2؉dissociates 5-29 times faster from the N-terminal in these CaM⅐peptide complexes and both lobes are required for activation, Ca 2؉ dissociation from the N-terminal would control target protein inactivation. Ca

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...

To better understand the mechanism of divergent electron transfer from ubiquinol to the iron-sulfur protein and cytochrome b L within the cytochrome bc 1 complex, we have examined the effects of antimycin on the presteady state reduction kinetics of the bc 1 complex in the presence or absence of endogenous ubiquinone. When ubiquinone is present, antimycin slows the rate of cytochrome c 1 reduction by ϳ10-fold but had no effect upon the rate of cytochrome c 1 reduction in bc 1 complex lacking endogenous ubiquinone. In the absence of endogenous ubiquinone cytochrome c 1 , reduction was slower than when ubiquinone was present and was similar to that in the presence of ubiquinone plus antimycin. These results indicate that the low potential redox components, cytochrome b H and b L , exert negative control on the rate of reduction of cytochrome c 1 and the Rieske iron-sulfur protein at center P. If electrons cannot equilibrate from cytochrome b H and b L to ubiquinone, partial reduction of the low potential components slows reduction of the high potential components. We also examined the effects of decreasing the midpoint potential of the iron-sulfur protein on the rates of cytochrome b reduction. As the midpoint potential decreased, there was a parallel decrease in the rate of b reduction, demonstrating that the rate of b reduction is dependent upon the rate of ubiquinol oxidation by the iron-sulfur protein. Together these results indicate that ubiquinol oxidation is a concerted reaction in which both the low potential and high potential redox components control ubiquinol oxidation at center P, consistent with the protonmotive Q cycle mechanism.Although the protonmotive Q 1 cycle mechanism of the cytochrome bc 1 complex is generally understood (1-3), the mechanism of ubiquinol oxidation at center P has not been fully elucidated. With the determination of the crystal structure of the cytochrome bc 1 complex (4 -5), a more extensive examination of the structure-function relationships of the Q cycle mechanism is possible.It is generally accepted that the mechanism of ubiquinol oxidation at center P involves a divergent oxidation in which the iron-sulfur protein oxidizes ubiquinol to semiquinone and the semiquinone reduces cytochrome b L (1, 3). It is unclear, however, whether the oxidation of ubiquinol occurs through semiquinone in a sequential mechanism or whether ubiquinol is oxidized by the iron-sulfur protein and cytochrome b L in a concerted reaction. Earlier experiments suggested the presence of a transient semiquinone at center P (6), consistent with a sequential mechanism, although recent experiments suggest otherwise (7).There have been two proposals for concerted reaction mechanisms at center P. Link (8) proposed a "proton-gated affinity change" mechanism in which stabilization of ubisemiquinone by anti-ferromagnetic coupling to the reduced iron-sulfur protein raises the potential of the iron-sulfur cluster such that the cluster cannot be oxidized by cytochrome c 1 until the semiquinone is oxidized. Jü nemann...

We have examined the pre-steady state reduction kinetics of the Saccharomyces cerevisiae cytochrome bc 1 complex by menaquinol in the presence and absence of endogenous ubiquinone to elucidate the mechanism of triphasic cytochrome b reduction. With cytochrome bc 1 complex from wild type yeast, cytochrome b reduction was triphasic, consisting of a rapid partial reduction phase, an apparent partial reoxidation phase, and a slow rereduction phase. Absorbance spectra taken by rapid scanning spectroscopy at 1-ms intervals before, during, and after the apparent reoxidation phase showed that this was caused by a bona fide reoxidation of cytochrome b and not by any negative spectral contribution from cytochrome c 1 . With cytochrome bc 1 complex from a yeast mutant that cannot synthesize ubiquinone, cytochrome b reduction by either menaquinol or ubiquinol was rapid and monophasic. Addition of ubiquinone restored triphasic cytochrome b reduction, and the duration of the reoxidation phase increased as the ubiquinone concentration increased. When reduction of the cytochrome bc 1 complex through center P was blocked, cytochrome b reduction through center N was biphasic and was slowed by the addition of exogenous ubiquinone. These results show that ubiquinone residing at center N in the oxidized cytochrome bc 1 complex is responsible for the triphasic reduction of cytochrome b.Although the protonmotive Q cycle mechanism of the cytochrome bc 1 complex is generally well understood (1-3), the redox behavior of cytochrome b during pre-steady state reduction of the bc 1 complex is not fully understood. Cytochrome b reduction is triphasic, consisting of a rapid partial reduction phase, a partial reoxidation phase, and a slow rereduction phase. This behavior is puzzling, because the reoxidation phase occurs while reduced substrate is still available, and continued reduction of cytochrome b would be expected.Previous examinations of the pre-steady state reduction kinetics of the bc 1 complex were limited to single wavelength kinetics, and the spectral data, when collected, extended over time ranges that were long relative to the half-times of the reactions (4 -9). The substrates used in these studies, succinate, duroquinol, trimethylquinol, and ubiquinol, have relatively high redox potentials and reduce only a small percentage of cytochrome b. This is of concern because the high redox potential may predispose these substrates to oxidize cytochrome b and thus introduce artifacts into the pre-steady state kinetics in the absence of a low potential reductant.Several explanations for the triphasic reduction have been put forth. One proposal is that ubiquinone formed at center P is not in rapid equilibration with the quinone pool and oxidizes cytochrome b at center N (5, 10). Crystal structures of the mitochondrial cytochrome bc 1 complexes show a pear-shaped and dimeric integral membrane protein that extends ϳ80 Å into the matrix and ϳ30 Å into the intermembrane space (11, 12). There are two large cavities within the bc 1 dimer that lin...