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Surface-modulated motion switch: capture and release of iron-sulfur protein in the cytochrome bc1 complex.

ABSTRACT: In the cytochrome bc(1) complex, the swivel motion of the iron-sulfur protein (ISP) between two redox sites constitutes a key component of the mechanism that achieves the separation of the two electrons in a substrate molecule at the quinol oxidation (Q(o)) site. The question remaining is how the motion of ISP is controlled so that only one electron enters the thermodynamically favorable chain via ISP. An analysis of eight structures of mitochondrial bc(1) with bound Q(o) site inhibitors revealed that the presence of inhibitors causes a bidirectional repositioning of the cd1 helix in the cytochrome b subunit. As the cd1 helix forms a major part of the ISP binding crater, any positional shift of this helix modulates the ability of cytochrome b to bind ISP. The analysis also suggests a mechanism for reversal of the ISP fixation when the shape complementarity is significantly reduced after a positional reorientation of the reaction product quinone. The importance of shape complementarity in this mechanism was confirmed by functional studies of bc(1) mutants and by a structure determination of the bacterial form of bc(1). A mechanism for the high fidelity of the bifurcated electron transfer is proposed.

SUBMITTER: Esser L

PROVIDER: S-EPMC1551902 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:One of the important elements of operation of cytochrome bc1 (mitochondrial respiratory complex III) is a large scale movement of the head domain of iron-sulfur protein (ISP-HD), which connects the quinol oxidation site (Qo) located within the cytochrome b, with the outermost heme c(1) of cytochrome c(1). Several mitochondrial disease-related mutations in cytochrome b are located at the cytochrome b-ISP-HD interface, thus their molecular effects can be associated with altered motion of ISP-HD. Using purple bacterial model, we recently showed that one of such mutations - G167P shifts the equilibrium position of ISP-HD towards positions remote from the Qo site as compared to the native enzyme [Borek et al., J. Biol. Chem. 290 (2015) 23781-23792]. This resulted in the enhanced propensity of the mutant to generate reactive oxygen species (ROS) which was explained on the basis of the model evoking "semireverse" electron transfer from heme bL to quinone. Here we examine another mutation from that group - G332D (G290D in human), finding that it also shifts the equilibrium position of ISP-HD in the same direction, however displays less of the enhancement in ROS production. We provide spectroscopic indication that G332D might affect the electrostatics of interaction between cytochrome b and ISP-HD. This effect, in light of the measured enzymatic activities and electron transfer rates, appears to be less severe than structural distortion caused by proline in G167P mutant. Comparative analysis of the effects of G332D and G167P confirms a general prediction that mutations located at the cytochrome b-ISP-HD interface influence the motion of ISP-HD and indicates that "pushing" ISP-HD away from the Qo site is the most likely outcome of this influence. It can also be predicted that an increase in ROS production associated with the "pushing" effect is quite sensitive to overall severity of this change with more active mutants being generally more protected against elevated ROS. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Project description:The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P 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 stigmatellin. 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 stigmatellin, 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 stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, 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, stigmatellin, freezes the Rieske protein iron-sulfur 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 iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c2, when it is proximal to cytochrome c1.

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Surface-modulated motion switch: capture and release of iron-sulfur protein in the cytochrome bc1 complex.

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