Heterogeneous Rieske Proteins in the Cytochrome b6 f Complex of Synechocystis PCC6803?

Abstract

The completely sequenced genome of the cyanobacterium Synechocystis PCC6803 contains three open reading frames, petC1, petC2, and petC3, encoding putative Rieske iron-sulfur proteins. After heterologous overexpression, all three gene products have been characterized and shown to be Rieske proteins as typified by sequence analysis and EPR spectroscopy. Two of the overproduced proteins contained already incorporated iron-sulfur clusters, whereas the third one formed unstable aggregates, in which the FeS cluster had to be reconstituted after refolding of the denatured protein.Although EPR spectroscopy showed typical FeS signals for all Rieske proteins, an unusual low midpoint potential was revealed for PetC3 by EPR redox titration. Detailed characterization of Synechocystis membranes indicated that all three Rieske proteins are expressed under physiological conditions. Both for PetC1 and PetC3 the association with the thylakoid membrane was shown, and both could be identified, although in different amounts, in the isolated cytochrome b 6 f complex. The considerably lower redox potential determined for PetC3 indicates heterogeneous cytochrome b 6 f complexes in Synechocystis and suggests still to be established alternative electron transport routes.The functional core of cytochrome bc complexes consists of three subunits, which are a b-type cytochrome, a c-type cytochrome, and the Rieske iron-sulfur protein (1). In contrast to the cytochrome bc 1 complex of mitochondria and bacteria with three subunits, cytochrome b, cytochrome c 1 , and the Rieske protein, the homologous cytochrome b 6 f complex of chloroplasts and cyanobacteria contains four core subunits named cytochrome b 6 , subunit IV, cytochrome f, and the Rieske protein (2).The Rieske protein consists of an N-terminal transmembrane ␣-helix, which anchors the protein in the membrane (3, 4), and a large soluble domain on the luminal side of the membrane. The three-dimensional structure of this luminal part has been solved both for the Rieske protein of a cytochrome b 6 f complex (5) and of a cytochrome bc 1 complex (6) to a resolution of 1.8 and 1.5 Å, respectively. The redox active [2Fe-2S] cluster, which is bound by the luminal domain, is ligated by two cysteine and two histidine residues with one iron being ligated by the two cysteines and the other by the two histidines. It is likely that this unusual coordination causes the high midpoint potential of about ϩ100 to ϩ400 mV. Besides these coordinating amino acids there are two additional cysteine residues, which form a disulfide bridge close to the ironsulfur cluster. In the amino acid sequence the coordinating amino acids as well as the additional cysteines are organized in two boxes with highly conserved sequences in all cytochrome bc complexes. It was shown that substitutions of single amino acids within or around these boxes affect the midpoint potential of the Rieske proteins (7) and that this midpoint potential can be predicted to some extent from the amino acid sequence. Proteins with the sequence CP...

PetC1 Is the Major Rieske Iron-Sulfur Protein in the Cytochrome b6f Complex of Synechocystis sp. PCC 6803

“…From these data we conclude that the Rieske protein PetC2 can indeed replace the Rieske protein PetC1 in the cytochrome b 6 f complex to some extent. This is consistent with the relative high sequence similarity and especially with the similar midpoint potential of these two proteins (5). Also the fact that the petC2 gene is functionally expressed in Synechocystis supports the view of various co-existing Rieske proteins in the cytochrome b 6 f complex of this organism (5).…”

“…It was shown that all three genes encode functional D1 proteins, although only psbD1 and psbD2 are expressed at a significant level. Similarly, three potential petC genes are present encoding for the Rieske subunit of the cytochrome b 6 f complex (5).…”

Ssr2998 of Synechocystis sp. PCC 6803 Is Involved in Regulation of Cyanobacterial Electron Transport and Associated with the Cytochrome b6f Complex

“…if the subunit is not essential for the overall function of the complex (2). This has previously been shown for the small subunit PetM as well as for Rieske iron-sulfur proteins (PetC1-3), the corresponding genes of which have been deleted in Synechocystis (23,37,38). Also the ORF ssr2998 can be completely disrupted in Synechocystis, which suggests that the encoded protein is not essential for the survival of Synechocystis under moderate culture conditions.…”

Functional Insensitivity of the Cytochrome b6f Complex to Structure Changes in the Hinge Region of the Rieske Iron-Sulfur Protein

“…However, a BLAST search in the recently deposited genome (NCBI accession number NC_003488) indicated that, as in Synechocystis sp. PCC 6803 (39), there are two additional petC genes (petC2 and petC3) in the Synechococcus genome. In Synechocystis, the petC gene located in the petCA operon was shown to be the dominant species in membranes and in isolated b 6 f complex by immunoblotting (39).…”

“…PCC 6803 (39), there are two additional petC genes (petC2 and petC3) in the Synechococcus genome. In Synechocystis, the petC gene located in the petCA operon was shown to be the dominant species in membranes and in isolated b 6 f complex by immunoblotting (39). To study the function of PetC1 and its hinge region, a ⌬petC1 mutant with the entire petC1 gene deleted and nine hinge region mutants with changes in the composition, flexibility, and length of the hinge region were constructed (Table II).…”