On the basis of x-ray diffraction data to a resolution of 2.9 angstroms, atomic models of most protein components of the bovine cytochrome bc1 complex were built, including core 1, core 2, cytochrome b, subunit 6, subunit 7, a carboxyl-terminal fragment of cytochrome c1, and an amino-terminal fragment of the iron-sulfur protein. The positions of the four iron centers within the bc1 complex and the binding sites of the two specific respiratory inhibitors antimycin A and myxothiazol were identified. The membrane-spanning region of each bc1 complex monomer consists of 13 transmembrane helices, eight of which belong to cytochrome b. Closely interacting monomers are arranged as symmetric dimers and form cavities through which the inhibitor binding pockets can be accessed. The proteins core 1 and core 2 are structurally similar to each other and consist of two domains of roughly equal size and identical folding topology.

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...

Abstract. The electron paramagnetic resonance signals of the soluble P-450 cytochrome from Pseudomonas putida were observed at temperatures from 4.2 to 80'K. As isolated, P450 has a signal typical of a low spin ferric-heme compound with sulfur as one of the axial ligands (g = 2.45, 2.26, 1.915). We also detected a minor signal typical of high spin ferric heme (g = 8, 4, 1.8) equivalent to less than 7% of the heme at temperatures below 20'K. On titration with the substrate, (+)-camphor, the low spin signal decreased and the high spin signal increased, maximally representing about 60% of the heme. For reasons not thus far understood, 40% of the heme is not converted to high spin by either (+) or (-)-camphor. The high spin signal has a rhombic character which is stronger than any previously observed with a heme compound (E = 0.33 cm-'; D = 3.8 cm-'; E/D = 0.087).We conclude that P-450,,.m as isolated is equal to or more than 95% in a low spin form probably having sulfur as one of the axial ligands. The binding of substrate displaces this ligand sufficiently to allow for conversion from a low to a high spin form.Cytochrome P450 (henceforth called P-450) was discovered as a CO-binding pigment in isolated mammalian liver microsomes. 12 Since then similar cytochromes have been found in adrenal mitochondria and in bacteria.A5 Since the proteins from these sources were not readily solubilized and purified, we undertook a study of the pure bacterial protein on the hypothesis that an understanding of the structure and function of this class of heme protein, including the microsomal cytochrome, could be obtained.The visible absorption bands of P-450 from all sources are shifted upon addition of numerous compounds, many of which are known substrates.6 Thus the P450 is thought to be the site of substrate binding for the hydroxylation reactions catalyzed.7-'2 In general, aromatic primary amines and aromatic Nheterocyclic compounds shift the Soret band of the microsomal protein to the red (type II), whereas various drugs, either substrates or inhibitors, shift the Soret peak to the blue (type I). These and the other optical properties of P450 complexes with substrates, etc., prompted the suggestion that the spin state of these heme proteins shift from low spin to high spin on addition of substrates.'3 14 1157