Evidence for the intertwined dimer of the cytochrome bc(1) complex in solution.
To confirm that the cytochrome bc(1) complex exists as a dimer with intertwining Rieske iron-sulfur proteins in solution, four Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes containing two pairs of cysteine substitutions, one in the interface between the head domain of iron-sulfur protein (ISP) and cytochrome b and the other between the tail domain of ISP and cytochrome b, were generated and characterized. They are: K70C(ISP)/A185C(cytb).P33C(ISP)/G89C(cytb), K70C(ISP)/A185C(cytb).P33C(ISP)/M92C (cytb), K70C (ISP)/A185C(cytb).L34C(ISP)/V64C(cytb), and K70C(ISP)/A185C(cytb).N36C(ISP)/G89C(cytb). The K70C(ISP)/A185C(cytb) cysteine pair cross-links the head domain of ISP and cytochrome b; the P33C(ISP)/G89C(cytb), P33C(ISP)/M92C (cytb), L34C(ISP)/V64C(cytb), and N36C(ISP)/G89C(cytb) cysteine pairs cross-link the tail domain of ISP and cytochrome b. An adduct protein with an apparent molecular mass of 128 kDa containing two cytochrome b and two ISP proteins is detected in the K70C(ISP)/A185C(cytb).P33C(ISP)/G89C(cytb) and K70C(ISP)/A185C(cytb).N36C(ISP)/G89C(cytb) mutant complexes, confirming that the bc(1) complex exists as a dimer with intertwining ISPs. The loss of activity in these two double-cysteine-pair mutant complexes was attributed to the disulfide bond between the head domain of ISP and cytochrome b and not the one between the tail domain of ISP and cytochrome b.
Xiao
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Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
Chandrasekaran
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Yu
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Yu
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D001483:Base Sequence; D017931:DNA Primers; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008958:Models, Molecular; D010788:Photosynthesis; D012242:Rhodobacter sphaeroides / Q000201:enzymology / Q000502:physiology; D012996:Solutions
D017931:DNA Primers; D004220:Disulfides; D007506:Iron-Sulfur Proteins; D012996:Solutions; D014450:Electron Transport Complex III