Effects of ecologically relevant concentrations of Irgarol 1051 in tropical to subtropical coastal seawater on hermatypic coral Acropora tenuis and its symbiotic dinoflagellates.

The effects of ecologically relevant concentrations of Irgarol 1051, a representative PSII herbicide, on hermatypic corals were studied in the laboratory. The colour and chlorophyll fluorescence of Acropora tenuis were examined following exposure to around ambient concentrations of Irgarol 1051 (20 ng/L and 200 ng/L) for 7 days. While the colour of corals was stable throughout the experiment at both concentrations, the maximum effective quantum yield (ΔF/Fm') of symbiotic dinoflagellates decreased with increasing Irgarol 1051 concentration (day 7: 8%, 20 ng/L; 37%, 200 ng/L). The expression of heat shock protein (HSP) 70 and 90 in symbiotic dinoflagellates was upregulated after 7 days exposure to both Irgarol concentrations, whereas HSP90 in coral was not upregulated. The findings of the present study suggest that the threshold of chlorophyll fluorescence and HSP expression in symbiotic dinoflagellates is lower than 20 ng/L, which is around ecologically relevant concentrations in tropical to subtropical waters.

Creation of a binuclear purple copper site within a de novo coiled-coil protein.

Although various kinds of metal binding proteins have been constructed by de novo design, the creation of a binuclear metal binding site remains especially challenging. The purple copper site in subunit II of COX, referred to as the Cu(A) site, has two copper ions bridged by two Cys residues. We constructed the Cu(A) site consisting of two Cys and two His residues in a de novo designed four-helical coiled-coil protein. The protein bound two copper ions and exhibited a purple color, with relatively intense absorption bands at 488 and 530 nm in the UV-vis spectrum. The EPR spectrum displayed unresolved hyperfine splittings in the g(∥) region, which was similar to the native or engineered Cu(A) site with an A(∼480)/A(∼530) > 1. The extended X-ray absorption structure analyses of the protein revealed the presence of the Cu(2)S(2) core structure, with two typical N(His)-Cu bonds per core at 1.90 Å, two S (Cys)-Cu bonds at 2.21 Å, and the Cu-Cu bond at 2.51 Å, which are also characteristic structures of a purple copper site.

Tuning the geometries of a de novo blue copper protein by axial interactions.

The axial interactions of Cu(2+) in type 1 copper proteins control the physical characteristics of the proteins. We tuned the geometries of a de novo designed blue copper protein with a four-helical bundle structure. The designed protein axially bound various ligands, such as chloride, phosphate, sulfate, acetate, azide, and imidazole, to Cu(2+), exhibiting a blue or green color. The UV-vis spectral bands were observed at approximately 600 nm and approximately 450 nm, with the A (~450)/A (~600) ratios between 0.14 and 1.58. The stronger axial interaction shifted the geometry of the type 1 copper site from trigonal planar geometry (blue copper) toward a tetrahedral-like geometry (green copper). Resonance Raman spectral analyses showed that the phosphate-bound type had the highest-strength Cu-S bond, similar to that of plastocyanin. The chloride-bound type exhibited features similar to those of stellacyanin and nitrite reductase, and the imidazole-bound type exhibited features similar to those of azurin M121E mutant.