**6. Results**

### **6.1 Verification of genomic DNA sequence**

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

Mutations in the CP43 Protein of Photosystem II

**A** 

**B** 

**C** 

Fig. 2. Photoautrophic Growth of Mutant and Control Strains.

Growth curves showing control (diamonds) and mutant strains (R320K squares; R320D triangles) grown in either **A.** complete media, **B.** chloride-deficient media, or **C.** chloridedeficient media supplemented with bromide. These results are the average of at least 3 independent experiments. The error bars represent plus and minus one standard deviation.

Affect PSII Function and Cytochrome C550 Binding 65

containing the AccI/AccI fragment of *psbC*, the DNA sequence included between these restriction sites in the genomic DNA was also sequenced. This segment consists of 1221 base pairs, along with an inserted kanamycin resistance cartridge. DNA sequencing confirmed the presence of the desired point mutations, as well as the integrity of the sequence within the AccI/AccI fragment.

Additional mutants were created for the insertion of a poly-histidine tag on the CP47 protein of control and mutant strains. This was done by inserting a series of six consecutive histidine-encoding codons, along with a spectinomycin resistance cartridge, into *psb*B. The poly-histidine encoding sequence was verified by DNA sequencing for each strain.

#### **6.2 Growth measurements**

The mutant and control strains were grown in the absence of glucose to determine the effect of each mutation on the photoautotrophic growth characteristics of the organism. Each strain was grown in complete BG-11 media, in BG-11 media deficient in chloride, or in chloride-deficient BG-11 media supplemented with 480 M sodium bromide, in order to determine any effect chloride had on the growth of the strains. In complete media, both R320K and R320D grew at rates comparable to the control strain (Fig. 2A). In chloridedeficient media, the control strain exhibited a normal growth rate. (Fig. 2B). The mutant strains, however, were affected under these conditions, with R320K growing at a lower rate than it did in complete media and R320D showing virtually no photoautotrophic growth. When the strains were grown in chloride-deficient media supplemented with bromide (Fig. 2C), growth rates for each mutant strain were restored to near those observed for the strains grown in complete media. It is well known that bromide can substitute for chloride at the PSII active site (Sandusky & Yocum, 1983; Yachandra et al., 1993).

#### **6.3 Oxygen evolution**

Oxygen evolution rates were measured for control and mutant strains grown either photoautotrophically or in the presence of glucose. When grown photoautrophically (Fig. 3), the control strain evolved oxygen at similar rates regardless of the media conditions. In complete media, the R320K strain evolved oxygen at a rate about 80% that of the control. When chloride was lacking in the growth media, this rate fell to 35% of the control. The R320D strain was the least successful of these strains for evolving oxygen. When grown photoautotrophically in complete media, the rate of oxygen evolution for this strain was depressed to 60% of the control. The effect on oxygen evolution was greatly exacerbated when the strain was grown under chloride-limiting conditions, where the observed rate of oxygen evolution was only 5% of the control rate. For both mutants, the addition of bromide restored this rate to levels near those observed for cells grown in complete media. These same results were observed when the strains were grown photomixotrophically (not shown).

#### **6.4 Variable fluorescence yields**

Variable fluorescence yield assays are used to make qualitative estimates of the number of PSII centers present in the thylakoid membranes of a particular strain, and also allow for a determination of the functionality of these centers. The results of these experiments for cells grown in complete media are shown in Table 1. Both mutant strains assemble relatively

containing the AccI/AccI fragment of *psbC*, the DNA sequence included between these restriction sites in the genomic DNA was also sequenced. This segment consists of 1221 base pairs, along with an inserted kanamycin resistance cartridge. DNA sequencing confirmed the presence of the desired point mutations, as well as the integrity of the sequence within

Additional mutants were created for the insertion of a poly-histidine tag on the CP47 protein of control and mutant strains. This was done by inserting a series of six consecutive histidine-encoding codons, along with a spectinomycin resistance cartridge, into *psb*B. The

The mutant and control strains were grown in the absence of glucose to determine the effect of each mutation on the photoautotrophic growth characteristics of the organism. Each strain was grown in complete BG-11 media, in BG-11 media deficient in chloride, or in chloride-deficient BG-11 media supplemented with 480 M sodium bromide, in order to determine any effect chloride had on the growth of the strains. In complete media, both R320K and R320D grew at rates comparable to the control strain (Fig. 2A). In chloridedeficient media, the control strain exhibited a normal growth rate. (Fig. 2B). The mutant strains, however, were affected under these conditions, with R320K growing at a lower rate than it did in complete media and R320D showing virtually no photoautotrophic growth. When the strains were grown in chloride-deficient media supplemented with bromide (Fig. 2C), growth rates for each mutant strain were restored to near those observed for the strains grown in complete media. It is well known that bromide can substitute for chloride at the

