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Sequence alignment of the Rieske iron-sulfur protein (ISP) of cytochrome bc1 complex from various sources reveals that bacterial ISPs contain an extra fragment. To study the role of this fragment in bacterial cytochrome bc1 complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with deletion or single- or multiple-alanine substitution at various positions of this fragment (residues 96-107) were generated and characterized. The ISPA(96- 107), ISP(96- 107)A, and ISP( 104- 107)A mutant cells, in which residues 96-107 of ISP are deleted, and residues 96-107 and 104-107 are substituted with alanine, respectively, do not grow photosynthetically and show no bc1 complex activity in intracytoplasmic membranes prepared from these mutant cells. The ISP(96-99)A, in which residues 96-99 are substituted with alanine, grows photosynthetically at a rate comparable to that of the complement cells, whereas ISP(100-103)A, in which residues 100-103 are substituted with alanine, has a longer lag period prior to photosynthetic growth. Chromatophores prepared from these two mutant cells have 48% and 9% of the bc1 activity found in the complement chromatophores. The loss (or decrease) of bc1 activity in these mutant membranes results from a lack (or decrease) of ISP in the membrane due to ISP protein instability and not from mutations affecting the assembly of cytochromes b and c1 into the membrane, the binding affinity of cytochrome b to cytochrome c1, or the ability of these two cytochromes to interact with ISP or subunit IV. The order of essentiality of residues in this fragment is residues 104-107 > residues 100-103 > residues 96-99. [ABSTRACT FROM AUTHOR]