| Abstract
| - The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and itsK93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured byplasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin.The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 μM) but bindsmuch more weakly to the oxidized form (Kd = 3.1 μM). In contrast, oxidized cytochrome c2 binds tooxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 μM. Such a biphasicinteraction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 μM) and reducedcytochrome c2 binds less strongly (Kd = 0.11 μM) but ∼30-fold more tightly than in the absence ofstigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have ledto the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1components during turnover. In one conformation, the Rieske protein binds near the heme of cytochromec1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite sidefrom the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor,stigmatellin, freezes the Rieske protein iron−sulfur cluster in a conformation proximal to cytochrome band distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein isprimarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizesturnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron−sulfur clusteris proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c2, when it isproximal to cytochrome c1.
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