Cl- channel inhibitors of the arylaminobenzoate type act as photosystem II herbicides: a functional and structural study.

The Cl- channel blocker NPPB (5-nitro-2-(3-phenylpropylamino) benzoic acid) inhibited photosynthetic oxygen evolution of isolated thylakoid membranes in a pH-dependent manner with a K(i) of about 2 microM at pH 6. Applying different electron acceptors, taking electrons either directly from photosystem II (PS II) or photosystem I (PS I), the site of inhibition was localized within PS II. Measurements of fluorescence induction kinetics and thermoluminescence suggest that the binding of NPPB to the QB binding site of PS II is similar to the herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). The effects of different arylaminobenzoate derivatives and other Cl- channel inhibitors on photosynthetic electron transport were investigated. The structure--activity relationship of the inhibitory effect on PS II shows interesting parallels to the one observed for the arylaminobenzoate block of mammalian Cl- channels. A molecular modeling approach was used to fit NPPB into the QB binding site and to identify possible molecular interactions between NPPB and the amino acid residues of the binding site in PS II. Taken together, these data give a detailed molecular picture of the mechanism of NPPB binding.

Resonance Raman and surface-enhanced resonance Raman spectra of LH2 antenna complex from Rhodobacter sphaeroides and Ectothiorhodospira sp. excited in the Qx and Qy transitions.

Well-resolved vibrational spectra of LH2 complex isolated from two photosynthetic bacteria, Rhodobacter sphaeroides and Ectothiorhodospira sp., were obtained using surface-enhanced resonance Raman scattering (SERRS) exciting into the Qx and the Qy transitions of bacteriochlorophyll a. High-quality SERRS spectra in the Qy region were accessible because the strong fluorescence background was quenched near the roughened Ag surface. A comparison of the spectra obtained with 590 nm and 752 nm excitation in the mid- and low-frequency regions revealed spectral differences between the two LH2 complexes as well as between the LH2 complexes and isolated bacteriochlorophyll a. Because peripheral modes of pigments contribute mainly to the low-frequency spectral region, frequencies and intensities of many vibrational bands in this region are affected by interactions with the protein. The results demonstrate that the microenvironment surrounding the pigments within the two LH2 complexes is somewhat different, despite the fact that the complexes exhibit similar electronic absorption spectra. These differences are most probably due to specific pigment-pigment and pigment-protein interactions within the LH2 complexes, and the approach might be useful for addressing subtle static and dynamic structural variances between pigment-protein complexes from different sources or in complexes altered chemically or genetically.

Release of lipid vesicle contents by the bacterial protein toxin alpha-haemolysin.

alpha-Haemolysin is a protein toxin (107 kDa) secreted by some pathogenic strains of E. coli. It binds to mammalian cell membranes, disrupting cellular activities and lysing cells. This paper describes the mechanism of alpha-haemolysin-induced membrane leakage, from experiments in which extrusion large unilamellar vesicles, loaded with fluorescent solutes, are treated with purified toxin. The results show that the toxin does not require of any membrane receptor to exert its activity, that vesicles become leaky following an 'all-or-none' mechanism, and that leakage occurs through a non-osmotic detergent-like bilayer disruption induced by the protein. Small pores formed by monomeric alpha-haemolysin, as described by other authors, do not appear to be related to the process of membrane disruption. Instead, the experimental data would be in agreement with the idea of oligomeric assemblies being required to produce release of solutes from a single vesicle.

Precise location of the Cu(II)-inhibitory binding site in higher plant and bacterial photosynthetic reaction centers as probed by light-induced absorption changes.

Light-dependent absorption change at 325 nm, ascribed to QA activity, was strongly reduced in the presence of Cu(II) in oxygen-evolving core complex. This change was much less affected in the presence of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), indicating that the Cu(II)-binding site is different from that of the DCMU and that Cu(II) blocks QA reduction. Cu(II) did not eliminate the absorption change at 545 nm, ascribed to pheophytin reduction, in Na2S2O4-treated oxygen-evolving core and D1-D2-cytochrome b559 complexes. This indicates that Cu(II) does not affect the electron transport between P680 and pheophytin. Moreover, the activity of the bacterial reaction center probed by the absorption change at 790 nm was inhibited by Cu(II), but the signal at 530 nm, associated to the reduction of bacteriopheophytin in Na2S2O4-treated reaction center, was not inhibited. We conclude that Cu(II) impaired the photosynthetic electron transport between pheophytin and QA in both higher plants and photosynthetic bacteria. Cu(II) would bind to an amino acid(s) highly conserved in non-oxygenic and oxygenic reaction centers, which is(are) necessary for the electron transfer between pheophytin and QA. Based on the atomic structure of the bacterial reaction center several schemes of possible Cu(II) binding are shown.