Algal Heme Oxygenase from Cyanidium caldarium

Enzymatic heme oxygenase activity has been partially purified from extracts of the unicellular red alga Cyanidium caldarium, and the macromolecular components have been separated into three protein fractions, referred to as Fractions I, 11, and 111, by serial column chromatography through DEAE-cellulose and Reactive Blue 2-Sepharose. Fraction I is retained by DEAE-cellulose at low salt concentration and eluted by 1 M NaCl. Fraction I1 is retained by Blue Sepharose at low salt concentration and eluted by 1 M NaCl. Fraction I11 is retained on 2’,5’-ADP-agarose and eluted by 1 mM NADPH, while Fraction I1 is not retained on ADP-agarose. Fractions 1-111, have M, values of 22,000, 38,000, and 37,000, respectively (all +2,000), as determined by Sephadex gel filtration chromatography. In vitro heme oxygenase activity requires the presence of all three fractions, plus substrate, 0 2 , reduced pyridine nucleotide, and another reductant. Ascorbate, isoascorbate, and phenylenediamine serve equally well as the second reductant, but hydroquinone can also be used, with lower activity resulting. Fractions 1-111 are heat sensitive and inactive by Pronase digestion. Fraction I has a visible absorption spectrum similar to that of ferredoxin and is bleached by dithionite reduction or incubation with p-hydroxymercuribenzoate. Fraction I can be replaced by commercially available ferredoxin derived from the red alga Porphyra umbilicalis, and to a smaller extent, by spinach ferredoxin. Fraction I11 contains ferredoxin-linked cytochrome c reductase activity and can be partially replaced by spinach ferredoxin-NADP+ oxidoreductase. Reconstituted heme oxygenase and ferredoxin-linked cytochrome c reductase activities are both abolished if Fraction I or I11 is preincubated with 0.1 mM p-hydroxymercuribenzoate, but heme oxygenase activity is only slightly affected if Fraction I1 is preincubated with p-hydroxymercuribenzoate. Preincubation of Fraction I1 with 0.5 mM diethylpyrocarbonate inactivates heme oxygenase in the reconstituted system, and 10 p~ mesohemin partially protects this Fraction against diethylpyrocarbonate inactivation. Algal heme oxygenase is inhibited 80% by 2 p~ Sn-protoporphyrin even in the presence of 20 p~ mesohemin. Fraction I1 is rate limiting in unfractionated and reconstituted incubation mixtures. None of the three cell fractions could be replaced by bovine spleen microsomal heme oxygenase or NADPH-cytochrome Prao reductase.