Phycoerythrobilin is a linear tetrapyrrole molecule found in cyanobacteria, red algae, and cryptomonads. Together with other bilins such as phycocyanobilin it serves as a light-harvesting pigment in the photosynthetic light-harvesting structures of cyanobacteria called phycobilisomes. The biosynthesis of both pigments starts with the cleavage of heme by heme oxygenases to yield biliverdin IXalpha, which is further reduced at specific positions by ferredoxin-dependent bilin reductases (FDBRs), a new family of radical enzymes. The biosynthesis of phycoerythrobilin requires two subsequent two-electron reductions, each step being catalyzed by one FDBR. This is in contrast to the biosynthesis of phycocyanobilin, where the FDBR phycocyanobilin: ferredoxin oxidoreductase (PcyA) catalyzes a four-electron reduction. The first reaction in phycoerythrobilin biosynthesis is the reduction of the 15,16-double bond of biliverdin IXalpha by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA). This reaction reduces the conjugated pi -electron system thereby blue-shifting the absorbance properties of the linear tetrapyrrole. The second FDBR, phycoerythrobilin:ferredoxin oxidoreductase (PebB), then reduces the A-ring 2,3,3(1),3(2)-diene structure of 15,16-dihydrobiliverdin to yield phycoerythrobilin. Both FDBRs from the limnic filamentous cyanobacterium Fremyella diplosiphon and the marine cyanobacterium Synechococcus sp. WH8020 were recombinantly produced in Escherichia coli and purified, and their enzymatic activities were determined. By using various natural bilins, the substrate specificity of each FDBR was established, revealing conformational preconditions for their unique specificity. Preparation of the semi-reduced intermediate, 15,16-dihydrobiliverdin, enabled us to perform steady state binding experiments indicating distinct spectroscopic and fluorescent properties of enzyme.bilin complexes. A combination of substrate/product binding analyses and gel permeation chromatography revealed evidence for metabolic channeling.