Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced @entity10052 cytochrome c2 to the cytochrome bc1 complex mediated by the conformation of the Rieske XXXX protein.

@entity72749 @entity5985 @entity10052 @entity72748
The dissociation constants for the binding of @entity10052 cytochrome c2 and its @entity72748 mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor @entity72749 . The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 microM) but binds much more weakly to the oxidized form (Kd = 3.1 microM). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 microM. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of @entity72749 , we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 microM) and reduced cytochrome c2 binds less strongly (Kd = 0.11 microM) but approximately 30-fold more tightly than in the absence of @entity72749 . Structural studies with cytochrome bc1, with and without the inhibitor @entity72749 , have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, @entity72749 , freezes the Rieske protein @entity5985 cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the @entity5985 cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c2, when it is proximal to cytochrome c1.
