Phycocyanobilin:ferredoxin oxidoreductase of Anabaena sp. PCC 7120. Biochemical and spectroscopic

J Biol Chem. 2003 Mar 14;278(11):9219-26. doi: 10.1074/jbc.M211643200. Epub 2003 Jan 3.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Anabaena / enzymology*
  • Bacterial Proteins*
  • Bile Pigments / chemistry
  • Catalysis
  • Chromatography
  • Chromatography, High Pressure Liquid
  • Electrons
  • Electrophoresis, Polyacrylamide Gel
  • Models, Chemical
  • Oxidoreductases / chemistry*
  • Oxidoreductases / metabolism
  • Porphyrins / chemistry
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Spectrophotometry
  • Substrate Specificity
  • Time Factors
  • Ultraviolet Rays

Substances

  • Bacterial Proteins
  • Bile Pigments
  • Porphyrins
  • Recombinant Proteins
  • Oxidoreductases
  • phycocyanobilin ferredoxin oxidoreductase, Synechocystis