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 twostep 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 GHz 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.

His74 conservation in the bilin reductase PcyA family reflects an important role in protein-substrate structure and dynamics

“…In the substrate-free PcyA structure, the pro- BV [13]. Substitution of either residue with a non-proton-donating 89 amino acid prevents transfer of a second electron thereby stabiliz-90 ing the one-electron reduced BVHÅ radical [16]. In addition to D-91 ring exovinyl group reduction, His88 also plays a critical role in 92 A-ring endovinyl reduction since the H88Q variant is unable to re-93 duce the A-ring vinyl group of exogenous DHBV [12].…”

Structural Insights into Vinyl Reduction Regiospecificity of Phycocyanobilin:Ferredoxin Oxidoreductase (PcyA)

“…1B) (17, 18). More recent EPR spectroscopy and density-functional theory computations on the D105N mutant of PcyA revealed potential protonation states of the semireduced BV radical intermediate (25), which, in turn, indirectly implicated the role of proton donors within the active site.…”

First computational step towards the understanding of the antioxidant activity of the Phycocyanobilin:Ferredoxin Oxidoreductase in complex with biliverdin IXα

“…These computations were performed on the BV, Glu76, His88 and Asp105 system (structure extracted from the D105N diffraction data) in gas phase. In that model system, all heteroatoms were frozen and the propionate groups were replaced by methyl groups [7]. The new intermediate proposed in Ref.…”

“…The new intermediate proposed in Ref. [7] presents the C-OH of the A-ring oriented towards His88 while the C-OH of the D-ring is directed to the oxygen atom of the A-ring. In 2010, the group of Higawara suggested that Glu76 is playing a role in the proton donation process while His88 would be important for the second reduction step, in agreement with the previous works [8].…”

“…Indeed, only a few works were performed to explore the BV:PcyA complex, and most of them were focused on spectroscopic properties (absorption spectra and g-tensor) only [4,7]. These studies were typically performed on quite small BV model systems with propionate groups replaced by methyl groups, without accounting for vicinal water molecules nor considering the BV relaxation in the PcyA surrounding.…”