Topography and function of cytochrome b(559) in oxygenic photosynthesis

Gun-Sik Tae, Purdue University

Abstract

Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the $M\sb{\rm r}$ 10,000 psbE gene product $\alpha$-subunit of the cytochrome $b\sb{559}$. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the $\alpha$-subunit to a slightly smaller polypeptide. The data shows that the $\alpha$-subunit of cytochrome $b\sb{559}$ crosses the membrane once and the NM$\sb2$ terminus of the $\alpha$-polypeptide is on the stromal side of the membrane. The single histidine residue in the $\alpha$-subunit is located on the stromal side of the hydrophobic domain. Preliminary data on the $\beta$-subunit orientation indicate that it is oriented with NH$\sb2$-terminus on the stromal side, consistent with heme ligation on this site. In the oxygen evolving PS II complex, removal of the extrinsic 33 kDa polypeptide increased the accessibility to trypsin of a COOH-terminal tridecapeptide epitope of the cytochrome $b\sb{559}\ \alpha$-subunit. Shielding of the COOH-terminal segment of the cytochrome $b\sb{559}\ \alpha$-subunit by the OEC extrinsic 33 kDa polypeptide implied that the COOH-terminal domain extends into the lumen space and forms a part of a protein network peripheral to the PS II reaction center. The COOH-terminal segment of the cytochrome $b\sb{559}\ \alpha$-subunit was sequentially truncated in the cyanobacterium, Synechocystis sp. PCC 6803, in order to determine the minimum length of the COOH-terminal domain of the $\alpha$-subunit required for function(s) and the possible role of this domain in the binding of ions such as manganese, calcium, and chloride that are critical for O$\sb2$ evolution. Slow photoautotrophic growth rates of $\alpha$-R50-stop and $\alpha$-R59-stop mutants comes from a defect in PSII, not PSI. Chemical difference spectra of cytochrome $b\sb{559}$ also show a substantial decrease of cytochrome $b\sb{559}$ in the mutant. These results imply that an $\alpha$-R50-stop mutant can assemble an intact PSII complex, but that the number of the properly assembled PSII complexes in the membrane decreases. The existence of a portion of properly assembled PS II complexes in the $\alpha$-R50-stop mutant implies that most (31 residues) of the COOH-terminal domain of the cytochrome $b\sb{559}\ \alpha$-subunit is not required for the binding of ions critical to O$\sb2$ evolution although the presence of this domain greatly increases the probability of correct or stable assembly of the PS II reaction center. (Abstract shortened with permission of author.)

Degree

Ph.D.

Advisors

Cramer, Purdue University.

Subject Area

Biophysics|Biochemistry

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