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...