The role of cytochrome c-550 as studied through reverse genetics and mutant characterization in Synechocystis sp. PCC 6803

J. R. Shen, Willem Vermaas, Y. Inoue

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Abstract

The gene coding for cytochrome c-550 in Synechocystis sp. PCC 6803 was cloned based on the N-terminal sequence of the mature polypeptide. Using the most probable translation start codon, the gene is expected to code for 160 amino acid residues. This includes a cleavable N-terminal leader sequence of 25 residues. This leader sequence has an Arg-Asn-Arg sequence immediately before the cleavage site; this is characteristic for transit peptides in prokaryotes. Comparison of this sequence with the leader sequence of the photosystem II-associated extrinsic 33-kDa protein from the same cyanobacterium showed an identity of 13 out of 25 residues. These results suggest that after synthesis of the apoprotein, cytochrome c-550 is transported into the thylakoid lumen. Using the cloned gene, insertion and deletion mutants of Synechocystis sp. PCC 6803 were constructed. In the absence of cytochrome c-550, both mutants were capable of photoautetrophic growth but at a significantly reduced rate. Atrazine binding and Western blot analysis showed that these mutants on a per-chlorophyll basis contained 53- 67% of the amount of photosystem II as compared with wild type. The photosystem II-specific oxygen-evolving activity at saturating light intensity was reduced to about 40% of that in the wild type strain. Taken together, these results indicate that the cytochrome c-550 is transported into the thylakoid lumen and contributes to optimal functional stability of photosystem II in cyanobacteria. This supports our biochemical evidence that cytochrome c-550 is associated with the lumenal side of photosystem II as one of the extrinsic proteins enhancing oxygen evolution (Shen, J.-R., Ikeuchi, M., and Inoue, Y. (1992) FEBS Lett. 301, 145-149; Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 18251832). Based on these results, the gene for cytochrome c-550 was named psbV. The possible evolutionary relationship among extrinsic proteins of the photosystem II donor side is discussed.

Original languageEnglish (US)
Pages (from-to)6901-6907
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number12
DOIs
StatePublished - 1995
Externally publishedYes

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Synechocystis
Reverse Genetics
Photosystem II Protein Complex
Cytochromes c
Genes
Thylakoids
Cyanobacteria
Oxygen
Atrazine
Peptides
Biochemistry
Proteins
Apoproteins
Initiator Codon
Insertional Mutagenesis
Gene Deletion
Chlorophyll
Genetics
Western Blotting
Light

ASJC Scopus subject areas

  • Biochemistry

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The role of cytochrome c-550 as studied through reverse genetics and mutant characterization in Synechocystis sp. PCC 6803. / Shen, J. R.; Vermaas, Willem; Inoue, Y.

In: Journal of Biological Chemistry, Vol. 270, No. 12, 1995, p. 6901-6907.

Research output: Contribution to journalArticle

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title = "The role of cytochrome c-550 as studied through reverse genetics and mutant characterization in Synechocystis sp. PCC 6803",
abstract = "The gene coding for cytochrome c-550 in Synechocystis sp. PCC 6803 was cloned based on the N-terminal sequence of the mature polypeptide. Using the most probable translation start codon, the gene is expected to code for 160 amino acid residues. This includes a cleavable N-terminal leader sequence of 25 residues. This leader sequence has an Arg-Asn-Arg sequence immediately before the cleavage site; this is characteristic for transit peptides in prokaryotes. Comparison of this sequence with the leader sequence of the photosystem II-associated extrinsic 33-kDa protein from the same cyanobacterium showed an identity of 13 out of 25 residues. These results suggest that after synthesis of the apoprotein, cytochrome c-550 is transported into the thylakoid lumen. Using the cloned gene, insertion and deletion mutants of Synechocystis sp. PCC 6803 were constructed. In the absence of cytochrome c-550, both mutants were capable of photoautetrophic growth but at a significantly reduced rate. Atrazine binding and Western blot analysis showed that these mutants on a per-chlorophyll basis contained 53- 67{\%} of the amount of photosystem II as compared with wild type. The photosystem II-specific oxygen-evolving activity at saturating light intensity was reduced to about 40{\%} of that in the wild type strain. Taken together, these results indicate that the cytochrome c-550 is transported into the thylakoid lumen and contributes to optimal functional stability of photosystem II in cyanobacteria. This supports our biochemical evidence that cytochrome c-550 is associated with the lumenal side of photosystem II as one of the extrinsic proteins enhancing oxygen evolution (Shen, J.-R., Ikeuchi, M., and Inoue, Y. (1992) FEBS Lett. 301, 145-149; Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 18251832). Based on these results, the gene for cytochrome c-550 was named psbV. The possible evolutionary relationship among extrinsic proteins of the photosystem II donor side is discussed.",
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