**5.3 Oxygen evolution assays and photoinactivation assays**

Oxygen evolution activity was measured by O2 polarography with a Hansatech oxygen electrode (Knoepfle et al., 1998). Assays were performed at 25°C on whole cells in complete or chloride-depleted BG-11 media with 1 mM 2,6-dichlorobenzoquinone (DCBQ) added as

Mutations in the CP43 Protein of Photosystem II

stored immediately at –70 C.

**6. Results** 

**5.6 Spectrophotometric assays** 

**6.1 Verification of genomic DNA sequence** 

Excess Buffer A was decanted and the pellet was frozen at –70 °C.

Affect PSII Function and Cytochrome C550 Binding 63

combined into a single container, and centrifuged again at 10000 x *g* for 10 min at 4°C.

To purify the His-tagged PSII, the procedure of Bricker et al. (1998) was used. Frozen cells were thawed and resuspended in 5 ml of Buffer A. The cells were then assayed for chlorophyll concentration and brought to 1 mg/ml of chlorophyll. Then, the cell suspension was brought to 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 1.0 mM benzamidine, 1 mM -amino caproic acid, and 50 g/ml DNAase. The sample was added to an ice water cooled bead beater apparatus (Bio-Spec Products, Inc.) and glass beads (1.0 mm) were added at a 1:1 ratio to the cell suspension. The cells were then broken at 4ºC over 12-14 break cycles, each consisting of 15 seconds of homogenization followed by 4 minutes of cooling. The cell homogenate and glass beads were then transferred to a beaker and the glass beads allowed to settle. The cell homogenate was decanted and the beads were washed several times with Buffer A to recover additional homogenate. The cell homogenate was brought to 1% n-dodecyl--D-maltoside by the addition of a 10% ndodecyl--D-maltoside stock solution (freshly prepared) and was immediately centrifuged at 36000 x *g* for 10 minutes to remove unbroken cells and residual glass beads. The solubilized cell homogenate was then loaded onto a 25 ml cobalt metal affinity column (Clontech, Inc.) which had been pre-equilibrated with Buffer A + 0.04% n-dodecyl--Dmaltoside at 4ºC. The column was washed with several bed volumes of Buffer A + 0.04% ndodecyl--D-maltoside and the bound His-tagged PSII particles were eluted with Buffer A + 0.04% n-dodecyl--D-maltoside + 50 mM L-histidine. The eluted fractions were then pooled and the PSII complex was precipitated by addition of an equal volume of 25% PEG-8000 in 50 mM MES-NaOH, pH 6.0, and incubation at 4ºC for 30 minutes. The precipitated PSII particles were harvested by centrifugation at 36000 x *g* for 30 minutes. The precipitated PSII particles were resuspended in 1.0 ml of Buffer A + 0.04% n-dodecyl--D-maltoside and

For the determination of absorbance spectra, the procedure of Bricker and coworkers (2002) was employed. First, purified PSII particles were assayed for chlorophyll concentration by methanol extraction, and samples were diluted to 20 g/ml chlorophyll in a solution of 50 mM MES-NaOH, pH 6.5, 10 mM MgCl2, 5 mM CaCl2, 50 mM NaCl, 0.04% -D-dodecyl maltoside, and 25% glycerol. After performing a baseline correction on a Cary 100 dualbeam spectrophotometer (Varian, Inc.), the samples were either oxidized with a few crystals of potassium ferricyanide, for the reference cuvette, or reduced with a few crystals of sodium dithionite, for the sample cuvette. After a 10-minute dark incubation, reduced minus oxidized difference spectra were collected by scanning the samples from 540-580 nm for the cytochrome-specific spectra, and from 400-800 nm for the overall absorbance spectra.

The charge-switching mutants constructed for this study were created by site-directed mutagenesis of Arg320 of CP43 to either lysine or aspartate. DNA sequencing of the portion of the *psbC* gene surrounding the mutation site was used to verify each point mutation. Additionally, because the mutant sequence was engineered within a plasmid insert

an artificial electron acceptor. Cells were allowed to grow for 5 days under the appropriate growth conditions, as above, and were harvested on the fifth day by centrifuging at 12000 x *g* for 5 minutes at 4C. The cells were then resuspended in 1-2 ml of complete BG-11 media. Next, the chlorophyll concentration of each sample was determined by taking absorbance readings at 678 nm and 710 nm and calculating the concentration of chlorophyll (in g/ml) by the equation: (OD678-OD710) \* 14.96 \* dilution factor (modified from Williams, 1988). The light intensity used during these assays was 2500 mol photons\*m-2\*s-1 of white light, verified with a spectroradiometer equipped with a quantum probe (Li-Cor, Inc.).

