (Fig. 4), and the thermoluminescence properties of the acceptor side of PSII (Fig. 5). These measurements were used to calculate the binding constants of quinone at the QB site, the individual rate constants of the electron transfer, and the overall equilibrium constant for the QAQB electron transfer (Crofts et. al. 1993, Petrouleas et. al. 2005 and see the Kinetics section).

References:

Bouges-Bocquet, B. (1973). "Electron transfer between the two photosystems in spinach chloroplasts." Biochimica et Biophysica Acta 314(2): 250-256.Crofts, A., I. Baroli, et al. (1993). "Kinetics of Electron Transfer Between QA and QB in Wild Type and Herbicide-Resistant Mutants of Chlamydomonas reinhardtii." Z. Naturforsch 48c: 259-266.Crofts, A., H. Robinson, et al., Eds. (1987). Catalytic Sites for Reduction and Oxidation of Quinones. Cytochrome Systems: Molecular Biology and Bioenergetics, Plenum Publishing Corporation, New York.Michel H, Epp O, J. Deisenhofer (1986) “Pigment-protein interactions in the photosynthetic reaction centre from Rhodopseudomonas viridis.” EMBO J. 5(10):2445–2451.Golden, S. S. and R. Haselkorn (1985). "Mutation to herbicide resistance maps within the psbA gene of Anacystis nidulans R2." Science 229(4718): 1104-1107.Guskov, A., J. Kern, et al. (2009). "Cyanobacterial photosystem II at 2.9-A resolution and the role of quinones, lipids, channels and chloride." Nat Struct Mol Biol 16(3): 334-342.Heifetz, P. B., B. Forster, et al. (2000). "Effects of Acetate on Facultative Autotrophy in Chlamydomonas reinhardtii Assessed by Photosynthetic Measurements and Stable Isotope Analyses." Plant Physiol. 122(4): 1439-1446.Loll, B., J. Kern, et al. (2005). "Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II." Nature 438(7070): 1040-1044.Paddock, M. L., S. H. Rongey, et al. (1989). "Pathway of proton transfer in bacterial reaction centers: replacement of glutamic acid 212 in the L subunit by glutamine inhibits quinone (secondary acceptor) turnover.“ PNAS 86(17): 6602-6606.Petrouleas, V. and A. Crofts, Eds. (2005). The Iron-Quinone Acceptor Complex. Photosystem II: The Light-Driven Water:Plastoquinone Oxidoreductase. Dordrecht, The Netherlands, Springer.Robinson, H. H. and A. R. Crofts (1983). "Kinetics of the oxidation--reduction reactions of the photosystem II quinone acceptor complex, and the pathway for deactivation." FEBS Letters 153(1): 221.Rutherford, A. W., A. R. Crofts, et al. (1982). "Thermoluminescence as a probe of Photosystem II photochemistry. The origin of the flash-induced glow peaks." Biochimica et Biophysica Acta (682(3): 457.Stowell, M. H. B., T. M. McPhillips, et al. (1997). "Light-Induced Structural Changes in Photosynthetic Reaction Center: Implications for Mechanism of Electron-Proton Transfer." Science 276(5313): 812-816.Velthuys, B. and J. Amesz (1974). "Charge accumulation at the reducing side of system 2 of photosynthesis." Biochimica et Biophysica Acta 333(1): 85-94.Velthuys, B. R. (1981). "Electron-dependent competition between plastoquinone and inhibitors for binding to photosystem II." FEBS Letters 126(2): 277-281.Wraight, C. A. (2004). "Proton and Electron Transfer in the Acceptor Quinone Complex of Photosynthetic Reaction Centers from Rhodobacter sphaeroides." Frontiers in Biosciences 9: 309-337.

