stigmatellin and hydroquinone

Multiple instances of low-potential electron-transport pathway inhibitors that affect the structure of the cytochrome (cyt) bc(1) complex to varying degrees, ranging from changes in hydroquinone (QH(2)) oxidation and cyt c(1) reduction kinetics to proteolytic accessibility of the hinge region of the iron-sulfur-containing subunit (Fe/S protein), have been reported. However, no instance has been documented of any ensuing change on the environment(s) of the [2Fe-2S] cluster. In this work, this issue was addressed in detail by taking advantage of the increased spectral and spatial resolution obtainable with orientation-dependent electron paramagnetic resonance (EPR) spectroscopic analysis of ordered membrane preparations. For the first time, perturbation of the low-potential electron-transport pathway by Q(i)-site inhibitors or various mutations was shown to change the EPR spectra of both the cyt b hemes and the [2Fe-2S] cluster of the Fe/S protein. In particular, two interlinked effects of Q(i)-site modifications on the Fe/S subunit, one changing the local environment of its [2Fe-2S] cluster and a second affecting the mobility of this subunit, are revealed. Remarkably, different inhibitors and mutations at or near the Q(i) site induce these two effects differently, indicating that the events occurring at the Q(i) site affect the global structure of the cyt bc(1). Furthermore, occupancy of discrete Q(i)-site subdomains differently impede the location of the Fe/S protein at the Q(o) site. These findings led us to propose that antimycin A and HQNO mimic the presence of QH(2) and Q at the Q(i) site, respectively. Implications of these findings in respect to the Q(o)-Q(i) sites communications and to multiple turnovers of the cyt bc(1) are discussed.

Topics: Antimycin A; Benzoquinones; Binding Sites; Binding, Competitive; Cell Membrane; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Enzyme Inhibitors; Enzyme Stability; Heme; Hydroquinones; Hydroxyquinolines; Iron-Sulfur Proteins; Oxidation-Reduction; Polyenes; Quinones; Rhodobacter capsulatus

We have previously reported that mutant strains of Rhodobacter capsulatus that have alanine insertions (+nAla mutants) in the hinge region of the iron sulfur (Fe-S) containing subunit of the bc(1) complex have increased redox midpoint potentials (E(m)) for their [2Fe2S] clusters. The alteration of the E(m) in these strains, which contain mutations far from the metal binding site, implied that the local environment of the metal center is indirectly altered by a change in the interaction of this subunit with the hydroquinone oxidizing (Q(o)) site [Darrouzet, E., Valkova-Valchanova, M., and Daldal, F. (2002) J. Biol. Chem. 277, 3464-3470]. Subsequently, the E(m) changes have been proposed to be predominantly due to a stronger or more stabilized hydrogen bonding between the reduced [2Fe2S] cluster and the Q(o) site inhabitant ubiquinone (Q) [Shinkarev, V. P., Kolling, D. R. J., Miller, T. J., and Crofts, A. R. (2002) Biochemistry 41, 14372-14382]. To further investigate this issue, Fe-S protein-Q interactions were monitored by electron paramagnetic resonance (EPR) spectroscopy and the findings indicated that the wild type and mutant proteins interactions with Q are similar. Moreover, when the Q(pool) was chemically depleted, the E(m) of the [2Fe2S] cluster in mutant bc(1) complexes remained more positive than a similarly treated native enzyme (e.g., the [2Fe2S] E(m) of the +2Ala mutant was 55 mV more positive than the wild type). These data suggest that the increased E(m) of the [2Fe2S] cluster in the +nAla mutants is in part due to the cluster's interaction with Q, and in part to additional factors that are independent of hydrogen bonding to Q. One such factor, the possibility of a different position of the Fe-S at the Q(o) site of the mutant proteins versus the native enzyme, was addressed by determining the orientation of the [2Fe2S] cluster in the membrane using EPR spectroscopy. In the case of the +2Ala mutant, the [2Fe2S] cluster orientation in the absence of inhibitor is different than that seen in the native enzyme. However, the +2Ala mutant cluster shared a similar orientation with the native enzyme when both samples were exposed to either stigmatellin or myxothiazol. In addition, Q(pool) extracted membranes of +2Ala mutant exhibited fewer overall orientations, with the predominant one being more similar to that observed in the non-Q-depleted membranes of the +2Ala mutant than the Q-depleted membranes of a wild-type strain. Therefore, additional comp

Topics: Alanine; Benzoquinones; Cell Membrane; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Enzyme Stability; Hydrogen Bonding; Hydroquinones; Iron-Sulfur Proteins; Mutagenesis, Site-Directed; Oxidation-Reduction; Polyenes; Potentiometry; Protein Structure, Tertiary; Protein Subunits; Rhodobacter capsulatus

Quinol oxidation by the bc(1) complex of Rhodobacter sphaeroides occurs from an enzyme-substrate complex formed between quinol bound at the Q(o) site and the iron-sulfur protein (ISP) docked at an interface on cytochrome b. From the structure of the stigmatellin-containing mitochondrial complex, we suggest that hydrogen bonds to the two quinol hydroxyl groups, from Glu-272 of cytochrome b and His-161 of the ISP, help to stabilize the enzyme-substrate complex and aid proton release. Reduction of the oxidized ISP involves H transfer from quinol. Release of the proton occurs when the acceptor chain reoxidizes the reduced ISP, after domain movement to an interface on cytochrome c(1). Effects of mutations to the ISP that change the redox potential and/or the pK on the oxidized form support this mechanism. Structures for the complex in the presence of inhibitors show two different orientations of Glu-272. In stigmatellin-containing crystals, the side chain points into the site, to hydrogen bond with a ring hydroxyl, while His-161 hydrogen bonds to the carbonyl group. In the native structure, or crystals containing myxothiazol or beta-methoxyacrylate-type inhibitors, the Glu-272 side chain is rotated to point out of the site, to the surface of an external aqueous channel. Effects of mutation at this residue suggest that this group is involved in ligation of stigmatellin and quinol, but not quinone, and that the carboxylate function is essential for rapid turnover. H(+) transfer from semiquinone to the carboxylate side chain and rotation to the position found in the myxothiazol structure provide a pathway for release of the second proton.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Chickens; Cytochrome b Group; Cytochromes c1; Electron Transport Complex III; Enzyme Stability; Hydrogen Bonding; Hydroquinones; Kinetics; Mitochondria, Heart; Models, Chemical; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Polyenes; Protein Conformation; Rhodobacter sphaeroides