title: The role of an extra fragment of cytochrome b (residues 309-326) in the cytochrome bc1 complex from Rhodobacter sphaeroides.
abstract: In bacterial cytochrome b of the cytochrome bc(1) complex, there is an extra fragment located between the amphipathic helix ef and the transmembrane helix F compared to the mitochondrial counterparts. In this work, mutants at various positions of this extra fragment were generated in Rhodobacter sphaeroides in an effort to investigate its specific role in the bacterial bc(1) complex. The total deletion [cytb-Delta(309-326)] and alanine substitution [cytb-(309-326)A] mutant complexes have about 20% of the bc(1) activity found in the wild-type complex. Mutant complexes of cytb-(309-311)A, cytb-(312-314)A, cytb-(315-317)A, cytb-(318-321)A, cytb-(322-323)A, cytb-(324-326)A, cytb-(F323A), and cytb-(S322A) have respectively 87%, 85%, 89%, 100%, 32%, 90%, 100%, and 32% of the bc(1) activity, indicating that the S322 of cytochrome b is important. EPR spectral analysis reveals that the [2Fe-2S] cluster in the cytb-(S322A) mutant complex has a broadened and shifted g(x)() signal (g = 1.76). The rate of superoxide anion (O(2)(*)(-)) generation is 4 times higher in the cytb-(S322A) mutant complex than in the wild-type or mutant complexes of S322T, S322Y, or S322C. These results support the idea that alanine substitution at S322 of cytochrome b causes conformational changes at the Q(o) site by weakening the binding between cytochrome b and ISP through hydrogen bonding provided by the hydroxyl group of this residue. This change facilitates electron leakage from the Q(o) site for reaction with molecular oxygen to form superoxide anion, thus decreasing bc(1) activity.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
authors.forename: Xing
authors.initials: X
authors.lastname: Gong
authors.forename: Linda
authors.initials: L
authors.lastname: Yu
authors.forename: Chang-An
authors.initials: CA
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D045303:Cytochromes b / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017382:Reactive Oxygen Species / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D010446:Peptide Fragments; D017382:Reactive Oxygen Species; D045303:Cytochromes b; D014450:Electron Transport Complex III