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.