A Conserved Histidine-Aspartate Pair Is Required for Exovinyl Reduction of Biliverdin by a Cyanobacterial Phycocyanobilin:Ferredoxin Oxidoreductase

Abstract

Phycocyanobilin:ferredoxin oxidoreductase is a member of the ferredoxin-dependent bilin reductase family and catalyzes two vinyl reductions of biliverdin IX␣ to produce phycocyanobilin, the pigment precursor of both phytochrome and phycobiliprotein chromophores in cyanobacteria. Atypically for ferredoxin-dependent enzymes, phycocyanobilin:ferredoxin oxidoreductase mediates direct electron transfers from reduced ferredoxin to its tetrapyrrole substrate without metal ion or organic cofactors. We previously showed that bound bilin radical intermediates could be detected by low temperature electron paramagnetic resonance and absorption spectroscopies (Tu, S., Gunn, A., Toney, M. D., Britt, R. D., and Lagarias, J. C. (2004) J. Am. Chem. Soc. 126, 8682-8693). On the basis of these studies, a mechanism involving sequential electroncoupled proton transfers to protonated bilin substrates buried within the phycocyanobilin:ferredoxin oxidoreductase protein scaffold was proposed. The present investigation was undertaken to identify catalytic residues in phycocyanobilin:ferredoxin oxidoreductase from the cyanobacterium Nostoc sp. PCC7120 through site-specific chemical modification and mutagenesis of candidate proton-donating residues. These studies identified conserved histidine and aspartate residues essential for the catalytic activity of phycocyanobilin:ferredoxin oxidoreductase. Spectroscopic evidence for the formation of stable enzyme-bound biliverdin radicals for the H85Q and D102N mutants support their role as a "coupled" protondonating pair during the reduction of the biliverdin exovinyl group.

Mechanistic Studies of the Phytochromobilin Synthase HY2 from Arabidopsis

“…Two organic radical intermediates have also been detected in the two double bond reduction steps, strongly supporting the proposed mechanism for sequential electron-coupled proton transfer in the PcyA-catalyzed BV reduction (11). Mutagenesis studies have shown a histidine/aspartate ion pair playing the essential role in the active site, which is further confirmed by the crystal structure of PcyA (12)(13)(14). Combining the structural information and biochemical data, a more detailed mechanism for PcyA reaction has been proposed (12).…”

“…So far, many ionizable residues in the active site of PcyA have been selected to investigate their functions in binding and catalysis. Critical residues have been identified (12,13). None of the mutations of these critical residues give a significant loss of the 650 nm band.…”

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

“…Since biliproteins are necessary both for energy cap-54 ture and for adaptation to the light environment, production of 55 these linear tetrapyrroles is critical to photoautotrophic organisms 56 in which they are present. 57 The linear tetrapyrrole phycocyanobilin (PCB), 3 comprising the 75 With each electron transfer, a proton must be transferred to the 76 bilin substrate [9,12,14]. Site directed mutagenesis has been a 77 powerful tool to help identify residues that mediate this proton 78 donation to the tetrapyrrole substrate.…”

“…Previously, His88 and 79 Asp105 (Synechocystis sp. PCC 6803 PcyA numbering) were shown 80 to be critical for the reduction of both vinyl groups [14]. Crystal 81 structures reveal that these residues interact in the absence of sub-82 strate, but move apart upon BV binding to interact with the bilin 83 substrate [11,12,15].…”

Homology Modeling and Site-Directed Mutagenesis Reveal Catalytic Key Amino Acids of 3β-Hydroxysteroid-Dehydrogenase/C4-Decarboxylase from Arabidopsis

“…The pKa of His is likely to rise upon interaction with the substrate carboxyl group during binding (Anderson et al, 1990), and the acid form, the imidazolium ion, is able to interact with the substrate by forming a H-bonded ion pair with its carboxyl group. Stabilizing hydrogenbonded ion pairing between His and Asp residues has been shown to be critical in several enzymatic reactions (Sundaramoorthy et al, 1998;Chen et al, 2004;Tu et al, 2006). Finally, sequence alignments show that aromatic and hydrophobic amino acid residues that are confining the steroid nucleus and side chain in the binding hydrophobic cavity remain conserved or similar across the 3bHSD/D phylogeny (Rahier et al, 2006).…”