**6.6 Visible absorption spectra**

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 (Bricker et al., 1998).

Mutations in the CP43 Protein of Photosystem II

Fig. 5. Reduced Minus Oxidized Difference Spectra

**7. Discussion** 

absorbance at 560 nm measured approximately 0.10 absorbance units.

Affect PSII Function and Cytochrome C550 Binding 69

**A.** 

**B.** 

**C.** 

Reduced minus oxidized absorbance difference spectra for **A.** control, **B.** R320K, and **C.** R320D His-tagged PSII particles purified from cells grown photomixotrophically in complete media. Samples were diluted to 20 g/ml chlorophyll in 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 Varian Cary 100 dual-beam spectrophotometer, 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 580-540 nm. The peak

The purpose of the present study was to elucidate further the interaction of CP43 with the extrinsic cytochrome c550 protein of PSII by studying the effect of additional alterations of the arginine residue at position 320 of the CP43 large extrinsic loop. Two additional mutations, R320K and R320D, were introduced into the large extrinsic loop and the effects of

Fig. 4. Visible Absorption Spectra

Visible absorbance spectra for **A**. control, **B.** R320K, and **C.** R320D PSII histidine-tagged particles purified from cells grown photomixotrophically in complete BG-11 media.

#### **6.7 Reduced-minus oxidized difference spectra**

Reduced minus oxidized difference spectra were collected in the wavelength range of 540- 580 nm for PSII particles isolated from the His-tagged control and mutant strains grown photomixotrophically in complete media (Fig. 5) Plots of the data reveal a non-symmetrical absorbance curve for the control strain. This non-symmetrical curve is also present in control PSII particles derived from cells grown under chloride depletion (not shown). This curve reveals a maximum absorbance at 559 nm that tapers off into a wide shoulder toward 550 nm, while toward 570 nm, there is no indication of a shoulder. The signal at 559 nm indicates the presence of cytochrome b559, while the broadened shoulder arises due to the absorbance properties of cytochrome c550 (Bricker et al., 2002). The plots of data derived from the mutant strains, like the control, exhibit an absorbance maximum at 559 nm. However, unlike the control strain, the plots for the mutant strains are symmetrical, giving no indication of a broad shoulder on either side of the peak, regardless of the media, suggesting the absence of cytochrome c550 within these preparations. The same symmetrical peaks are observed for both mutants grown under chloride-limited conditions (not shown).

**A.**

**B.** 

**C.**

Reduced minus oxidized difference spectra were collected in the wavelength range of 540- 580 nm for PSII particles isolated from the His-tagged control and mutant strains grown photomixotrophically in complete media (Fig. 5) Plots of the data reveal a non-symmetrical absorbance curve for the control strain. This non-symmetrical curve is also present in control PSII particles derived from cells grown under chloride depletion (not shown). This curve reveals a maximum absorbance at 559 nm that tapers off into a wide shoulder toward 550 nm, while toward 570 nm, there is no indication of a shoulder. The signal at 559 nm indicates the presence of cytochrome b559, while the broadened shoulder arises due to the absorbance properties of cytochrome c550 (Bricker et al., 2002). The plots of data derived from the mutant strains, like the control, exhibit an absorbance maximum at 559 nm. However, unlike the control strain, the plots for the mutant strains are symmetrical, giving no indication of a broad shoulder on either side of the peak, regardless of the media, suggesting the absence of cytochrome c550 within these preparations. The same symmetrical peaks are observed for both mutants grown under chloride-limited conditions (not shown).

Visible absorbance spectra for **A**. control, **B.** R320K, and **C.** R320D PSII histidine-tagged particles purified from cells grown photomixotrophically in complete BG-11 media.

Fig. 4. Visible Absorption Spectra

**6.7 Reduced-minus oxidized difference spectra** 

Fig. 5. Reduced Minus Oxidized Difference Spectra

Reduced minus oxidized absorbance difference spectra for **A.** control, **B.** R320K, and **C.** R320D His-tagged PSII particles purified from cells grown photomixotrophically in complete media. Samples were diluted to 20 g/ml chlorophyll in 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 Varian Cary 100 dual-beam spectrophotometer, 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 580-540 nm. The peak absorbance at 560 nm measured approximately 0.10 absorbance units.