For photoinactivation experiments, cells were incubated at 25°C in BG-11 media, at a chlorophyll concentration of 10 g/ml, and exposed to a light intensity of 5000 mol photons\*m-2\*s-1. Cells were removed from exposure to the photoinactivating light in increments of 2 minutes and assayed for oxygen-evolving activity, as above. The t0.5 for photoinactivation was calculated by fitting the data to a single exponential decay.

#### **5.4 Fluorescence measurements**

Fluorescence yield measurements were performed on a Waltz PAM 101 fluorometer as described previously (Nixon & Diner; 1992, Chu et al., 1994). Samples were incubated in the dark for 5 minutes in the presence of 1 mM potassium ferricyanide and 330 M DCBQ. Next, DCMU was added to a final concentration of 40 M followed one minute later by the addition of hydroxylamine hydrochloride, pH 6.5, to a concentration of 20 mM. After 20 s, the weak monitoring flashes were turned on, followed 1 s later by continuous actinic illumination (1000 mol photons\*m-2\*s-1). The variable fluorescence was measured and a second set of assays was performed, this time omitting the hydroxylamine, in order to measure the variable fluorescence with water as the electron donor. For both assays, Fmax was measured 5 s after the onset of actinic illumination.

#### **5.5 Introduction of poly-histidine tags and purification of photosystem II**

A recombinant pTZ18 plasmid was created previously that contained the *psbB* gene encoding CP47 with six histidyl codons at the 3' end of the gene (Young et al., 2002). An antibiotic resistance gene encoding resistance to spectinomycin was cloned into the 3' noncoding portion of the *psb*B gene downstream of the His-tag. This plasmid was transformed into *Synechocystis* as previously described (Williams, 1988). Briefly, 5 x 108 cells in 100 l of BG-11 media were added to a sterile 15 ml centrifuge tube. To these cells, 2.5 g of plasmid DNA was added. The tubes were incubated for 4-6 hours at room temperature and exposure to a light intensity of 25 mol photons\*m-2\*s-1. After incubation, the entire volume of cells was plated onto solid BG-11/glucose/DCMU to which no antibiotic had been added. After 48 hours, the plates were underlaid with antibiotic solution (196 l of BG-11 media + 8 l spectinomycin (50 mg/ml stock)). Transformants were allowed to sort out by serial streaking on BG-11 plates containing glucose, DCMU, and spectinomycin. The insertion of the histidyl codons was confirmed by DNA sequencing, as above.

Cells were grown in either complete BG-11 media or in BG-11 media deficient in chloride. Cultures were supplemented with 5 mM glucose and 5 g/ml spectinomycin. The cells were grown at 25°C, with aeration, at a light intensity of 25 mol photons\*m-2\*s-1, for 7-9 days. Cells from 15 l cultures were centrifuged 10000 x *g* for 10 minutes at 4C. The cells were resuspended in an adequate amount of Buffer A (50 mM 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH, pH 6.0, 10 mM MgCl2, 5 mM CaCl2, 25% glycerol) (~10 ml per pellet),

an artificial electron acceptor. Cells were allowed to grow for 5 days under the appropriate growth conditions, as above, and were harvested on the fifth day by centrifuging at 12000 x *g* for 5 minutes at 4C. The cells were then resuspended in 1-2 ml of complete BG-11 media. Next, the chlorophyll concentration of each sample was determined by taking absorbance readings at 678 nm and 710 nm and calculating the concentration of chlorophyll (in g/ml) by the equation: (OD678-OD710) \* 14.96 \* dilution factor (modified from Williams, 1988). The light intensity used during these assays was 2500 mol photons\*m-2\*s-1 of white light,

For photoinactivation experiments, cells were incubated at 25°C in BG-11 media, at a chlorophyll concentration of 10 g/ml, and exposed to a light intensity of 5000 mol photons\*m-2\*s-1. Cells were removed from exposure to the photoinactivating light in increments of 2 minutes and assayed for oxygen-evolving activity, as above. The t0.5 for

Fluorescence yield measurements were performed on a Waltz PAM 101 fluorometer as described previously (Nixon & Diner; 1992, Chu et al., 1994). Samples were incubated in the dark for 5 minutes in the presence of 1 mM potassium ferricyanide and 330 M DCBQ. Next, DCMU was added to a final concentration of 40 M followed one minute later by the addition of hydroxylamine hydrochloride, pH 6.5, to a concentration of 20 mM. After 20 s, the weak monitoring flashes were turned on, followed 1 s later by continuous actinic illumination (1000 mol photons\*m-2\*s-1). The variable fluorescence was measured and a second set of assays was performed, this time omitting the hydroxylamine, in order to measure the variable fluorescence with water as the electron donor. For both assays, Fmax