Results: Thermoluminesence:

Thermoluminescence was measured on the D1H252 mutants and the wild type cells. A major observation was that the B-band of thermoluminescence (due to S2/QB

recombination, Rutherford et al. (1982)), from the mutants was shifted to lower temperatures (Fig. 5A, Table IV),while the position of the Q-band remained unchanged (Fig. 5B, Table IV). When the WT data was subtracted from the mutant data, scaled and normalized, the position of the Q-Band did not change, and the height of the peaks represent the population of electrons left on QA

- (Fig 5C). When the WT curve was subtracted from the mutant curve in the presence of DCMU (Fig 5D) it was observed that the height of the Q-band relative to the WT was increased when a negative charge was placed in the QB-site (D1H252D) leaving a positive peak, and decreased when a positive charge was placed in the QB-site (D1H252K), forming a negative trough. The D1H252Q strain retains a Q-band which has a Tm much lower than the other two mutants, and also displays a B-band indicating DCMU resistance.

Fig. 4. Variable Chl a fluorescence decayWhole cells of four mutants (H252D, H252K, H252N, H252Q) and the wild type (pBA158) 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 seconds. F t is fluorescence at time t; and F0 is the initial minimal fluorescence. [Chl], 7 -10 μg/mL. Half-times are reported in Table 3. curves in the presence of DCMU.

Figure 3: Binary oscillation in chlorophyll a fluorescence decay, measured at 195 µs after the flash, as a function of flash number in mutant strains of Chlamydomonas reinhardtii (ac-µ-ε pBA158 and pBA155). Flashes were given at 1 Hz. Panel A: D1H252D; panel B: D1H252K; panel C: D1H252N; and panel D: D1H252Q. Whole cells of the mutant strains were treated as in Figure 1, with the inclusion of 100 µM NH4Cl as done with the WT. For panel A, the delay between the actinic flash and the first flash is: 0.8 s (black squares), 0.9 s (open squares), 1 s (black circles), and 5s (open circles). For panels B, C and D, the delay is 1s (black squares), 5 s (open squares), 10 s (black circles), 20 s (open circles), and 30 s (black triangles). Each curve is an average of 3 samples from 7 day old secondary cultures, with an average [Chl] of 7 - 10 µg/mL. All binary oscillation curves are offset for clarity.

‘The Acetate Effect’:

The acetate effect on the growth of Chlamydomonas is of paramount importance to the study of the D1H252 mutations. As E. coli preferentially chooses glucose over fructose as a carbon source when mixed, Chlamydomonas will choose acetate over photosynthetic growth (Heifitz, 2000). Since all D1H252 mutations are lethal (even the D1H252D) once the acetate is consumed the mutants begin a downward spiral towards death. This makes analysis of the kinetics of the two electron gate problematic . The acetate effect may even have an effect on the WT strain, as the photosynthetic capacity of the organism will mature as the acetate is consumed.

Fig 2 The [QA-] fluorescence decay of the C.

reinhardtii pBA158 strain and the D1H252 mutants where the first flash is a black line with black squares and the second flash is a red line with red circles. Panel A: pBA158 (WT); panel B D1H252D; panel C D1H252K; panel D D1H252N; panel E D1H252Q.

AO and BO are the amplitudes of the slow and fast phase respectively obtained directly from the fluorescence decay curves (Fig. 1 panel A – E). Ft-(AO+BO) is the residual of the curves and is equal to “C” in the binary decay equation: y = AO(e-x*r

1) + BO(e-x*r2) + C. KO is the ratio (AO/BO), r1 and r2 are the rates of electron transfer for the slow and fast phase,

while t1 and t2 are the half-times for the slow and fast phase respectively. (Crofts et. al. 1993, Petrouleas, 2005)

C. reinhardtii strainsO2- uptake

µmol O2 mg[Chl] -1 / hr

pBA158 262 +/-5

H252D 145+/-10

H252K 5+/-5

H252N nd

H252Q nd

ac-µ-ε nd

Temperature and TL PeaksMutant No DCMU, +/- 2oC DCMU (1 µM), +/- 2oC

Temp Max. Peak Temp Max. PeakpBA158 39 88 15 44H252D 19 99 15 142H252K 30 30 13 53H252Q 32 39 17 70

