The role of the chromophore in the biological photoreceptor phytochrome: an approach using chemically synthesized tetrapyrroles.

In plants and bacteria, phytochromes serve as light-inducible, red-/far-red light sensitive photoreceptors that control a wide range of photomorphogenetic processes. Phytochromes comprise a protein moiety and a covalently bound bilin chromophore. Bilins are open-chain tetrapyrrole compounds that derive biosynthetically from ubiquitous porphyrins. The investigations of phytochromes reveal that precise interactions between the protein moiety and its bilin chromophore are essential for the proper functioning of this photoreceptor; accordingly, synthetic manipulation of the parts is an important method for studying the whole. Although variations in the protein structure are readily accomplished by routine mutagenesis protocols, the generation of structurally modified bilins is a laborious, multistep process. Recent improvement in the synthesis of open-chain tetrapyrroles now permits the generation of novel, structurally modified (and even selectively isotope-labeled) chromophores. Furthermore, by using the capability of recombinant apo-phytochrome to bind the chromophore autocatalytically, researchers can now generate novel chromoproteins with modified functions. In the protein-bound state, the phytochrome chromophore is photoisomerized at one double bond, in the bridge between the last two of the four pyrrole rings (the C and D rings), generating the thermally stable, physiologically active P(fr) form. This conversion--photoisomerization from the form absorbing red light (P(r)) to the form absorbing far-red light (P(fr))--covers 12 orders of magnitude, from subpicoseconds to seconds. Such spectroscopic and kinetic studies yield a wealth of time-resolved spectral data, even more so, if proteins with changed sequence or chromophore structure are utilized. In particular, bilins with a changed substitution pattern at the photoisomerizing ring D have shed light on the chromophore-protein interactions during the photoisomerization. The mechanisms generating and stabilizing the light-induced P(fr) form of phytochromes are now seen in greater detail. On the other hand, the use of bilins with selective incorporation of stable isotopes identify light-induced conformational motions when studied by vibrational (FTIR and Raman) and NMR spectroscopy. In this Account, we present spectroscopic investigations that provide structural details in these biological photoreceptors with great precision and document the dynamics elicited by light excitation. This approach yields important information that complements the data deduced from crystal structure.

Interactions between chromophore and protein in phytochrome identified by novel oxa-, thia- and carba-chromophores.

Six new bilin chromophores of the plant photoreceptor phytochrome have been synthesized, carrying at the photoisomerizing ring D an oxygen or a sulfur atom or a methylene group instead of the pyrrole nitrogen atom. These furanone-, thiophenone- or cyclopentenone-containing compounds bound covalently to the recombinant apophytochrome phyA of Avena sativa. The novel chromoproteins showed hypsochromically shifted absorption spectra with respect to native phytochrome and a strongly diminished photochemical activity, but a three- to four-fold higher fluorescence quantum yield. These results demonstrate that, on the one hand, also ring D-modified chromophores can be forced into a partially extended structure, required for incorporation into the apoprotein binding pocket and covalent binding. On the other hand, the modifications introduced into ring D of the chromophores strongly impede the formation of stable far red-absorbing forms of plant photoreceptor phytochrome (P(fr)-form) of the chromoproteins, highlighting especially the role of the pyrrole nitrogen atom and hydrogen bonding for the precise interactions between that part of the chromophore and the protein for the P(fr)-formation.

Synthesis of water-soluble bile pigments bound to amine-ended monomethoxypolyethyleneglycol: thiol addition and attempted enzymatic reduction of a bilindione derivative.

The water-soluble amide to an NH2-ended monomethoxypolyethyleneglycol (MPEG-NH2, molecular mass of about 2000) of the dipyrrinone xanthobilirubic acid (XBR, 1) and the bis-amides of mesobiliverdin-XIII alpha (MBV, 2) and mesobilirubin-XIII alpha (MBR, 3) have been prepared with high yields. Contrary to what is observed with biliverdin-IX alpha, 4, the enzymatic reduction of the mesobiliverdin derivative 2-MPEGA to the corresponding mesobilirubin 3-MPEGA by the soluble biliverdin reductase/NADPH system in pH 7.4 aqueous phosphate does not occur. In contrast, thiol addition to 2-MPEGA and to 4 under similar conditions is immediate, although this equilibrium is slightly less favourable for 2-MPEGA. These results enable us to discount the intrinsically low reactivity of 2-MPEGA towards thiols as the reason for its lack of enzymatic reduction, and suggest instead that this particular mesobiliverdin cannot fit properly into the enzyme binding site, either because of steric hindrance or the lack of the two propionic acid groups.

Radical intermediates in the oxidation of octaethylheme to octaethylverdoheme.

Iron(III) oxyoctaethylporphyrin was isolated and purified as a dimer. The addition of tosylmethyl isocyanide to a solution of the dimer produced a monomer species, which was isolated and identified as bis(tosylmethyl isocyanide)iron(II) 5-oxyoctaethylporphyrin pi-neutral radical. The product of dissociation of the dimer by imidazole was bis(imidazole)iron(III) 5-oxyoctaethylporphyrin. The spectral properties of the product of dissociation of the dimer by pyridine and published data on bis(pyridine)oxymesoheme and bis(pyridine)oxyprotoheme were consistent with its identification as bis(pyridine)iron(II) 5-oxyoctaethylporphyrin pi-neutral radical. When this product was exposed to oxygen, a weak radical signal appeared in its electron spin resonance spectrum, which was attributed to the displacement of one of its pyridine ligands by O2 to form (pyridine)(dioxygen)iron(II) 5-oxyoctaethylporphyrin pi-neutral radical. The pyridine oxygen radical converted spontaneously to octaethylverdohemochrome, which was purified and identified as bis-(tosylmethyl isocyanide)iron(II) octaethylverdohemochrome hydroxide. The yield of verdohemochrome from iron oxyporphyrin was increased by the addition of phenylhydrazine or ascorbate. A scheme for the oxidation of iron(III) oxyporphyrin to iron(II) verdoheme by O2 that proposes a mechanism for the expulsion of CO and the replacement of a methene bridge of the porphyrin ring by an oxa bridge is presented.

The structure of verdohemochrome and its implications for the mechanism of heme catabolism.

The synthesis, purification as a tetrafluoroborate salt and structural elucidation of the verdohemochrome 2a derived from the coupled oxidation of octaethylhemochrome 1 is described. Based on elemental analyses, spectroscopic studies (visible and infrared absorption, 1H-NMR) and fast atom bombardment mass spectrometry, the assignment of the iron(II) oxaporphyrin structure for the verdohemochrome 2a and the blue monocarbonyl species 2b, obtained upon treatment of 2a with carbon monoxide, has been accomplished. This assignment raises a number of questions regarding the iron oxidation state of intermediates in the pathway of heme catabolism both in vitro and in vivo. Furthermore, the implications of the occurrence of an iron oxaporphyrin intermediate in the pathway of heme metabolism, which is suggested by the similarity of the visible absorption spectrum of the CO species 2b with that of a new intermediate recently observed in the heme oxygenase-catalyzed degradation of heme and mesoheme, is considered.