CHROMOPHORE TOPOGRAPHY AND SECONDARY STRUCTURE OF 124-KILODALTON AVENA PHYTOCHROME PROBED BY ZN-2+-INDUCED CHROMOPHORE MODIFICATION

The relative extent of chromophore exposure of the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of 124-kDa oat phytochrome and the secondary structure of the phytochrome apoprotein have been investigated by using zinc-induced modification of the phytochrome chromophore. The absence of bleaching of Pr in the presence of a 1:1 stoichiometric ratio of zinc ions, in contrast to extensive spectral bleaching of the Pfr form, confirms previous reports of differential exposure of the Pfr chromophore relative to the Pr chromophore [Hahn et al. (1984) Plant Physiol. 74, 755-758]. The emission of orange fluorescence by zinc-chelated Pfr indicates that the Pfr chromophore has been modified from its native extended/semiextended conformation to a cyclohelical conformation. Circular dichroism (CD) analyses of native phytochrome in 20 mM Tris buffer suggests that the Pr-to-Pfr phototransformation is accompanied by a photoreversible change in the far-UV region consistent with an increase in the α-helical folding of the apoprotein. The secondary structure of phytochrome in Tris buffer, as determined by CD, differs slightly from that of phytochrome in phosphate buffer, suggesting that phytochrome is a conformationally flexible molecule. Upon the addition of a 1:1 molar ratio of zinc ions to phytochrome, a dramatic change in the CD of the Pfr form is observed, while the CD spectrum of the Pf form is unaffected. Analysis of the bleached Pfr CD spectrum by the method of Chang et al. (1978) reveals that chelation with zinc ions significantly alters the secondary structure of the phytochrome molecule, specifically by increasing the β-sheet content primarily at the expense of α-helical folding. Further evidence of a zinc-induced conformational change in phytochrome has been obtained with proteolytic digestions of chelated and nonchelated Pfr. We propose that chelation with zinc ions at the phytochrome chromophore cyclizes the semiextended Pfr chromophore, which reduces/eliminates the interactive forces between the chromophore and the polypeptide. Once these forces are attenuated, the phytochrome molecule undergoes a conformational rearrangement, resulting in a secondary structure similar to that of the phytochrome apoprotein, which appears to differ substantially from that of the holoprotein. © 1990, American Chemical Society. All rights reserved.