The Role of the Chromophore in the Biological Photoreceptor Phytochrome: An Approach Using Chemically Synthesized Tetrapyrroles

摘要

Inn plants and bacteria, phytochromes serve as light-inducible, red-far-red light sensitive photoreceptors that control a wide range ofnphotomorphogenetic processes. Phytochromes comprise a proteinnmoiety and a covalently bound bilin chromophore. Bilins are open-nchain tetrapyrrole compounds that derive biosynthetically from ubi-nquitous porphyrins. The investigations of phytochromes reveal thatnprecise interactions between the protein moiety and its bilin chro-nmophore are essential for the proper functioning of this photore-nceptor; accordingly, synthetic manipulation of the parts is annimportant method for studying the whole. Although variations in thenprotein structure are readily accomplished by routine mutagenesisnprotocols, the generation of structurally modified bilins is a labori-nous, multistep process. Recent improvement in the synthesis of open-nchain tetrapyrroles now permits the generation of novel, structurallynmodified (and even selectively isotope-labeled) chromophores. Furthermore, by using the capability of recombinant apo-phyto-nchrome to bind the chromophore autocatalytically, researchers can now generate novel chromoproteins with modified functions.nIn the protein-bound state, the phytochrome chromophore is photoisomerized at one double bond, in the bridge between thenlast two of the four pyrrole rings (the C and D rings), generating the thermally stable, physiologically active Pfr form. This con-nversion photoisomerization from the form absorbing red light (Pr) to the form absorbing far-red light (Pfr) covers 12 ordersnof magnitude, from subpicoseconds to seconds. Such spectroscopic and kinetic studies yield a wealth of time-resolved spectral data,neven more so, if proteins with changed sequence or chromophore structure are utilized. In particular, bilins with a changed sub-nstitution pattern at the photoisomerizing ring D have shed light on the chromophore protein interactions during the photo-nisomerization. The mechanisms generating and stabilizing the light-induced Pfr form of phytochromes are now seen in greater detail.nOn the other hand, the use of bilins with selective incorporation of stable isotopes identify light-induced conformational motionsnwhen studied by vibrational (FTIR and Raman) and NMR spectroscopy.nIn this Account, we present spectroscopic investigations that provide structural details in these biological photoreceptors withngreat precision and document the dynamics elicited by light excitation. This approach yields important information that comple-nments the data deduced from crystal structure.