Single Hydrogen Bond Donation from Flavin N5 to Proximal Asparagine Ensures FAD Reduction in DNA Photolyase.

The spread of the absorbance of the stable FADH(•) radical (300-700 nm) allows CPD photolyase to highly efficiently form FADH(-), making it functional for DNA repair. In this study, FTIR spectroscopy detected a strong hydrogen bond, from FAD N5-H to the carbonyl group of the Asn378 side chain, that is modulated by the redox state of FAD. The observed characteristic frequency shifts were reproduced in quantum-mechanical models of the flavin binding site, which were then employed to elucidate redox tuning governed by Asn378. We demonstrate that enhanced hydrogen bonding of the Asn378 side chain with the FADH(•) radical increases thermodynamic stabilization of the radical state, and further ensures kinetic stabilization and accumulation of the fully reduced FADH(-) state.

Flavin adenine dinucleotide chromophore charge controls the conformation of cyclobutane pyrimidine dimer photolyase α-helices.

Observations of light-receptive enzyme complexes are usually complicated by simultaneous overlapping signals from the chromophore, apoprotein, and substrate, so that only the initial, ultrafast, photon-chromophore reaction and the final, slow, protein conformational change provide separate, nonoverlapping signals. Each provides its own advantages, whereas sometimes the overlapping signals from the intervening time scales still cannot be fully deconvoluted. We overcome the problem by using a novel method to selectively isotope-label the apoprotein but not the flavin adenine dinucleotide (FAD) cofactor. This allowed the Fourier transform infrared (FTIR) signals to be separated from the apoprotein, FAD cofactor, and DNA substrate. Consequently, a comprehensive structure-function study by FTIR spectroscopy of the Escherichia coli cyclobutane pyrimidine dimer photolyase (CPD-PHR) DNA repair enzyme was possible. FTIR signals could be identified and assigned upon FAD photoactivation and DNA repair, which revealed protein dynamics for both processes beyond simple one-electron reduction and ejection, respectively. The FTIR data suggest that the synergistic cofactor-protein partnership in CPD-PHR linked to changes in the shape of FAD upon one-electron reduction may be coordinated with conformational changes in the apoprotein, allowing it to fit the DNA substrate. Activation of the CPD-PHR chromophore primes the apoprotein for subsequent DNA repair, suggesting that CPD-PHR is not simply an electron-ejecting structure. When FAD is activated, changes in its structure may trigger coordinated conformational changes in the apoprotein and thymine carbonyl of the substrate, highlighting the role of Glu275. In contrast, during DNA repair and release processes, primary conformational changes occur in the enzyme and DNA substrate, with little contribution from the FAD cofactor and surrounding amino acid residues.

Detection of distinct α-helical rearrangements of cyclobutane pyrimidine dimer photolyase upon substrate binding by Fourier transform infrared spectroscopy.

Photolyases (PHRs) utilize near-ultraviolet (UV)-blue light to specifically repair the major photoproducts (PPs) of UV-induced damaged DNA. The cyclobutane pyrimidine dimer PHR (CPD-PHR) from Escherichia coli binds flavin adenine dinucleotide (FAD) as a cofactor and 5,10-methenyltetrahydrofolate as a light-harvesting pigment and specifically repairs CPD lesions. By comparison, a second photolyase known as (6-4) PHR, present in a range of higher organisms, uniquely repairs (6-4) PPs. To understand the repair mechanism and the substrate specificity that distinguish CPD-PHR from (6-4) PHR, we applied Fourier transform infrared (FTIR) spectroscopy to bacterial CPD-PHR in the presence or absence of a well-defined DNA substrate, as we have studied previously for vertebrate (6-4) PHR. PHRs show light-induced reduction of FAD, and photorepair by CPD-PHR involves the transfer of an electron from the photoexcited reduced FAD to the damaged DNA for cleaving the dimers to maintain the DNA's integrity. Here, we measured and analyzed difference FTIR spectra for the photoactivation and DNA photorepair processes of CPD-PHR. We identified light-dependent signals only in the presence of substrate. The signals, presumably arising from a protonated carboxylic acid or the DNA substrate, implicate conformational rearrangements of the protein and substrate during the repair process. Deuterium exchange FTIR measurements of CPD-PHR highlight potential differences in the photoactivation and photorepair mechanisms in comparison to those of (6-4) PHR. Although CPD-PHR and (6-4) PHR appear to exhibit similar overall structures, our studies indicate that distinct conformational rearrangements, especially in the α-helices, are initiated within these enzymes upon binding of their respective DNA substrates.