Structural and computational analysis of the quinone-binding site of complex II (succinate-ubiquinone oxidoreductase): a mechanism of electron transfer and proton conduction during ubiquinone reduction.

The transfer of electrons and protons between membrane-bound respiratory complexes is facilitated by lipid-soluble redox-active quinone molecules (Q). This work presents a structural analysis of the quinone-binding site (Q-site) identified in succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli. SQR, often referred to as Complex II or succinate dehydrogenase, is a functional member of the Krebs cycle and the aerobic respiratory chain and couples the oxidation of succinate to fumarate with the reduction of quinone to quinol (QH(2)). The interaction between ubiquinone and the Q-site of the protein appears to be mediated solely by hydrogen bonding between the O1 carbonyl group of the quinone and the side chain of a conserved tyrosine residue. In this work, SQR was co-crystallized with the ubiquinone binding-site inhibitor Atpenin A5 (AA5) to confirm the binding position of the inhibitor and reveal additional structural details of the Q-site. The electron density for AA5 was located within the same hydrophobic pocket as ubiquinone at, however, a different position within the pocket. AA5 was bound deeper into the site prompting further assessment using protein-ligand docking experiments in silico. The initial interpretation of the Q-site was re-evaluated in the light of the new SQR-AA5 structure and protein-ligand docking data. Two binding positions, the Q(1)-site and Q(2)-site, are proposed for the E. coli SQR quinone-binding site to explain these data. At the Q(2)-site, the side chains of a serine and histidine residue are suitably positioned to provide hydrogen bonding partners to the O4 carbonyl and methoxy groups of ubiquinone, respectively. This allows us to propose a mechanism for the reduction of ubiquinone during the catalytic turnover of the enzyme.

Synthesis and agrochemical screening of a library of natural product-like bicyclo[2,2,2]octenones.

A general route to a series of differentially substituted bicyclo[2,2,2]octenones has been developed, making use of the in situ intramolecular Diels Alder reaction of masked ortho-benzoquinones. This approach was used to synthesize a series of thirteen key acid-containing templates from which a solution phase discovery library of 1126 diverse amides was then constructed. The rigid polycyclic nature of the templates and the prevalence of oxygenated functionality confer natural product-like qualities and three-dimensional diversity. The library was screened in HTS in vivo against a number of weed, insect and fungal model organisms leading to the discovery of a novel series of herbicidally active compounds. The development, production and biological activity of the library are described.

Modeling the Qo site of crop pathogens in Saccharomyces cerevisiae cytochrome b.

Saccharomyces cerevisiae has been used as a model system to characterize the effect of cytochrome b mutations found in fungal and oomycete plant pathogens resistant to Q(o) inhibitors (QoIs), including the strobilurins, now widely employed in agriculture to control such diseases. Specific residues in the Q(o) site of yeast cytochrome b were modified to obtain four new forms mimicking the Q(o) binding site of Erysiphe graminis, Venturia inaequalis, Sphaerotheca fuliginea and Phytophthora megasperma. These modified versions of cytochrome b were then used to study the impact of the introduction of the G143A mutation on bc(1) complex activity. In addition, the effects of two other mutations F129L and L275F, which also confer levels of QoI insensitivity, were also studied. The G143A mutation caused a high level of resistance to QoI compounds such as myxothiazol, axoxystrobin and pyraclostrobin, but not to stigmatellin. The pattern of resistance conferred by F129L and L275F was different. Interestingly G143A had a slightly deleterious effect on the bc(1) function in V. inaequalis, S. fuliginea and P. megasperma Q(o) site mimics but not in that for E. graminis. Thus small variations in the Q(o) site seem to affect the impact of the G143A mutation on bc(1) activity. Based on this observation in the yeast model, it might be anticipated that the G143A mutation might affect the fitness of pathogens differentially. If so, this could contribute to observed differences in the rates of evolution of QoI resistance in fungal and oomycete pathogens.

Hydrogen bond basicity of the chlorogroup; hexachlorocyclohexanes as strong hydrogen bond bases.

A simple chloroalkane or chlorocycloalkane has a very small hydrogen bond basicity, B = 0.1 units. Since B is often an additive function, it is possible that polychloro-alkanes or -cycloalkanes could have quite large hydrogen bond basicities. Literature data on the 1,2,3,4,5,6-hexachlorocyclohexanes (HCHs) have been analyzed by Abraham's linear free energy relationships to obtain solvation descriptors. These are not extraordinary except for the hydrogen bond basicity, B, which is indeed very large. Values of B for the HCHs are larger than many functionally substituted aliphatic compounds and as large as that of aliphatic amines. We find that B is 0.62-0.72 for the HCHs compared to 0.45 for propanone and 0.70 for ethylamine, the first time that such large hydrogen bond basicities have been identified in compounds with no functional groups. Hydrogen bond basicities are analyzed in order to examine what types of polychlorocompounds give rise to these elevated B values.