Oxygen evolution rates were measured for control and mutant strains grown either photoautotrophically or in the presence of glucose. When grown photoautrophically (Fig. 3), the control strain evolved oxygen at similar rates regardless of the media conditions. In complete media, the R320K strain evolved oxygen at a rate about 80% that of the control. When chloride was lacking in the growth media, this rate fell to 35% of the control. The R320D strain was the least successful of these strains for evolving oxygen. When grown photoautotrophically in complete media, the rate of oxygen evolution for this strain was depressed to 60% of the control. The effect on oxygen evolution was greatly exacerbated when the strain was grown under chloride-limiting conditions, where the observed rate of oxygen evolution was only 5% of the control rate. For both mutants, the addition of bromide restored this rate to levels near those observed for cells grown in complete media. These same results were observed when the strains were grown photomixotrophically (not

Variable fluorescence yield assays are used to make qualitative estimates of the number of PSII centers present in the thylakoid membranes of a particular strain, and also allow for a determination of the functionality of these centers. The results of these experiments for cells grown in complete media are shown in Table 1. Both mutant strains assemble relatively

poly-histidine encoding sequence was verified by DNA sequencing for each strain.

PSII active site (Sandusky & Yocum, 1983; Yachandra et al., 1993).

the AccI/AccI fragment.

**6.2 Growth measurements** 

**6.3 Oxygen evolution** 

shown).

**6.4 Variable fluorescence yields** 

Fig. 2. Photoautrophic Growth of Mutant and Control Strains. Growth curves showing control (diamonds) and mutant strains (R320K squares; R320D triangles) grown in either **A.** complete media, **B.** chloride-deficient media, or **C.** chloridedeficient media supplemented with bromide. These results are the average of at least 3 independent experiments. The error bars represent plus and minus one standard deviation.

Mutations in the CP43 Protein of Photosystem II

Table 1. Variable Fluorescence Yields.

Table 2. t0.5 Values for Photoinactivation.

(\* Results from Young et al., 2002)

**6.6 Visible absorption spectra** 

(Bricker et al., 1998).

and R320D, and R320S\* strains.

closely mirror those of the R320S mutant (Young et al., 2002).

Affect PSII Function and Cytochrome C550 Binding 67

mutant strains exhibited large increases in photoinactivation demonstrated by t0.5 values of 2.9 and 0.9 min, respectively, for R320K and R320D. These data indicate that the PSII complexes of the mutants are less stable, and are much more susceptible to the effects of photoinactivating light intensities. It should be noted that the t0.5 values for the R320K

> **Strain Hydroxylamine Water**  Control 0.46 ± 0.069 0.45 ± 0.056 *psb*C Deletion 0.02 ± 0.000 -0.02 ± 0.029 R320K 0.37 ± 0.034 0.35 ± 0.022 R320D 0.38 ± 0.029 0.33 ± 0.020 R320S\* 0.41 ± 0.030 0.46 ± 0.050

Results of variable fluorescence yield assays performed on control, *psbC* deletion, R320K,

(\* Results for the R320S (R320S) strain obtained from Young et al., 2002. The control values

**Strain Complete Media Chloride-Deficient Media** 

t0.5 values for photoinactivation for each strain after growth in either complete or chloridedeficient media and exposure to photoinactivating light (5000 mol photons\*m-2\*s-1). The

Absorbance spectra in the range of 400-800 nm were obtained for PSII particles purified from His-tagged control and mutant strains grown photomixotrophically in complete media. As can be seen in Fig. 4, there were no appreciable differences in these spectra, regardless of the strain, which indicates that no major disruption of the pigment-containing portion of PSII occurred within the mutant strains. The major peaks located at 410, 430, and 680 nm represent absorption by chlorophyll-a molecules, with the major peak at 680 nm representing the reaction center chlorophyll of PSII, P680. The minor peaks arise from accessory pigment molecules associated with the photosystem. These spectra are similar to spectra obtained from PSII preparations of non-His-tagged control strains (Noren et al., 1991; Tang & Diner, 1994) and to spectra obtained from other His-tagged PSII preparations

for these experiments were hydroxylamine = 0.58 and water = 0.67.)

