Photolyases and cryptochromes (CRY) are structurally homologous flavoproteins with divergent functions. While photolyases repair UV-damaged DNA by photoinduced electron transfer from their FAD cofactor, CRY are involved in varied cellular processes, including light-dependent plant growth, regulation of mammalian circadian rhythm, and possibly magnetoreception. Despite their importance in Nature and human health, little is known about how they tune their FAD redox properties to achieve remarkable functional diversity. In this study, we reveal a kinetic mechanism, exploited by cyclobutane pyrimidine dimer photolyase (PL), for regulating the stability of its FAD semiquinone (sq). We find that the sq in CRY-DASH (Synechocystis) is substantially more reactive toward oxidation than in PL (Anacystis nidulans) and, using deuterium isotope and pH effects, show that rate-limiting proton transfer contributes to the exceptional kinetic stability of the PL sq. Through mutagenesis, we identify two PL-specific residues in the flavin binding pocket, Trp392 and Gly389 (Try398 and Asn395 in CRY-DASH, respectively), that ensure this kinetic stability, possibly through interactions with the adenine moiety of FAD and/or adjusting the polarity of the binding site. Significantly, these relatively distal residues have a much more profound impact than two amino acids closer to the FAD. By quantifying sq stability in a series of PL-CRY exchange mutants, our findings pave the way for investigations aimed at correlating sq stability with function in these proteins. As is being recognized with other flavoproteins, we expect that kinetic tuning of the rates of electron transfer will play a function-defining role in photolyases and cryptochromes.

Flavoproteins can dramatically adjust the thermodynamics and kinetics of electron transfer at their flavin cofactor. A versatile regulatory tool is proton transfer. Here, we demonstrate the significance of proton-coupled electron transfer to redox tuning and semiquinone (sq) stability in photolyases (PLs) and cryptochromes (CRYs). These light-responsive proteins share homologous overall architectures and FAD-binding pockets, yet they have evolved divergent functions that include DNA repair, photomorphogenesis, regulation of circadian rhythm, and magnetoreception. We report the first measurement of both FAD redox potentials for cyclobutane pyrimidine dimer PL (CPD-PL, Anacystis nidulans). These values, E 1 (hq/sq) ‫؍‬ ؊140 mV and E 2 (sq/ox) ‫؍‬ ؊219 mV, where hq is FAD hydroquinone and ox is oxidized FAD, establish that the sq is not thermodynamically stabilized (⌬E ‫؍‬ E 2 ؊ E 1 ‫؍‬ ؊79 mV). Results with N386D CPD-PL support our earlier hypothesis of a kinetic barrier to sq oxidation associated with proton transfer. Both E 1 and E 2 are upshifted by ϳ100 mV in this mutant; replacing the N5-proximal Asn with Asp decreases the driving force for sq oxidation. However, this Asp alleviates the kinetic barrier, presumably by acting as a proton shuttle, because the sq in N386D CPD-PL oxidizes orders of magnitude more rapidly than wild type. These data clearly reveal, as suggested for plant CRYs, that an N5-proximal Asp can switch on proton transfer and modulate sq reactivity. However, the effect is context-dependent. More generally, we propose that PLs and CRYs tune the properties of their N5-proximal residue to adjust the extent of proton transfer, H-bonding patterns, and changes in protein conformation associated with electron transfer at the flavin. Electron transfer (ET)2 within and between proteins is a ubiquitous and essential molecular process in biology. Proteins must tightly control the rates of ET and lifetimes of radical intermediates to use this fundamental chemical reaction for a vast array of cellular functions. A significant control mechanism involves coupling of the ET to a proton transfer (PT) (1-3). The PT ensures heightened discrimination toward subtle changes in protein structure. As compared with ET, it has a much shorter range and is very sensitive to the relative orientation of a few atoms (the proton, donor, and acceptor), and its kinetics and thermodynamics can be adjusted dramatically by tuning pK a values over a very large range (3). Consequently, proton-coupled ET (PCET) is widespread in nature, notably in biological energy conversion and redox catalysis. Understanding the mechanisms for PCET and identifying its role in protein evolution are central problems in chemical biology.Flavoproteins are major players in cellular redox reactions (4). The flavin cofactor can exist in three redox states: the two-electron reduced hydroquinone (hq), which is often anionic; the one-electron reduced semiquinone (sq), as a neutral or anionic radical; and the fully oxidized form (ox) (Fig. 1a). Flavoprotein...

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