Results: Kinetic Analysis of the D1H252 Mutants:

Kinetic analysis of the two-electron gate in Photosystem II, based on the data in Figs. 2, 3, and 4 for the WT and the D1H252 mutants, is presented in Table 1 above. The collected data shows the D1H252 mutants affect the slow and fast rates of electron transfer; where the rate for electron transfer for the fast phase fit of the D1H252K, D1H252N and D1H252Q mutants at pH 7.0 was similar to that of WT, with similar half-times; whereas in the D1H252D mutant the half time of the fast phase was more than doubled. In the case of the slow phase, the D1H252D, D1H252N, and D1H252Q had similar half times, whereas the D1H252K increased the half time more than five times longer than the WT.

Table 4: Determination of maximal peak height at temperature of the thermoluminescence bands

Table 3: Values for the production of oxygen, with DCPIP as an electron acceptor, in cells of C. reinhardtii

Results: Chlorophyll a fluorescence decay.

[When the D1H252 mutants were struck on media without a reduced carbon source, the WT strain showed colonies in ~ 14 days, the D1H252D mutation in ~ 21 days, while the D1H252K, N, and Q mutants failed to show colonies out to ~28 days (data not shown). ]

After dark adaptation, the D1H252D mutant showed electron transfer from QA

- to QB on both the odd and even flashes (Fig. 2. panel B), but the rate of the electron transfer after the first flash was slower than after the second flash, leading to a reversed phase pattern of the binary oscillation at the two-electron gate (Fig.3 panel A). The D1H252N and D1H252Q mutants showed electron transfer from QA

- to QB with reduced yield on the first flash; and both were severely inhibited on the second flash (Fig. 2 panel D, E). The D1H252K mutant was severely inhibited even on the first flash. (Fig. 2, panel C). In addition to D1H252D only the D1H252Q mutation had any binary oscillation present. (Fig. 3, panel d) in the same phase as the WT (WT curve not shown). The data derived from the fluorescence decay is presented in Table 1 in the Kinetics section)

Results: Oxygen Evolution

Steady-state oxygen evolution activity of the cells was measured in TAP medium with a Clark-type oxygen electrode in the presence of 0.5 mM 2,5-dimethyl-p-benzoquinone (DMBQ) and 2 mM K3Fe(CN)6 at 25°C under illumination at 3,000 μmol photons m−2 s−1 using dichlorophenol indophenol (DCPIP) as an electron acceptor. Each strain was assayed in at least two separate cultures, with an average of three measurements for a single culture.

Oxygen evolution data show that the pBA158 strain produced the most oxygen at 262 µmol O2 mg[Chl]-1 / hr, while the D1H252D mutant produced ~50% of the WT value, ~145 µmol O2 mg[Chl]-1 / hr. The D1H252N and D1H252Q mutants failed to produce any oxygen. (Table 3). We conclude that the disruption of oxygen production was a result of the D1H252 mutations on the equilibrium constant between QA and S2 of the OEC.

Results: The QA/S2 Back Reaction

When the WT and mutants strains were treated with 10 µM DCMU the timing of the back-reaction from the acceptor side to the oxygen evolving complex was modified (Fig. 4, Table 3) in each of the mutants. The D1H252D and D1H252K mutants had the fastest back reaction, ~5X sooner than WT. The D1H252N had a back reaction ~ one-half that of WT, where the D1H252Q mutant had the most similar time.

Strain AO

(slow)

BO

(fast)

ResidualFt-(AO+BO)

r1

(ms-1)

t1

(ms)

r2

(ms-1)

t2

(µs)

H252D 0.182 0.186 0.74 0.42 1.66 2.60 270

H252K 0.214 0.063 0.78 0.13 5.47 6.26 110

H252N 0.177 0.618 0.35 0.42 1.65 7.69 90

H252Q 0.176 0.269 0.52 0.28 2.47 5.55 120

Table 1: Kinetic parameters determined from first flash of the fluorescence decay curves of the WT and mutant strains of C. reinhardtii

Results : Kinetic Analysis of the D1 H252 mutants (cont.)

