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

ABSTRACT: The cyanobacterial enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the two-step four-electron reduction of biliverdin IXalpha 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).

SUBMITTER: Stoll S 

PROVIDER: S-EPMC2753389 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature