Structure of the biliverdin radical intermediate in phycocyanobilin: Ferredoxin oxidoreductase identified by high-field EPR and DFT

Stefan Stoll, Alexander Gunn, Marcin Brynda, Wesley Sughrue, Amanda C. Kohler, Andrew Ozarowski, Andrew J Fisher, J. Clark Lagarias, R. David Britt

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

The cyanobacterial enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the two-step four-electron reduction of biliverdin IXα to phycocyanobilin, the precursor of biliprotein chromophores found in phycobilisomes. It is known that catalysis proceeds via paramagnetic radical intermediates, but the structure of these intermediates and the transfer pathways for the four protons involved are not known. In this study, high-field electron paramagnetic resonance (EPR) spectroscopy of frozen solutions and single crystals of the one-electron reduced protein-substrate complex of two PcyA mutants D105N from the cyanobacteria Synechocystis sp. PCC6803 and Nostoc sp. PCC7120 are examined. Detailed analysis of Synechocystis D105N mutant spectra at 130 and 406 GHz reveals a biliverdin radical with a very narrow g tensor with principal values 2.00359(5), 2.00341(5), and 2.00218(5). Using density-functional theory (DFT) computations to explore the possible protonation states of the biliverdin radical, it is shown that this g tensor is consistent with a biliverdin radical where the carbonyl oxygen atoms on both the A and the D pyrrole rings are protonated. This experimentally confirms the reaction mechanism recently proposed (Tu, et al. Biochemistry 2007, 46, 1484).

Original languageEnglish (US)
Pages (from-to)1986-1995
Number of pages10
JournalJournal of the American Chemical Society
Volume131
Issue number5
DOIs
StatePublished - Feb 11 2009

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Biliverdine
Ferredoxins
Electron Spin Resonance Spectroscopy
Density functional theory
Tensors
Paramagnetic resonance
Oxidoreductases
Synechocystis
Biochemistry
Electrons
Protonation
Chromophores
Catalysis
Protons
Enzymes
Phycobilisomes
Nostoc
Single crystals
Spectroscopy
Proteins

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry
  • Medicine(all)

Cite this

Structure of the biliverdin radical intermediate in phycocyanobilin : Ferredoxin oxidoreductase identified by high-field EPR and DFT. / Stoll, Stefan; Gunn, Alexander; Brynda, Marcin; Sughrue, Wesley; Kohler, Amanda C.; Ozarowski, Andrew; Fisher, Andrew J; Lagarias, J. Clark; Britt, R. David.

In: Journal of the American Chemical Society, Vol. 131, No. 5, 11.02.2009, p. 1986-1995.

Research output: Contribution to journalArticle

Stoll, Stefan ; Gunn, Alexander ; Brynda, Marcin ; Sughrue, Wesley ; Kohler, Amanda C. ; Ozarowski, Andrew ; Fisher, Andrew J ; Lagarias, J. Clark ; Britt, R. David. / Structure of the biliverdin radical intermediate in phycocyanobilin : Ferredoxin oxidoreductase identified by high-field EPR and DFT. In: Journal of the American Chemical Society. 2009 ; Vol. 131, No. 5. pp. 1986-1995.
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