PubMed · 8753575
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T Yamaguchi, A Sugimoto. 1995. [Biliverdin].. https://pubmed.ncbi.nlm.nih.gov/8753575/
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Compared with phytochromes isolated from etiolated higher plant tissues and a number of lower plant species, the absorption spectrum of phytochrome isolated from the unicellular green alga Mesotaenium caldariorum is blue-shifted (Kidd, D. G., and Lagarias, J. C. (1990) J. Biol. Chem. 265, 7029-7035). The present studies were undertaken to determine whether this blue shift is due to a chromophore other than phytochromobilin or reflects a different protein environment for the phytochromobilin prosthetic group. Using reversed phase high performance liquid chromatography, we show that soluble protein extracts prepared from algal chloroplasts contain the enzyme activities for ferredoxin-dependent conversions of biliverdin IXalpha to (3Z)-phytochromobilin and (3Z)-phytochromobilin to (3Z)-phycocyanobilin. In vitro assembly of recombinant algal apophytochrome was undertaken with (3E)-phytochromobilin and (3E)-phycocyanobilin. The difference spectrum of the (3E)-phycocyanobilin adduct was indistinguishable from that of phytochrome isolated from dark-adapted algal cells, while the (3E)-phytochromobilin adduct displayed red-shifted absorption maxima relative to purified algal phytochrome. These studies indicate that phycocyanobilin is the immediate precursor of the green algal phytochrome chromophore and that phytochromobilin is an intermediate in its biosynthesis in Mesotaenium.
The oxidation of heme to biliverdin IXalpha by heme oxygenase involves regiospecific alpha-meso-hydroxylation followed by extrusion of the alpha-meso-carbon as CO. In an earlier study, enzymatic oxidation of the four meso-methylmesoheme isomers suggested that the reaction regiospecificity is sensitive to the electronic properties of the meso-methyl group (Torpey, J. W., and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 26067-26073), although we could not exclude the possibility that the altered reaction regiochemistry was due to perturbation of the porphyrin structure by the meso-substituent. To examine this possibility, we have synthesized the four meso-formylmesoporphyrin isomers and have examined their oxidation by heme oxygenase. The meso-formyl and meso-methyl substituents differ in that the former is electron withdrawing and the latter is electron donating. In contrast to alpha-meso-methylmesoheme, which is exclusively oxidized at the methyl-substituted position, alpha-meso-formylmesoheme is exclusively oxidized at a non-formyl-substituted meso-carbon. The finding that the methyl and formyl groups channel the reaction regiospecificity in opposite directions establishes that the regiochemistry of the heme oxygenase reaction is primarily under electronic rather than steric control. It also confirms that the oxidation involves electrophilic addition of the oxygen to the porphyrin ring.
The cDNAs encoding full-length type A and B phytochromes (phyA and phyB, respectively) from potato were expressed in inducible yeast systems (Saccharomyces cerevisiae and Pichia pastoris). In addition, a deletion mutant of phyB (delta 1-74) was expressed. The apoproteins were reconstituted into chromoproteins by incorporation of the native chromophore, phytochromobilin (P phi B), and of phycocyanobilin (PCB). The incorporation of P phi B yielded chromoproteins with difference absorptions lambda max at 660 and 712 nm (Pr and Pfr, respectively) for phyA, and at 665 and 723 nm for phyB. All difference maxima of PCB phytochromes are blue-shifted by several nanometers with respect to those obtained with the P phi B chromophore. The deletion construct with PCB shows difference absorption maxima at 652 and 705 nm with the Pfr absorbance considerably reduced. Time-resolved kinetic analysis of a phyB-type phytochrome by nanosecond flash photolysis was performed for the first time. Recombinant full-length phyB afforded transient absorbance changes similar (but not identical) to those of phyA from Avena, whereas the kinetic behavior of these intermediates was very different. Contrary to phyA from Avena, the I700 intermediate from phyB reconstituted with either PCB or P phi B decayed following single exponential kinetics with a lifetime of 87 or 84 microseconds, respectively, at 10 degrees C. The formation of Pfr of PCB-containing recombinant phyB (phyB-PCB) could be fitted with three lifetimes of 9, 127, and 728 ms. The corresponding lifetimes of phyB-P phi B are 22.5, 343, and 2083 ms. Whereas for phyB-PCB all three millisecond lifetimes are related to the formation of Pfr, the 2 s component of phyB-P phi B is concomitant with a rapid recovery of Pr. For recombinant potato phyA and delta 1-74 phyB, no time-resolved data could be obtained due to the limited quantities available. As described for phytochromes of other dicotelydons, the Pfr forms of full-length phyA and PhyB of potato underwent rapid dark conversion to Pr.