PubMed · 8753575
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T Yamaguchi, A Sugimoto. 1995. [Biliverdin].. https://pubmed.ncbi.nlm.nih.gov/8753575/
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Heme oxygenase catalyzes the NADPH, O2, and cytochrome P450 reductase dependent oxidation of heme to biliverdin and carbon monoxide. One of two primary isozymes, HO-1, is anchored to the endoplasmic reticulum membrane via a stretch of hydrophobic residues at the C-terminus. While full-length human HO-1 consists of 288 residues, a truncated version with residues 1-265 has been expressed as a soluble active enzyme in Escherichia coli. The recombinant enzyme crystallized from ammonium sulfate solutions but the crystals were not of sufficient quality for diffraction studies. SDS gel analysis indicated that the protein had undergone proteolytic degradation. An increase in the use of protease inhibitors during purification eliminated proteolysis, but the intact protein did not crystallize. N-terminal sequencing and mass spectral analysis of dissolved crystals indicated that the protein had degraded to two major species consisting of residues 1-226 and 1-237. Expression of the 1-226 and 1-233 versions of human HO-1 provided active enzyme that crystallizes in a form suitable for diffraction studies. These crystals belong to space group P2(1), with unit cell dimensions a = 79.3 A, b = 56.3 A, c = 112.8 A, and beta = 101.5 degrees.
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.