Mitochondrial cytochrome bc1 complex performs two functions: It is a respiratory multienzyme complex and it recognizes a mitochondrial targeting presequence. Refined crystal structures of the 11-subunit bc1 complex from bovine heart reveal full views of this bifunctional enzyme. The "Rieske" iron-sulfur protein subunit shows significant conformational changes in different crystal forms, suggesting a new electron transport mechanism of the enzyme. The mitochondrial targeting presequence of the "Rieske" protein (subunit 9) is lodged between the two "core" subunits at the matrix side of the complex. These "core" subunits are related to the matrix processing peptidase, and the structure unveils how mitochondrial targeting presequences are recognized.

The high-resolution structure supports the proposed coordination pattern involving histidine ligands and provides a basis for a detailed analysis of the spectroscopic and electrochemical properties. As the cluster is located at the tip of the protein, it might come into close contact with cytochrome b. The exposed N epsilon atoms of the histidine ligands of the cluster are readily accessible to quinones and inhibitors within the hydroquinone oxidation (QP) pocket of the bc1 complex and may undergo redox-dependent protonation/deprotonation.

The crystal structure of the bovine Rieske iron-sulfur protein indicates a sulfur atom (S-1) of the iron-sulfur cluster and the sulfur atom (S ␥ ) of a cysteine residue that coordinates one of the iron atoms form hydrogen bonds with the hydroxyl groups of Ser-163 and Tyr-165, respectively. We have altered the equivalent Ser-183 and Tyr-185 in the Saccharomyces cerevisiae Rieske ironsulfur protein by site-directed mutagenesis of the ironsulfur protein gene to examine how these hydrogen bonds affect the midpoint potential of the iron-sulfur cluster and how changes in the midpoint potential affect the activity of the enzyme.Eliminating the hydrogen bond from the hydroxyl group of Ser-183 to S-1 of the cluster lowers the midpoint potential of the cluster by 130 mV, and eliminating the hydrogen bond from the hydroxyl group of Tyr-185 to S ␥ of Cys-159 lowers the midpoint potential by 65 mV. Eliminating both hydrogen bonds has an approximately additive effect, lowering the midpoint potential by 180 mV. Thus, these hydrogen bonds contribute significantly to the positive midpoint potential of the cluster but are not essential for its assembly. The activity of the bc 1 complex decreases with the decrease in midpoint potential, confirming that oxidation of ubiquinol by the iron-sulfur protein is the rate-limiting partial reaction in the bc 1 complex, and that the rate of this reaction is extensively influenced by the midpoint potential of the iron-sulfur cluster.The Rieske iron-sulfur protein is a ubiquitous component of cytochrome bc 1 complexes (1-4) and has been shown to be essential for electron transfer and energy transduction by purification of the protein in a reconstitutively active form and reconstitution to iron-sulfur protein depleted bc 1 complex (5, 6). The electronic environment of the [2Fe-2S] cluster in the Rieske iron-sulfur protein differs from that in plant type [2Fe-2S] ferredoxins as evidenced by a distinct EPR spectrum (1) and a redox midpoint potential of the Rieske protein (e.g. ϩ280 mV) that is much more positive than the midpoint potentials typical of the ferredoxins (e.g. Ϫ420 mV; Ref. 2). The high midpoint potential of the iron-sulfur cluster is essential for the function of the Rieske protein in the Q cycle mechanism of the bc 1 complex (7,8), in which the Rieske protein is the primary electron acceptor and drives the electron transfer reaction by oxidizing ubiquinol and divergently transferring one electron to cytochrome c 1 , while the ubisemiquinone that is formed from ubiquinol reduces the low potential b heme.Recently, the crystal structure of the water-soluble part of the Rieske iron-sulfur protein of bovine heart mitochondrial bc 1 complex has been elucidated at 1.5 Å (9, 10). Ten ␤ strands form three layers of anti-parallel ␤ sheets in a flat spherical molecule as shown in Fig. 1A. The cluster binding fold is a small domain-like structure comprising approximately 46 residues; it consists of a distorted four-stranded antiparallel ␤-sheet and three loops. The loops between the strands ␤4...

We report here, for the first time, the primary structure of uncoupling protein as established by amino acid sequencing. Like the ADP/ATP carrier, this protein has a tripartite structure comprising three similar sequences of approximately 100 residues each. These six ‘repeats’ exhibit striking conservation of several residues, in particular glycine and proline, at possible structurally strategic positions. Although the two proteins differ strongly in their amino acid composition, their sequences are distantly homologous. Three membrane‐spanning alpha‐helices can be deduced from hydropathy plots. A modified plot accounting for amphiphilic helices indicates 5‐6 such alpha‐segments. In addition an amphiphilic beta‐strand of membrane‐spanning length can be discerned. The tripartite sequence structure is also distinctly reflected in the hydropathy distribution. Based on the membrane disposition of the segments of the ADP/ATP carrier, a model for the transmembrane folding path of the polypeptide chain of the uncoupling protein is proposed.