Evidence for the head domain movement of the Rieske iron-sulfur protein in electron transfer reaction of the cytochrome bc1 complex

Abstract

The three-dimensional structure of the mitochondrial cytochrome bc1 complex suggests that movement of the extramembrane domain (head) of the Rieske iron-sulfur protein (ISP) may play an important role in electron transfer. Such movement requires flexibility in the neck region of ISP, since the head and transmembrane domains of the protein are rather rigid. To test this hypothesis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with cysteine substitution at various positions in the ISP neck (residues 39-48) were generated and characterized. The mutants with a single cysteine substitution at Ala42 or Val44 and a double cysteine substitution at Val44 and Ala46 (VQA-CQC) or at Ala42 and Ala46 (ADVQA-CDVQC) have photosynthetic growth rates comparable with that of complement cells. Chromatophore membrane and intracytoplasmic membrane (ICM) prepared from these mutants have cytochrome bc1 complex activity similar to that in the complement membranes, indicating that flexibility of the neck region of ISP was not affected by these cysteine substitutions. Mutants with a double cysteine substitution at Ala42 and Val44 (ADV-CDC) or at Pro40 and Ala42 (PSA-CSC) have a retarded (50%) or no photosynthetic growth rate, respectively. The ADV-CDC or PSA-CSC mutant ICM contains 20 or 0% of the cytochrome bc1 complex activity found in the complement ICM. However, activity can be restored by the treatment with β- mercaptoethanol (β-ME). The restored activity is diminished upon removal of β-ME but is retained if the β-ME-treated membrane is treated with the sulfhydryl reagent N-ethylmaleimide or p-chloromercuribenzoic acid. These results indicate that the loss of bc1 complex activity in the ADV-CDC or PSA-CSC mutant membranes is due to disulfide bond formation, which increases the rigidity of ISP neck and, in turn, decreases the mobility of the head domain. Using the conditions developed for the isolation of His-tagged complement cytochrome bc1 complex, a two-subunit complex (cytochromes b and c1) is obtained from all of the double cysteine-substituted mutants. This suggests that introduction of two cysteines in the neck region of ISP weakens the interactions between cytochromes b, ISP, and subunit IV.