CHROMOPHORE ROTATION IN 124-kD ALTON A vena sativa PHYTOCHROME AS MEASURED BY LIGHT-INDUCED CHANGES IN LINEAR DICHROISM

Abstract— From light-induced changes in linear dichroism, we have calculated the rotation of the long-wavelength-absorbing transition moment that occurs during phototransformation of 124-kilodalton Avena sativa phytochrome. Phytochrome was purified to homogeneity and immobilized onto Sepharose beads covalently coated with antibodies against A. sativa phytochrome. Changes in linear dichroism were induced by plane-polarized red or far-red light and measured by the absorbance differences at 660 and 730 nm using a dual-wavelength spectrophotometer equipped with polarizing filters in the measuring beams. From such measurements, we calculate a rotation angle of 31^o (or 149^o) during photoconversion of Pr to Pfr and 30^o (or 150^o) during photoconversion of Pfr to Pr. These values are similar to the value of 32^o (or 148^o) reported earlier for the rotation of the transition moment of "large" A. sativa phytochrome (∽ 120 kilodalton) isolated under conditions that did not preclude post homogenization proteolysis of the 124 kilodalton molecule.     

ROSEOFLAVIN AS A BLUE LIGHT RECEPTOR ANALOG: SPECTROSCOPIC CHARACTERIZATION

Abstract— The blue light absorption band of roseoflavin is polarized along the axis roughly connecting N₃-C₈ positions. A weak, second π→π* transition with a polarization angle of ca. 25° is hidden under the short wavelength side of the blue absorption band. The excited state of roseoflavin is somewhat more basic than the ground state, by a 1.5 p K _a unit. The fluorescence quantum yield and lifetime of roseoflavin are substantially lower than those of other flavins, thus making it kinetically less efficient as a blue light photoreceptor.     

Comparison of the protein conformations between different forms (Pr and Pfr) of native (124 kDa) and degraded (118/114 kDa) phytochromes from Avena sativa

Abstract— Circular dichroic properties of native, 124 kDa phytochrome from etiolated Avena sativa seedlings have been examined and compared with those of degraded phytochrome (118/114 kDa). The CD spectrum of the P_r form of 124 kDa phytochrome does not differ significantly in the visible region from that of 118/114 kDa P_r. In contrast, the CD spectrum of the P_fr form of 124 kDa phytochrome differs from that of the 118/114 kDa species in the far-red, red and blue regions of the spectrum. This result confirms that the NH₂-terminal polypeptide segment has a critical role in chromophore-protein interaction in the P_fr but not in the P_r form. In the UV region, 124 kDa phytochrome exhibits a photoreversible difference between the CD spectra of P_r and P_fr, whereas no such difference is observed for 118/114 kDa preparations. These data suggest a possible photoreversible change in secondary structure of the 124 kDa phytochrome polypeptide that requires the presence of the 6/10 kDa NH₂-terminal domain to occur.     

A BLUE LIGHT-SENSITIVE CYTOCHROME-FLAVIN COMPLEX FROM CORN COLEOPTILES. FURTHER CHARACTERIZATION*

Abstract— The partially purified blue light-sensitive membrane-associated flavin-cytochrome complex from etiolated corn coleoptiles shows a unique sharp α-band at 555 nm in its light-minus-dark difference spectrum at liquid nitrogen temperature. This band is clearly distinguishable from the α-bands found in fractions enriched for mitochondria and endoplasmic reticulum respectively. The photoactive membrane fraction is shown to have ATPase activity that is not stimulated by K⁺ and that is not inhibited by oligomycin. Other than flavin fluorescence at 525 nm obtained upon excitation at 450 nm, there is a second fluorescent component with emission at 430 nm on excitation at 350 nm. The mid-point potential of the Triton X-100 solubilized b-cytochrome, measured by simultaneously monitoring the reduction of the pyocyanine 600-800 nm peak and the appearance of the 427 nm Soret peak of the b-cytochrome upon titration with dithionite in the presence of ferricyanide, is estimated to be ?65 mV. The kinetics of the blue light-induced reduction and dark rcoxidation of the 6-cytochrome suggest that the mid-point potential of the b-cytochrome is not affected by Triton X-100 solubilization.     

Fluorescence Analysis of Oat phyA Deletion Mutants Expressed in Tobacco Suggests that the N-Terminal Domain Determines the Photochemical and Spectroscopic Distinctions between phyA' and phyA"†

Abstract— In vivo low-temperature (85 K) fluorescence spectroscopy has defined two phytochrome A (phyA) subpopulations, designated phyA' and phyA", in etiolated seedlings (V. A. Sineshchekov, J. Photochem. Photobiol. 28, 53–55, 1995). Phytochrome A' is the more abundant but light-labile species characterized by longer wavelength emission/absorption maxima (687/673 nm) and by a higher extent of the photoconversion of its red-absorbing form (Pr) into photoproduct (lumi-R) at 85 K (γ₁≈ 0.5). Phytochrome A" is the minor but relatively light-stable species, characterized by shorter wavelength maxima (682/668 nm) and by a lower γ₁ (<0.05). To help define domains within phyA responsible for these differences, the low-temperature spectral properties of transgenic tobacco expressing full-length (FL) oat phyA and C-and N-terminally truncated versions (CD [Δ786–1129] and NA [Δ7–69], respectively) were compared. Oat phytochrome expression was more pronounced than that of tobacco in the basal section of etiolated seedlings following 2 h irradiation with white light. Seedlings expressing FL and CD phyA had spectral properties for phyA' and phyA" that were indistinguishable from that of wild-type tobacco. Conversely, expression of NA phyA generated an abundant phy species that behaved like phyA". From this we conclude that the N-terminal domain of phyA is involved in determining the photochemical and spectroscopic distinctions between the native phyA' and phyA" species.