The effect of the D1H252 mutations can be seen in the equilibrium constants KO, KE and Kapp (Table 2 below, see scheme above for the description of the equilibrium constants). In the case of KO, it appears as if each of the mutations, except the D1H252N mutation, has an effect on the binding of plastoquinone to the QB site. The D1H252K mutation had the most dramatic effect, decreasing the binding of plastoquinone by almost twenty times, thereby practically eliminating the fast phase of kinetics (Fig. 2, panel B), and binary oscillation (Fig 3, panel B). The D1H252Q mutant inhibited plastoquinone binding by only a factor of ~3, whereas the D1H252D mutant was approximately five times worse than the WT. The D1H252N mutant showed a similar affinity for quinone binding, yet was non-photosynthetic. The effect of the mutations on the binding of the plastoquinone also had an impact on the equilibrium constant between QA

-QB and QAQB- (KE). As

seen in Table 2, in the WT the QAQB- state is heavily favored with a KE value of ~ 29, while both the

D1H252D and D1H252Q values for KE were much closer to equilibrium, 4.9 and 3.9 respectively. As the apparent equilibrium constant, Kapp is calculated from KO and KE, it follows that changes which affect the binding of plastoquinone in the QB-site along with an increase in the QA

-QB population relative to the QAQB

- population will lead to a decrease in Kapp.

Table 2: Parameters of the two electron gate in wild type and mutant strain of C. reinhardtii

Strain t1/2 QA

(s)

t1/2 QB

(s)

KO Kapp KE kAB

(ms-1)

kBA

(ms-1)

kAV

(ms-1)

kVA

(ms-1)

pBA158 0.97 25 0.18 24.7 29.3 6.74 0.23 0.13 0.72

H252D 0.27 0.95 0.98 2.47 4.88 7.37 1.51 1.41 1.44

H252K 0.20 N/D 3.42 N/A N/A N/A N/A N/A N/A

H252N 0.42 N/D 0.29 N/A N/A N/A N/A N/A N/A

H252Q 0.90 3.0 0.65 2.33 3.86 1.12 .29 .63 .97

The t1/2 QA is the half-time of the DCMU back reaction QA- S2 (Fig. 4), and the t1/2 QB is the half-time of the back

reaction from QB S2 obtained from the rephrasing of the binary oscillation (Fig. 3). KO is equilibrium constant for the binding of the semiquinone to the QB-site, KE is the equilibrium constant for the sharing of the electron between QA and QB, Kapp is the apparent equilibrium constant between QA and QB. The rate constants kAB, kBA, kAV, and kVA are for the forward and reverse rate constants of the KE and KO equilibrium constants respectively. The binary oscillation for the D1H252K and D1H252N mutants was not detected (N/D). Thus the kinetic parameters could not be calculated and are not available (N/A) (Crofts et. al. 1993, Petrouleas et al., 2005).

Concluding Remarks: A Hypothesis

The effect of the QB-site mutations on the timing of the back reaction QA/S2 is dependent on the charge, polarity and position of the mutation relative to the plastoquinone molecule. Under the assumption that DCMU binds similar to plastoquinone, the model interaction is presented in Fig 6. The WT model is presented in Fig. 6, panel a, and shows the ‘serine hydrogen bond switch’ in PSII where a hydrogen bond network travels from D1H252 to D1S264 and onto O1 carbonyl group of the plastoquinone (O1). The D1H252D (Fig. 6, panel B) mutation disrupts this network by forming a second hydrogen bond to O1, and eliminating the D1H252 to D1S264 hydrogen bond. The D1H252K (Fig. 6, panel C) mutation can form a hydrogen bond to O1, but the lysine’s pkA is not suited to lose a proton. Correspondingly both of these mutations introduce a charge in to QB-site and rapidly increase the rate of the QA