Control 5.4 min. 5.7 min. R320K 5.3 min. 2.9 min. R320D 3.6 min. 0.9 min. R320S\* 4.7 min. 2.6 min.

results are the average of at least two independent experiments.

Fig. 3. Oxygen Evolution Rates of Control and Mutant Strains Grown Photoautotrophically. The control rate in complete media was 391 μmol O2\*(mg chlorophyll)- 1 \*hr-1. Rates are expressed as the percentage of the control rate in cells grown in complete media. White bars, oxygen evolution rates from cells grown in complete media; Striped bars, oxygen evolution rates from cells grown in chloride deficient media; Black bars, oxygen evolution rates from cells grown in chloride depleted media with 480M bromide added. The results are the average of at least three independent experiments. The error bars represent plus and minus one standard deviation.

large numbers of PS II centers that can transfer electrons from hydroxylamine to QA. R320K and R320D assembled 80% and 83%, respectively, of the PS II centers assembled in the control strain. If water is used as an electron donor, the variable fluorescence yield is a measure of the amount of PSII centers that can oxidize water. The data from the water experiment indicate that the mutant centers remain mostly functional under normal growth conditions, with the fluorescence yields for both mutants being approximately 80% that of the control strain. Data is also included for the psbC deletion strain, which lacks any functional PSII centers. Data for the R320S mutant (Young et al., 2002) is also included and shows that this mutant, like R320D and R320K, contains substantial numbers of PSII centers that are efficient in water oxidation.

#### **6.5 Photoinactivation**

Photoinactivation assays are used to determine the relative stability of the PSII complex of a strain. As summarized in Table 2, when grown photoautotrophically, regardless of the media, the control strain had a t0.5 for photoinactivation of approximately 5.6 min. In complete media, R320K exhibited a t0.5 close to the control of 5.3 min and R320D a t0.5 slightly lower than the control of 3.6 min. When grown in media deficient in chloride, however, the

Fig. 3. Oxygen Evolution Rates of Control and Mutant Strains Grown Photoautotrophically.

expressed as the percentage of the control rate in cells grown in complete media. White bars, oxygen evolution rates from cells grown in complete media; Striped bars, oxygen evolution rates from cells grown in chloride deficient media; Black bars, oxygen evolution rates from cells grown in chloride depleted media with 480M bromide added. The results are the average of at least three independent experiments. The error bars represent plus and minus

large numbers of PS II centers that can transfer electrons from hydroxylamine to QA. R320K and R320D assembled 80% and 83%, respectively, of the PS II centers assembled in the control strain. If water is used as an electron donor, the variable fluorescence yield is a measure of the amount of PSII centers that can oxidize water. The data from the water experiment indicate that the mutant centers remain mostly functional under normal growth conditions, with the fluorescence yields for both mutants being approximately 80% that of the control strain. Data is also included for the psbC deletion strain, which lacks any functional PSII centers. Data for the R320S mutant (Young et al., 2002) is also included and shows that this mutant, like R320D and R320K, contains substantial numbers of PSII centers

Photoinactivation assays are used to determine the relative stability of the PSII complex of a strain. As summarized in Table 2, when grown photoautotrophically, regardless of the media, the control strain had a t0.5 for photoinactivation of approximately 5.6 min. In complete media, R320K exhibited a t0.5 close to the control of 5.3 min and R320D a t0.5 slightly lower than the control of 3.6 min. When grown in media deficient in chloride, however, the

1 \*hr-

1. Rates are

The control rate in complete media was 391 μmol O2\*(mg chlorophyll)-

one standard deviation.

that are efficient in water oxidation.

**6.5 Photoinactivation** 

mutant strains exhibited large increases in photoinactivation demonstrated by t0.5 values of 2.9 and 0.9 min, respectively, for R320K and R320D. These data indicate that the PSII complexes of the mutants are less stable, and are much more susceptible to the effects of photoinactivating light intensities. It should be noted that the t0.5 values for the R320K closely mirror those of the R320S mutant (Young et al., 2002).


Table 1. Variable Fluorescence Yields.

Results of variable fluorescence yield assays performed on control, *psbC* deletion, R320K, and R320D, and R320S\* strains.

(\* Results for the R320S (R320S) strain obtained from Young et al., 2002. The control values for these experiments were hydroxylamine = 0.58 and water = 0.67.)


Table 2. t0.5 Values for Photoinactivation.

t0.5 values for photoinactivation for each strain after growth in either complete or chloridedeficient media and exposure to photoinactivating light (5000 mol photons\*m-2\*s-1). The results are the average of at least two independent experiments.

(\* Results from Young et al., 2002)
