Impact of the N5-proximal Asn on the Thermodynamic and Kinetic Stability of the Semiquinone Radical in Photolyase

Abstract

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...

What accounts for the different functions in photolyases and cryptochromes: a computational study of proton transfers to FAD

“…13 Damiani et al suggested that water molecules can enter the FAD pocket and form a PT pathway from the bulk water to FAD. 21 The formation of the FAD x state occurs in 3.3 ms in PL-N378D. Compared to this, spectroscopic studies in CRYI show that the photo-reduction of FAD ox occurs within 31 ps, the subsequent PT from Asp396 to FAD À occurs in 1.7 ms, while the backward transfer is two orders of magnitude slower (690 ms).…”

“…19 In E. coli and A. nidulans PL, like in the entomic CRY, the mutation of Asn into Asp does not enable a PT to form FADH . 20,21 After photo-reduction, the E. coli PL Asn to Asp mutant (PL-N378D) shows a slightly different UV-vis absorption spectrum compared to the spectrum for FAD À . It is characteristic of the presence of a different FAD state, called FAD x , and the absence of FADH .…”

Proton Transfer to Flavin Stabilizes the Signaling State of the Blue Light Receptor Plant Cryptochrome

“…This unspecific pathway seems to be slow because, in photolyase from E. coli, protonation is not observed within hundreds of microseconds after anion radical formation (59). A kinetic barrier has been identified for the respective deprotonation step from the neutral radical to the oxidized state, which can be lowered by the N386D mutation close to flavin (60). A role of the aspartate as a proton shuttle in this photolyase mutant has accordingly been suggested.…”

Binding of Substrate Locks the Electrochemistry of CRY-DASH into DNA Repair

“…The CRY-DASH proteins have Asn, not Asp, in the N5-proximal position, as shown with the published sequence alignment. 19 We also expect Asn to be proximal to N5 for VcCry1. From the literature, it appears plausible that the UV-p(dT) 10 molecule is acting to block proton transfer from the N5 position.…”