Enzymic Transformation of Biliverdin to Phycocyanobilin by Extracts of the Unicellular Red Alga Cyanidium caldarium

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Abstract
Cell-free extracts of the unicellular red alga C. caldarium catalyze the transformation of biliverdin to a product indistinguishable from phycocyanobilin, the free bilin derived from phycocyanin by methanolysis. Crude cell-free extract requires biliverdin as the only substrate, but after removal of low MW components by gel filtration, the reaction shows an additional requirement for a reduced pyridine nucleotide. Boiled extract is enzymically inactive, activity is not sedimented by high-speed centrifugation and mesobiliverdin cannot serve as a substrate. Incubation of cell extracts with biliverdin yields 2 products with very similar spectrophotometric properties in acidic methanol, but which are separable by reverse-phase high pressure liquid chromatography. The same 2 products are formed by methanolysis of protein-bound phycocyanin chromophore, with the late-eluting one predominating. The 2 products derived from either phycocyanin methanolysis or cell extract incubation with biliverdin are partially interconvertible and they form the same ethylidine-free isomeric derivative, mesobiliverdin. Their absorption spectra correspond to those of the Z- and E-ethylidine isomers of phycocyanobilin. Based on previous work showing that the major methanolysis product has the E-ethylidine configuration, the other product of methanolysis and enzymic biliveridin transformation is therefore the Z-ethylidine isomer. The time course for formation of the 2 products during incubation suggests that the early-eluting product is the precursor of the late-eluting one. Z-ethylidine phycocyanobilin is apparently the precursor of the E-ethylidine isomer, and the latter may be a normal cellular precursor to protein-bound phycocyanin chromophore.
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