Heme Oxygenase

Heme oxygenase catalyses the synthesis of BV IXa from protoheme in a reaction that requires O2 and reducing power, and liberates CO and Fe2+ (Figure 3) (34,42). In photosynthetic organisms, the most extensively characterized heme oxygenase system is that of C. caldarium (5). Heme oxygenase activity was first measured in Cyanidium extracts by Beale and Cornejo (7). The enzyme is soluble and has been partially purified using differential ammonium sulfate precipitation and DEAE-cel-lulose and blue-Sepharose affinity chro-matography steps (16). This procedure was subsequently extended to include a ferre-doxin-Sepharose affinity chromatography step to give a 200-fold purification of heme oxygenase activity (46).

The employment of a ferredoxin affinity purification step highlights one of the distinguishing features of heme oxygenases from photosynthetic organisms: the requirement for reduced ferredoxin, produced by ferredoxin-NADP+ oxidoreduc-tase and NADPH, for enzyme activity. In contrast, mammalian heme oxygenase (first described in Reference 53), found associated with microsomal membranes, utilizes an NADPH-cytochrome P450 reductase (35, 69). A second distinguishing feature is that the algal enzyme requires a second reduc-tant in addition to ferredoxin (16): ascor-bate appears to be the most effective with Trolox® (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) also proving suitable (46). The reason that the reaction requires a second reductant is currently unknown.

The heme oxygenase assay in extracts or partially purified fractions of Cyanidium is an HPLC-based assay that takes advantage

Figure 3. The reaction catalyzed by the enzyme heme oxygenase. Substrates and products are not shown stoichiometrically.

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