Effect of mutations in the cytochrome b ef loop on the electron-transfer reactions of the Rieske iron-sulfur protein in the cytochrome bc(1) complex
by Rajagukguk, Sany Clean Johnson; Yang, Shaoqing; Yu, Chang-An; Yu, Linda; Durham, Bill; Millett, Francis Spencer
Long-range movement of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c(1) redox centers plays a key role in electron transfer within the cyt bc(1) complex. A series of 21 mutants in the cyt b ef loop of Rhodobacter sphaeroides cyt bc(1) were prepared to examine the role of this loop in controlling the capture and release of the ISP from cyt b. Electron transfer in the cyt bc(1) complex was studied using a ruthenium dimer to rapidly photo-oxidize cyt c(1) within 1 mu s and initiate the reaction. The rate constant for electron transfer from the Rieske iron-sulfur center [2Fe2S] to cyt c(1) was k(1) = 60 000 s(-1). Famoxadone binding to the Q(o) site decreases k(1) to 5400 s(-1), indicating that a conformational change on the surface of cyt b decreases the rate of release of the ISP from cyt b. The mutation I292A on the surface of the ISP-binding crater decreased k(1) to 4400 s(-1), while the addition of famoxadone further decreased it to 3000 s(-1). The mutation L286A at the tip of the ef loop decreased k(1) to 33 000 s(-1), but famoxadone binding caused no further decrease, suggesting that this mutation blocked the conformational change induced by famoxadone. Studies of all of the mutants provide further evidence that the ef loop plays an important role in regulating the domain movement of the ISP to facilitate productive electron transfer and prevent short-circuit reactions.
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