The role of extra fragment at the C-terminal of cytochrome b (residues 421-445) in the cytochrome bc1 complex from Rhodobacter sphaeroides

Sequence alignment of cytochrome b of the cytochrome bc(1) complex from various sources reveals that bacterial cytochrome b contain an extra fragment at the C terminus. To study the role of this fragment in bacterial cytochrome bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with progressive deletion from this fragment ( residues 421 - 445) were generated and characterized. The cytb Delta-(433 - 445) bc(1) complex, in which 13 residues from the C-terminal end of this fragment are deleted, has electron transfer activity, subunit composition, and physical properties similar to those of the complement complex, indicating that this region of the extra fragment is not essential. In contrast, the electron transfer activity, binding of cytochrome b, ISP, and subunit IV to cytochrome c(1), redox potentials of cytochromes b and c(1) in the cytbDelta-(427 - 445), cytbDelta-( 425 - 445), and cytbDelta-(421-445) mutant complexes, in which 19, 21, or all residues of this fragment are deleted, decrease progressively. EPR spectra of the [2Fe-2S] cluster and the cytochromes b in these three deletion mutant bc(1) complexes are also altered; the extent of spectral alteration increases as this extra fragment is shortened. These results indicate that the first 12 residues ( residues 421 - 432) from the N-terminal end of the C-terminal extra fragment of cytochrome b are essential for maintaining structural integrity of the bc(1) complex.