SURFACE-TOPOGRAPHY OF PHYTOCHROME-A DEDUCED FROM SPECIFIC CHEMICAL MODIFICATION WITH IODOACETAMIDE

Phytochromes are a photoreversible photochromic light switch for photo morphogenesis in plants. The molecular structure and functional mechan ism of phytochromes are not fully understood. On the basis of complete mapping of total tryptic digest of the iodoacetamide-modified oat phy tochrome A (phyA), the molecular surface topography of phyA was probed by specific chemical modification of cysteine residues with [C-14]iod oacetamide. Under native conditions, only two cysteines (Cys-158 and C ys-311) of eleven half-cystines of the N-terminal chromophore binding domain were modified to a significant extent. In the C-terminal domain , six cysteine residues (Cys-715, Cys-774, Cys-809, Cys-869, Cys-961, Cys-995) were readily accessible to iodoacetamide. Among the reactive cysteine residues, only cysteine-311 displayed reactivity that was dep endent on the photochromic form (Pr reversible arrow Pfr) of the photo receptor. Surprisingly, the modification of Cys-311 in the vicinity of the chromophore attachment site (Cys-321) did not have any detectable effect on spectral properties of phyA. Most of the cysteines of the N -terminal domain (Cys-83, Cys-175, Cys-291, Cys-370, Cys-386, Cys-445, Cys-506) are deeply buried in the core of the chromophore binding dom ain, as they can be modified only after denaturation of the chromoprot ein. In the C-terminal domain, modification of only one cysteine resid ue (Cys-939) required protein denaturation. Since all 22 half-cystines can be modified with iodoacetamide without reduction of the chromopro tein, it follows that oat phyA does not have any disulfide bonds. We f ound that Cys-311, Cys-774, Cys-961, and Cys-995 could be easily parti ally oxidized under the conditions used for phytochrome isolation. The surface topography/conformation of oat phyA and its role in protein-p rotein recognition in phytochrome-mediated signal transduction are dis cussed in terms of the relative reactivity of cysteine residues.