Phycocyanobilin:ferredoxin oxidoreductase of Anabaena sp. PCC 7120. Biochemical and spectroscopic.
In cyanobacteria, the biosynthesis of the phycobiliprotein and phytochrome chromophore precursor phycocyanobilin is catalyzed by the ferredoxin-dependent enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA), which mediates an atypical four-electron reduction of biliverdin IXalpha. Here we describe the expression, affinity purification, and biochemical characterization of recombinant PcyA from Anabaena sp. PCC 7120. A monomeric protein with a native M(r) of 30,400 +/- 5,000, recombinant PcyA forms a tight and stable stoichiometric complex with its substrate biliverdin IXalpha. The enzyme exhibits a strong preference for plant type [2Fe-2S] ferredoxins; however, flavodoxin can also serve as an electron donor. HPLC analyses establish that catalysis proceeds via the two electron-reduced intermediate 18(1),18(2)-dihydrobiliverdin, indicating that exovinyl reduction precedes A-ring (endovinyl) reduction. Substrate specificity studies indicate that the arrangement of the A- and D-ring substituents alters the positioning of the bilin substrate within the enzyme, profoundly influencing the course of catalysis. Based on these observations and the apparent lack of a metal or small molecule cofactor, a radical mechanism for biliverdin IXalpha reduction by phycocyanobilin:ferredoxin oxidoreductase is envisaged.
Frankenberg
Nicole
N
Section of Molecular and Cellular Biology, University of California, Davis, California 95616, USA.
Lagarias
J Clark
JC
D017033:Anabaena / Q000201:enzymology*; D001426:Bacterial Proteins; D001654:Bile Pigments / Q000737:chemistry; D002384:Catalysis; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D008956:Models, Chemical; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D011166:Porphyrins / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D001654:Bile Pigments; D011166:Porphyrins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C442236:phycocyanobilin ferredoxin oxidoreductase, Synechocystis