-/S2 back reaction (Fig. 4), yet have an opposite effect on the height of the TL peak in the presence of DCMU (Fig. 5, panel D). The D1H252N was the only mutation to have two minimum energy states for the model (Fig. 6, panels d and e). The D1H252N mutant was also the least stable (hence no TL analysis). The two models show that the hydrogen bond between D1S264 and D1N252 is disrupted (Fig. 6, panel d) or weak (Fig. 6, panel e). In either case the D1H252N mutation will not release a proton to D1S264. The D1H252N mutation speeds up the QA

-/S2 back reaction by about 2x compared to WT (Fig. 4). The extra –CH2– group in the side chain of the D1H252Q mutant forces that mutant away from the O1, precluding the possibility of a hydrogen bond (Fig. 6, panel f). Without any quinone interaction, the half time of the QA/S2 back reaction is most similar to WT (Fig. 4). It is the lack of protonation of the anionic semiquinone in the D1H252K, D1H252N, and D1H252Q mutants which leads to the strains being non-photosynthetic and dependent on acetate for survival.

The effect of mutation of the ‘serine hydrogen bond shift’ (Wraight, 2004) is dependent on the position of the mutation. If the ‘front end’ of the switch is mutated (LD213 or D1H252 Fig. 1A and 1B) R. sphaeroides will resort to suppressor mutations in order to survive. No suppression mutants for the D1H252 mutations were seen in C. reinhardtii. Conversely, if the ‘back-end’ of the serine hydrogen bond switch (LS223 or D1S264 Fig 1A and 1B) is mutated there is an effect on the two-electron gate but both R. sphaeroides and C. reinhardtii survive without further modification. This relationship further emphasizes the importance of the ‘front-end’ of the ‘serine hydrogen bond switch’ in its relation to the first proton transfer to the anionic semiquinone at the QB-site.

Fig 1: VMD model of the ‘serine hydrogen bond switch’ (Wraight, 2004) . Final assembly of each figure was performed by Photoshop CS5 Extended.

1A: PDB 1AIG, (Stowell et. al. 1997). Bacterial reaction center. The green surf and coil (transparent) corresponds to the L sub-unit, the ice-blue surf represents the M sub-unit, and the blue surf shows the H sub-unit. The ubiquinone molecule is drawn as ‘licorice’, with the highlighted residues drawn as ball and stick. Carbon is yellow, oxygen red, nitrogen blue. The white dashed lines are modeled hydrogen bonds between the residues with the distance posted close by.

1B: PDB 3BZ1 (Guskov et. al. 2009). Photosystem II. The green surf and coil (transparent) corresponds to the D1 protein and the ice-blue surf represents the D2 protein. The plastoquinone molecule is drawn as ‘licorice’ with the highlighted residues drawn as ball and stick. Carbon is yellow, oxygen red, nitrogen blue. The white dashed lines are modeled hydrogen bonds between the residues with the distance posted close by.

Fig, 6: The PSII crystal structure 2AXT from (Loll et al., 2005) was used to model the QB site of PSII. Using the mutation analysis software within Swiss PBV Viewer 4.01 PC, the mutation was modeled in place of the WT histidine. The energy minimization was performed by Swiss PDB-viewer and the figures were re-modeled using POV-Ray 6.2 software. The images created were assembled and converted to .tiff format using Photoshop CS4 Extended. The golden helices indicate the helices d (with H215), and de (with H252 and S264) of the D1 protein of PSII with the random coil between the helices colored brown. Panel a: D1H252 (WT); panel b: D1H252D; panel c: D1H252K, panel d: D1H252N 1st minimization; panel e: D1H252N 2nd minimization; panel f: D1H252Q. The dashed green lines are modeled hydrogen bonds, the dashed purple line is a modeled steric hindrance (D1H252N panel d), and the dashed gray line is a weak hydrogen bond due to distance (D1H252N panel e). Only the D1H252N has two images as it was the only mutant to have two equal calculated energy minimizations.