Inhibitor binding changes domain mobility in the iron–sulfur protein of the mitochondrial bc 1 complex from bovine heart

Abstract

We Ubiquinol-cytochrome-c oxidoreductase (cytochrome bc1 complex; EC 1.10.2.2) is a segment of the respiratory chain in mitochondria and of the photosynthetic apparatus of purple bacteria. It catalyzes electron transfer (ET) from ubiquinol to cytochrome c, coupled to proton transport across a membrane (from the matrix space to the intermembrane space of mitochondria; from the cytoplasm to the periplasm of purple bacteria). The resulting electrochemical proton gradient drives ATP synthesis and transport processes (1, 2). Essential for the function of the bc1 complex are the three redox proteins cytochrome b, cytochrome c1, and the iron-sulfur protein (ISP). Two b-type hemes (b L and b H ) are attached to cytochrome b, one c-type heme is bound to cytochrome c1, and a Rieske-type iron-sulfur center (FeS) is bound to ISP (2). Whereas some bacterial bc1 complexes consist of only those redox subunits (3), their mitochondrial counterparts contain up to 8 additional protein subunits whose precise functions in the complex are largely unknown (4).The protonmotive Q cycle model (2, 5, 6) best explains experimental results on the ET pathway through the four redox centers of the bc1 complex. It postulates two separate ubiquinone binding sites, called Q o and Q i . In bc1 complexes of the inner membrane of mitochondria, Q o is located near the membrane surface facing the intermembrane space, and Q i is near the membrane surface facing the matrix space. The Q cycle model requires bifurcated electron flow from ubiquinol bound in the Q o site: The first electron of ubiquinol is sequentially transferred to the ISP, cytochrome c1, and eventually to the soluble cytochrome c. Protons are released into the intermembrane space, generating a ubisemiquinone anion in the Q o site. The second electron is transferred to hemes b L and b H and to a ubiquinone or a ubisemiquinone anion in the Q i site. The fully reduced quinone in the Q i site picks up two protons from the matrix space and moves to the Q o site for recycling.The discovery of different types of specific ET inhibitors of the bc1 complex was crucial for the development of this Q cycle hypothesis. The two major types of bc1 inhibitors are called Q o or Q i inhibitors, depending on their action in the cytochrome bc1 complex (7,8). All Q i inhibitors target specifically the ET path from heme b L to ubiquinone͞ubisemiquinone in the Q i site; they do not share common structural motifs. Q o inhibitors block binding of quinol to the Q o site and ET through this site. They can be classified further on the basis of common structural motifs and of their effects on the absorption spectrum of heme b L and on the electron paramagnetic resonance (EPR) spectrum and redox potential of the FeS (7). One Q o inhibitor subtype shares a methoxyacrylate (MOA) group (examples: myxothiazol, MOA-stilbene), another subtype resembles a hydroxyquinone molecule (example: 5-undecyl-6-hydroxy-4,7-dioxobenzothiazol or UHDBT), and a third subtype has a chromone group as the common motif (example: stigma...

Mechanism of Ubiquinol Oxidation by the bc1 Complex: Role of the Iron Sulfur Protein and Its Mobility

“…In the ISP B condition, a different set of weak forces constrains the system so that occupancy of the cyt b binding domain is higher, to favor formation of the reaction complex with quinol. The low Fe density for the cluster seen in the data of both the Zhang et al (7) and Kim et al (9) suggests that a significant fraction of the ISP head is not bound in either position, and this is confirmed by more detailed analysis of protein occupancy (22). The different positions may be thought of as mapping the trajectory of the displacement between the positions near cyt b and near cyt c 1 .…”

“…Lancaster The movement of the ISP provides a new perspective on these aspects of mechanism. For any mechanism in which dissociation of the reaction complex is the rate-limiting step and shows a high activation barrier (14,22,39), the reaction complex, either between quinol and the oxidized subunit in the ISP B position (22) or between semiquinone and reduced ISP (9,39), would also separate the ISP from its electron acceptor. Because of the high activation barrier, the reaction would proceed to the right only on removal of the semiquinone by oxidation through cyt b L .…”

Mechanism of Ubiquinol Oxidation by the bc1 Complex: Different Domains of the Quinol Binding Pocket and Their Role in the Mechanism and Binding of Inhibitors

“…Several different mechanisms have been proposed to resolve the paradox of the bifurcated reaction (see below), which combine some of the modifications suggested in i-iii above with a variety of switching mechanisms controlled by later redox events (6,10,(52)(53)(54)(55)(56).…”

“…For the distance of 11 Å from a distal domain occupant, a value for λ of 2 eV (higher values would give lower rates), and published values for E m of the redox centers, no obvious combination of reaction parameters would provide a rate for reduction of heme b L as fast as that measured. If the occupancy was determined by the activation barrier ([QH • -ISP red ] ∼ 2 × 10 -8 ), as seems to be implicit in the mechanism Kim et al (10), the maximal rate would be < 1 s -1 (depending on choice of λ), 3 orders of magnitude less than needed. The Marcus relationship would limit mechanisms to those in which electron transfer from the distal domain occurred after the activation barrier, allowing only those models in which the activation barrier is in the first electrontransfer reaction (21).…”

“…(4) Kim et al (10) have favored a model in which "the two electrons of ubiquinol in the complex are transferred simultaneously, one to FeS, and the second to heme b L ", but they did not suggest a mechanism by which this could happen.…”