Effect of Mutations in the Cytochrome b ef Loop on the Electron Transfer Reactions of the Rieske Iron-Sulfur Protein in the Cytochrome bc1 Complex

摘要

Long range movement of the iron-sulfur protein (ISP) between the cytochrome b (cyt b) and cyt c1 redox centers plays a key role in electron transfer within the cyt bc1 complex. A series of 21 mutants in the cyt b ef loop of Rhodobacter sphaeroides cyt bc1 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 bc1 complex was studied using a ruthenium dimer to rapidly photooxidize cyt c1 within 1 μs and initiate the reaction. The rate constant for electron transfer from the iron-sulfur center [2Fe2S] center to cyt c1 was k1 = 60,000 s⁻¹. Famoxadone binding to the Qo site decreases k1 to 5,400 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 k1 to 4,400 s⁻¹, while addition of famoxadone further decreased it to 3,000 s⁻¹. The mutation L286A at the tip of the ef loop decreased k1 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 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.