stigmatellin and quinone

The electrostatic potential in the secondary quinone (QB) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to QB. It is controlled by the ionization states of residues in a strongly interacting cluster around the QB site. Reduction of the QB induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the QB binding pocket. It binds to the QB site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the QB pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the QB site was modulated by a single mutation, L213Asp → Ala, or double mutations, L213Asp-L212Glu → Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn → AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the QB site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Benzoquinones; Binding Sites; Molecular Sequence Data; Photosynthetic Reaction Center Complex Proteins; Polyenes; Protein Binding; Rhodobacter sphaeroides; Static Electricity

We report here the first example of a reaction center mutant from Rhodobacter sphaeroides, where a single mutation (M266His --> Leu) taking place in the primary quinone protein pocket confers selective resistance to triazine-type inhibitors (terbutryn, ametryn, and atrazine), which bind in the secondary quinone protein pocket, at about 13 A from the mutation site. The M266His --> Leu mutation involves one of the iron atom ligands. Interestingly, neither the secondary quinone nor the highly specific inhibitor stigmatellin binding affinities are affected by the mutation. It is noticeable that in the M266His --> Ala mutant a nativelike behavior in observed. We suggest that the long side chain of Leu in position M266 may lack space to accommodate in the Q(A) pocket therefore transferring its hindrance to the Q(B) pocket. This may occur via the structural feature formed by the Q(A)-M219His-Fe-L190His-inhibitor (or Q(B)) connection, pushing L189Leu and/or L229Ile in closer contact to the triazine molecules, therefore decreasing their bindings. This opens the possibility to finely tune, in reaction center proteins, the affinity for herbicides by designing mutations distant from their binding sites.

Topics: Atrazine; Benzoquinones; Binding, Competitive; Drug Resistance, Bacterial; Histidine; Leucine; Methionine; Models, Chemical; Mutagenesis, Site-Directed; Photosystem II Protein Complex; Polyenes; Protein Binding; Rhodobacter sphaeroides; Triazines

The 3.0-3.1A X-ray structures of the cytochrome b(6)f complex from Mastigocladus laminosus and Chlamydomonas reinhardtii obtained in the presence of the p-side quinone-analogue inhibitor tridecyl-stigmatellin (TDS) are very similar. A difference occurs in the p-side binding position of TDS. In C.reinhardtii, TDS binds in the ring-in mode, as previously found for stigmatellin in X-ray structures of the cytochrome bc(1) complex. In this mode, the H-bonding chromone ring moiety of the TDS bound in the Q(p) niche is proximal to the ISP [2Fe-2S] cluster, and its 13 carbon tail extends through a portal to the large inter-monomer quinone-exchange cavity. However, in M.laminosus, TDS binds in an oppositely oriented ring-out mode, with the tail inserted toward the Q(p) niche through the portal and the ring caught in the quinone-exchange cavity that is 20A away from the [2Fe-2S] cluster. Site-directed mutagenesis of residues that might determine TDS binding was performed with the related transformable cyanobacterium Synechococcus sp. PCC 7002. The following changes in the sensitivity of electron transport activity to TDS and stigmatellin were observed: (a) little effect of mutation L193A in cytochrome b(6), which is proximal to the chromone of the ring-out TDS; (b) almost complete loss of sensitivity by mutation L111A in the ISP cluster binding region, which is close to the chromone of the ring-in TDS; (c) a ten and 60-fold increase associated with the mutation L81F in subunit IV. It was inferred that only the ring-in binding mode, in which the ring interacts with residues near the ISP, is inhibitory, and that residue 81 of subunit IV, which resides at the immediate entrance to the Q(p) niche, controls the relative binding affinity of inhibitor at the two different binding sites.