A recombinant pTZ18 plasmid was created previously that contained the *psbB* gene encoding CP47 with six histidyl codons at the 3' end of the gene (Young et al., 2002). An antibiotic resistance gene encoding resistance to spectinomycin was cloned into the 3' noncoding portion of the *psb*B gene downstream of the His-tag. This plasmid was transformed into *Synechocystis* as previously described (Williams, 1988). Briefly, 5 x 108 cells in 100 l of BG-11 media were added to a sterile 15 ml centrifuge tube. To these cells, 2.5 g of plasmid DNA was added. The tubes were incubated for 4-6 hours at room temperature and exposure to a light intensity of 25 mol photons\*m-2\*s-1. After incubation, the entire volume of cells was plated onto solid BG-11/glucose/DCMU to which no antibiotic had been added. After 48 hours, the plates were underlaid with antibiotic solution (196 l of BG-11 media + 8 l spectinomycin (50 mg/ml stock)). Transformants were allowed to sort out by serial streaking on BG-11 plates containing glucose, DCMU, and spectinomycin. The insertion of

Cells were grown in either complete BG-11 media or in BG-11 media deficient in chloride. Cultures were supplemented with 5 mM glucose and 5 g/ml spectinomycin. The cells were grown at 25°C, with aeration, at a light intensity of 25 mol photons\*m-2\*s-1, for 7-9 days. Cells from 15 l cultures were centrifuged 10000 x *g* for 10 minutes at 4C. The cells were resuspended in an adequate amount of Buffer A (50 mM 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH, pH 6.0, 10 mM MgCl2, 5 mM CaCl2, 25% glycerol) (~10 ml per pellet),

verified with a spectroradiometer equipped with a quantum probe (Li-Cor, Inc.).

photoinactivation was calculated by fitting the data to a single exponential decay.

**5.5 Introduction of poly-histidine tags and purification of photosystem II** 

the histidyl codons was confirmed by DNA sequencing, as above.

**5.4 Fluorescence measurements** 

was measured 5 s after the onset of actinic illumination.

combined into a single container, and centrifuged again at 10000 x *g* for 10 min at 4°C. Excess Buffer A was decanted and the pellet was frozen at –70 °C.

To purify the His-tagged PSII, the procedure of Bricker et al. (1998) was used. Frozen cells were thawed and resuspended in 5 ml of Buffer A. The cells were then assayed for chlorophyll concentration and brought to 1 mg/ml of chlorophyll. Then, the cell suspension was brought to 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 1.0 mM benzamidine, 1 mM -amino caproic acid, and 50 g/ml DNAase. The sample was added to an ice water cooled bead beater apparatus (Bio-Spec Products, Inc.) and glass beads (1.0 mm) were added at a 1:1 ratio to the cell suspension. The cells were then broken at 4ºC over 12-14 break cycles, each consisting of 15 seconds of homogenization followed by 4 minutes of cooling. The cell homogenate and glass beads were then transferred to a beaker and the glass beads allowed to settle. The cell homogenate was decanted and the beads were washed several times with Buffer A to recover additional homogenate. The cell homogenate was brought to 1% n-dodecyl--D-maltoside by the addition of a 10% ndodecyl--D-maltoside stock solution (freshly prepared) and was immediately centrifuged at 36000 x *g* for 10 minutes to remove unbroken cells and residual glass beads. The solubilized cell homogenate was then loaded onto a 25 ml cobalt metal affinity column (Clontech, Inc.) which had been pre-equilibrated with Buffer A + 0.04% n-dodecyl--Dmaltoside at 4ºC. The column was washed with several bed volumes of Buffer A + 0.04% ndodecyl--D-maltoside and the bound His-tagged PSII particles were eluted with Buffer A + 0.04% n-dodecyl--D-maltoside + 50 mM L-histidine. The eluted fractions were then pooled and the PSII complex was precipitated by addition of an equal volume of 25% PEG-8000 in 50 mM MES-NaOH, pH 6.0, and incubation at 4ºC for 30 minutes. The precipitated PSII particles were harvested by centrifugation at 36000 x *g* for 30 minutes. The precipitated PSII particles were resuspended in 1.0 ml of Buffer A + 0.04% n-dodecyl--D-maltoside and stored immediately at –70 C.
