PHYCOBILIN BIOSYNTHESIS - REDUCTANT REQUIREMENTS AND PRODUCT IDENTIFICATION FOR HEME OXYGENASE FROM CYANIDIUM-CALDARIUM

Algal heme oxygenase is a soluble enzyme from Cyanidium caldarium that catalyzes the first committed step of phycobilin biosynthesis by conv erting protoheme to biliverdin IX alpha. Although the physiological su bstrate (protoheme) of algal heme oxygenase is identical to that of mi crosomal heme oxygenase, which catalyzes heme catabolism in animals, t he two enzyme systems differ in several respects including the nature of the required reductants and solubility of the enzymes. Addition of the strong Fe3+ ion chelators, desferrioxamine and Tiron (4,5-dihydrox y-1,3-benzenedisulfonic acid), greatly increased the yield of solvent- extracted bilin product. The effect of the Fe3+ chelators was approxim ately equal whether they were added during or after the enzyme incubat ion. Postincubation treatment of the enzyme reaction mixture with stro ng acid also greatly increased the product yield. Addition of desferri oxamine to the reaction mixture after the incubation was terminated ca used the appearance of an absorption spectrum, indicating an increase in the concentration of free bilin product. Acid and Fe3+ chelators ar e known to cause dissociation of Fe(III)-bilin complexes. These result s indicate that the in vitro enzymic reaction product of algal heme ox ygenase is a nonenzyme-bound Fe(III)-biliverdin IX alpha complex that is poorly extracted and/or quantitated unless it is first dissociated. Algal heme oxygenase required the simultaneous presence of both reduc ed ferredoxin and a second reductant such as ascorbate for activity. T he requirement for L-ascorbate could be substituted by Trolox (6-hydro xy-2,5,7,8-tetramethylchroman-2-carboxylic acid) or D-ascorbate, but n ot by dehydroascorbate or dithiothreitol. Heme oxygenase was purified over 200-fold from C. caldarium by differential (NH4)(2)SO4 precipitat ion and serial column chromatography over reactive blue 2-Sepharose, D EAE - cellulose, Sephadex G-75, and ferredoxin-Sepharose. (C) 1995 Aca demic Press, Inc.