Subunit IV of Cytochrome bc1 Complex from Rhodobacter sphaeroides: LOCALIZATION OF REGIONS ESSENTIAL FOR INTERACTION WITH THE THREE-SUBUNIT CORE COMPLEX

Shih-Chia Tso,Sudha K Shenoy,Byron N Quinn,Linda Yu

doi:10.1074/jbc.m907367199

Shih-Chia Tso, Sudha K Shenoy + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m907367199

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Abstract

Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively.

Highlights

The cytochrome bc1 complex from Rhodobacter sphaeroides, which catalyzes electron transfer from ubiquinol to cytochrome c2 in the cyclic photosynthetic electron transfer chain, has been purified and characterized [1]
Since glutathione S-transferase (GST)-IV(86 –109) fusion protein has little reconstitutive activity, some region(s) in residues 1– 85 are required for bc1 activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex
The turnover rates for the cytochrome bc1 complexes purified from mitochondria of bovine heart and yeast and chromatophores of R. sphaeroides and R. capsulatus, by a single chromatographic procedure are: 1152, 219, 128, and 64 sϪ1 [4]

Summary

EXPERIMENTAL PROCEDURES

Generation of E. coli Strains Expressing GST-mutated IV Fusion Proteins—The expression vectors for mutants IV[1– 40], IV[1–76], IV[1– 85], and IV[1–109] were constructed by introducing the stop codon (TGA) at positions 121, 229, 256, and 328 base pairs downstream from the start codon of the fbcQ gene in the pSelect/IV plasmid, respectively. They were achieved by site-directed mutagenesis using the Altered Sites System from Promega Corp. The 100% reconstitutive activity is that restored by recombinant wild-type IV with or without fused GST

Wild type

RESULTS AND DISCUSSION

Recombinant IV