Topics: Amino Acid Sequence; Animals; Benzoquinones; Binding Sites; Chlamydomonas reinhardtii; Crystallography, X-Ray; Cyanobacteria; Cytochrome b6f Complex; Enzyme Inhibitors; Hydrogen Bonding; Kinetics; Models, Biological; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Plant Proteins; Polyenes; Sequence Homology, Amino Acid; Synechococcus

To determine the effect of the redox state of the Rieske protein on ligand binding to the quinol oxidation site of the bc(1) complex, we measured the binding rate constants (k(1)) for stigmatellin and myxothiazol, at different concentrations of decylbenzoquinone or decylbenzoquinol, in the bovine bc(1) complex with the Rieske protein in the oxidized or reduced state. Stigmatellin and myxothiazol bound tightly and competitively with respect to quinone or quinol, independently of the redox state of the Rieske protein. In the oxidized bc(1) complex, the k(1) values for stigmatellin ( approximately 2.6 x 10(6) m(-1)s(-1)) and myxothiazol ( approximately 8 x 10(5) m(-1)s(-1)), and the dissociation constant (K(d)) for quinone, were similar between pH 6.5 and 9, indicating that ligand binding is independent of the protonation state of histidine 161 of the Rieske protein (pK(a) approximately 7.6). Reduction of the Rieske protein increased the k(1) value for stigmatellin and decreased the K(d) value for quinone by 50%, without modifying the k(1) for myxothiazol. These results indicate that reduction of the Rieske protein and protonation of histidine 161 do not induce a strong stabilization of ligand binding to the quinol oxidation site, as assumed in models that propose the existence of a highly stabilized semiquinone as a reaction intermediate during quinol oxidation.

Topics: Animals; Benzoquinones; Cattle; Electron Transport Complex III; Enzyme Inhibitors; Hydroquinones; Iron-Sulfur Proteins; Kinetics; Methacrylates; Oxidation-Reduction; Polyenes; Thiazoles

The results are presented of an electrochemical and high-resolution spectral analysis of the heme prosthetic groups in the bc1 complex from mouse cells. To study the long-range interactions between the Qo and Qi quinone redox sites and the b heme groups, we analyzed the effects on the proximal and distal b heme groups, and the c1 heme, of inhibitors that tightly and specifically bind to the Qi or Qo redox site. A number of results emerged from these studies. (1) There is inhomogeneous broadening of the b heme alpha band absorption spectra. Furthermore, contrary to the conclusion from low-resolution spectral analysis, the higher energy transition in the split-alpha band spectrum of the bL heme is more intense than the lower energy transition. (2) Inhibitors that bind at the Qi site have significant effects upon the electronic environment of the distal bL heme. Conversely, Qo site inhibitors induced changes in the electronic environment of the distal bH heme. (3) In contrast, inhibitor binding at either site has little effect upon the midpoint potential of the distal heme. (4) Experiments in which both a Qi and a Qo inhibitor are bound at the redox sites indicate that the long-range effects of one inhibitor are not blocked by the second inhibitor; enhanced effects are often observed. (5) In the double-inhibitor titrations involving the Qo inhibitor myxothiazol, there is evidence for two electrochemically and spectrally distinct species of the bL heme group, a phenomenon not observed previously. (6) The high-resolution deconvolutions of alpha band absorption spectra allow an interpretation of these inhibitor-induced changes in terms of homogeneous broadening, inhomogeneous broadening, and changes in x-y degeneracy. The general conclusion from these experiments is that when an inhibitor binds to a quinone redox site of the cytochrome b protein, it produces local conformational changes that, in turn, are transmitted to distal regions of the protein. The ligation of the bH and bL hemes between two parallel transmembrane helices provides a mechanism by which long-distance interactions can be propagated. The lack of long-range effects upon the midpoint potentials of the heme groups suggests, however, that protein conformational changes are unlikely to be a major control mechanism for the transmembrane electron- and proton-transfer steps of the Q cycle.

Topics: Animals; Anthraquinones; Antimycin A; Benzoquinones; Binding Sites; Cell Line; Chromatography, Ion Exchange; Cytochrome b Group; Cytochromes c1; Electrochemistry; Electron Transport Complex III; Fibroblasts; Heme; Methacrylates; Mice; Oxidation-Reduction; Polyenes; Spectrophotometry; Thiazoles