cytochrome-c-t and stigmatellin

The modified Q cycle mechanism accounts for the proton and charge translocation stoichiometry of the bc(1) complex, and is now widely accepted. However the mechanism by which the requisite bifurcation of electron flow at the Q(o) site reaction is enforced is not clear. One of several proposals involves conformational gating of the docking of the Rieske ISP at the Q(o) site, controlled by the stage of the reaction cycle. Effects of different Q(o)-site inhibitors on the position of the ISP seen in crystals may reflect the same conformational mechanism, in which case understanding how different inhibitors control the position of the ISP may be a key to understanding the enforcement of bifurcation at the Q(o) site (Table 1). Here we examine the available structures of cytochrome bc(1) with different Q(o)-site inhibitors and different ISP positions to look for clues to this mechanism. The effect of ISP removal on binding affinity of the inhibitors stigmatellin and famoxadone suggest a "mutual stabilization" of inhibitor binding and ISP docking, however this thermodynamic observation sheds little light on the mechanism. The cd(1) helix of cytochrome b moves in such a way as to accommodate docking when inhibitors favoring docking are bound, but it is impossible with the current structures to say whether this movement of α-cd(1) is a cause or result of ISP docking. One component of the movement of the linker between E and F helices also correlates with the type of inhibitor and ISP position, and seems to be related to the H-bonding pattern of Y279 of cytochrome b. An H-bond from Y279 to the ISP, and its possible modulation by movement of F275 in the presence of famoxadone and related inhibitors, or its competition with an alternate H-bond to I269 of cytochrome b that may be destabilized by bound famoxadone, suggest other possible mechanisms. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

Topics: Binding Sites; Catalytic Domain; Crystallography, X-Ray; Cytochromes; Cytochromes c; Databases, Protein; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Enzyme Inhibitors; Hydrogen Bonding; Methacrylates; Models, Molecular; Molecular Structure; Mutation; Oxazoles; Polyenes; Principal Component Analysis; Protein Binding; Protein Conformation; Protein Structure, Secondary; Strobilurins; Tyrosine; Ubiquinone

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc(1) complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q(i) site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q(o) site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A(3) in a fashion similar to the Q(o) site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q(i) and the Q(o) sites of the fungal cytochrome bc(1) complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc(1) complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q(o) and Q(i) sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc(1) complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q(i) and Q(o) sites. Moreover, crystal structure of chicken cytochrome bc(1) complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q(i) and Q(o) sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc(1) inhibitor that acts at both of the active sites of this enzyme.

Topics: Alkenes; Animals; Anti-Bacterial Agents; Antimycin A; Catalytic Domain; Cattle; Chickens; Crystallography, X-Ray; Cytochromes b; Cytochromes c; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Male; Mitochondria, Liver; Mitochondrial Proteins; Oxidoreductases; Phenols; Pichia; Plant Proteins; Polyenes; Rats; Rats, Wistar; Respiration; Rhodobacter capsulatus; Superoxides; Ubiquinone

It has been shown that the intrinsic mitochondrial apoptotic cascade is activated in vascular hyperpermeability after conditions such as hemorrhagic shock. Studies from our laboratory demonstrated mitochondrial reactive oxygen species (ROS) formation in endothelial cells during vascular hyperpermeability. We hypothesized that the participation of mitochondrial ROS in the intrinsic apoptotic cascade results in microvascular endothelial cell hyperpermeability. The purpose of this study was to identify the site(s) of ROS formation in the mitochondrial complex(es) that leads to hyperpermeability. Rat lung microvascular endothelial cell monolayers were pretreated with inhibitors of the complex(es) (I-V) before the activation of the mitochondrial apoptotic cascade using the proapoptotic peptide BAK (BH3). Inhibitors of the xanthine oxidase, nicotinamide adenine dinucleotide phosphate (reduced form) oxidase, NOS, and cytochrome P-450 monooxygenase were also studied. The hyperpermeability was determined by the fluorescence of fluorescein isothiocyanate-albumin that leaked across endothelial cells and ROS production by 2',7& rime;-dichlorofluorescein diacetate. Cytochrome c levels were also measured. BAK (BH3)-transfected cells showed increased ROS, cytosolic cytochrome c, and hyperpermeability (P<0.05). Complex III inhibitors antimycin A (10 microM) and stigmatellin (10 microM) attenuated BAK (BH3)-mediated ROS formation and hyperpermeability (P<0.05). The complex III inhibition decreased BAK (BH3)-mediated cytochrome c release. The results suggest that mitochondrial ROS formation, particularly at respiratory chain complex III, is involved in BAK-induced monolayer hyperpermeability.

Topics: Animals; Antimycin A; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Cytochromes c; Electron Transport Complex III; Endothelial Cells; Lung; Microcirculation; Mitochondria; Models, Biological; Permeability; Polyenes; Rats; Reactive Oxygen Species