Structure, Function, and Dynamics of Mutant PcyA, the Archetypal Member of the Ferredoxin-Dependent Bilin Reductase Family

Ferredoxin oxidoreductase PcyA is a member of the ferredoxin-dependent bilin reductase family (FDBR). This enzyme serves as an archetype for the structural, functional and dynamics studies of FDBR. PcyA catalyzes the exovinyl and the endovinyl reductions of biliverdin IX; (BV) to produce phycocyanobilin (PCB). This dissertation presents the structures of PcyA variants (Asp105Asn, His88Gln) that have mutations in residues implicated in having a functional role in the reduction process of BV. In addition, we studied the structure of PcyA His74Ala variant, since His74 plays a critical role in the stability of PcyA active site. Based on Molecular Dynamics (MD) studies, we determined that the His74Ala mutation is responsible for increased flexibility of active site residues and loss of catalytic activity.;We also carried out number of mutational studies of PcyA utilizing biochemical end-point and UV/VIS spectroscopic assays of PcyA variants in complex with BV. End-point assays revealed the presence of 3E/3Z PCB product, the half reduced 181,182-dihydrobiliverdin (DHBV) intermediate, and unreduced BV starting material. UV/VIS time course measurements showed that the BV complexes of the PcyA variants under study displayed the characteristic spectra of that of the wild type. Upon addition of excess nicotinamide adenine dinucleotide phosphate (NADPH) reductant, the color of the BV in complexes with PcyA variants changes from green to blue, which results in a rapid decrease in peaks at 376 and 668 nm, along with an increase at 470 and 770 nm. This rapid change is followed by a slower disproportionation of the unstable radical intermediate to the stable 2e- reduced DHBV intermediate and BV. Addition of more NADPH (10 uM NADPH - up to 4 e- equivalents) affected a greater shift of wavelength from 470 to 370 nm (blue) and the increase of 590 nm peak.;We also characterized the mechanism of ferredoxin oxidoreductase PcyA, a unique cyanobacteria enzyme involved in the proton-coupled, four-electron reduction of BV to phycocyanobilin needed for the chromophores of phytochromes and phycobiliproteins. Interestingly, in our quest to uncover all the residues involved in the mechanism of BV reduction by PcyA, we discovered that the Glu76His PcyA variant is able to produce a non-native phycobilin product.;Lastly, rational sequence design of the PebA protein scaffold was carried out to convert its activity into that of PcyA. By introducing mutations into PebA, we attempt to change its reduction regiospecificity to that of PcyA. Instead of producing its normal product 3E/3Z-phycoerythrobilin (PEB), the new mutant was able to yield 3E/3Z-phycocyanobilin (PCB) - the normal product of PcyA.