Flexibility of the neck region of the rieske iron-sulfur protein is functionally important in the cytochrome bc1 complex.
The crystal structure of the mitochondrial cytochrome bc1 complex suggests that movement of the extramembrane (head) domain of the Rieske iron-sulfur protein (ISP) is involved in electron transfer. Such movement requires flexibility in the neck region of ISP. To test this hypothesis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with altered ISP necks (residues 39-48) were generated and characterized. Mutants with increased rigidity of the neck, generated by a double-proline substitution at Ala-46 and Ala-48 (ALA-PLP) or by a triple-proline substitution of ADV at residues 42-44 (ADV-PPP), have retarded (50%) or no photosynthetic growth, respectively. However, the mutant with a shortened neck, generated by deleting ADV (DeltaADV), has a photosynthetic growth rate comparable to that of complement cells, indicating that the length of the ISP neck is less critical than its flexibility in support of photosynthetic growth. The DeltaADV and ALA-PLP mutant membranes have 10 and 30% of the cytochrome bc1 complex activity found in the complement membrane, respectively, whereas the ADV-PPP mutant membrane contains no cytochrome bc1 complex activity. The loss of cytochrome bc1 complex activity in the DeltaADV membrane is attributed to improper docking of the head domain of ISP on cytochrome b, as indicated by a drastic change in the EPR characteristics of the Rieske iron-sulfur cluster. The loss of cytochrome bc1 complex activity in the ALA-PLP and ADV-PPP mutant membranes results from the decreased mobility of the ISP head domain due to the increased rigidity of the ISP neck. The ALA-PLP mutant complex has a larger activation energy than the wild-type complex, suggesting that movement of the head domain decreases the activation energy barrier of the cytochrome bc1 complex. Using the conditions developed for the isolation of the His-tagged complement cytochrome bc1 complex, a two-subunit complex (cytochromes b and c1) was obtained from the DeltaADV and ADV-PPP mutants, indicating that mutations at the neck region of ISP weaken the interactions among cytochrome b, ISP, and subunit IV.
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Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
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Mather
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D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D016296:Mutagenesis; D010788:Photosynthesis; D018583:Pliability; D011392:Proline / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides / Q000737:chemistry / Q000235:genetics*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D007506:Iron-Sulfur Proteins; C037989:Rieske iron-sulfur protein; D011392:Proline; D014450:Electron Transport Complex III