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 (ΔE = 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 ∼ 100 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.

FIGURE 1.

FAD redox reactions and environment in PLs and CRYs. a, kinetic and thermodynamic parameters for exchange among FAD redox states. The neutral sq is reduced to the anionic hq at reduction potential E₁. The hq reacts with O₂ to regenerate the sq with a pseudo first-order rate constant, k₁. ox is reduced to the sq radical at reduction potential E₂. Formation of the neutral sq radical requires a proton transfer. This radical is oxidized by O₂ with a rate constant, k₂, that requires deprotonation. The overall two-electron reduction of ox to hq is characterized by the midpoint potential, E_M; the complete conversion of hq to ox by reaction with O₂ is characterized by the rate constant k. b, sequence alignment of isoalloxazine-binding residues from species representing six classes of PLs and CRYs: Type 1 CPD-PL, 6-4 PL, CRY-DASH, plant CRY, and Type 1 and Type 2 animal CRY. Starred residues are strictly conserved, whereas residues marked with a closed circle differ between CPD-PL and plant CRY. An = A. nidulans; Ec = E. coli; Dm = Drosophila melanogaster; Dr = Danio rerio; At = A. thaliana; Hs = Homo sapiens. c, structure alignments of conserved (right) and non-conserved (left) isoalloxazine-binding residues in A. nidulans CPD-PL (Protein Data Bank (PDB): 1qnf) (26) and A. thaliana CRY1 (PDB: 1u3c) (27). For the non-conserved residues, those from A. thaliana CRY1 are labeled in bold.

FIGURE 2.

Thermodynamic and kinetic characterization of PCET in wild type and N386D CPD-PL. a, UV-visible absorption spectra taken during spectroelectrochemical reductive titrations of A. nidulans CPD-PL and N386D CPD-PL (inset) in the presence of mediators at pH 7 (see ”Experimental Procedures“). b, spectroelectrochemical reductive titration of CPD-PL (blue squares) and overlay of reductive (solid green circles) and oxidative (open green circles) titrations of N386D CPD-PL at pH 7 (all potentials reported versus normal hydrogen electrode, NHE). The plot shows the variation in extinction coefficients at 445 nm with potential (data from 5–7 replicates). The lines are fits to a modified Nernst equation (see ”Experimental Procedures“), for CPD-PL yielding E₁ = −140 ± 7 mV and E₂ = −219 ± 7 mV and for N386D CPD-PL yielding E₁ = −56 ± 7 mV and E₂ = −124 ± 7 mV. c, UV-visible absorption spectra of PL (dashed line) and N386D CPD-PL (solid line) in aqueous aerated buffer (pH 7) following complete purification. The concentration of each holoprotein was ∼25 μm. d, oxidation of FAD hq in N386D and wild type (inset) CPD-PL. Shown are time-dependent UV-visible absorption spectra following the introduction of O₂ into fully reduced N386D CPD-PL and CPD-PL (inset) over a period of ∼1100 and 2000 min, respectively. In N386D CPD-PL, the FAD fully oxidizes during this time, and <10% sq is detected. In CPD-PL, hq oxidizes quantitatively to the sq, which in turn is resistant to further oxidation. e, time-dependent change in absorbance at 450 nm following introduction of O₂ into fully reduced CPD-PL (squares), N386D CPD-PL (circles), and Synechocystis sp. PCC 6803 CRY-DASH (triangles). The absorption values have been adjusted to account for small differences in concentration of each protein sample (∼25 μm). For N386D CPD-PL and CRY-DASH, where very little sq accumulates, the growth at 450 nm is dominated by ox formation. These are well fit to a single exponential expression to yield the rate constant for oxidation, k (lines). For wild type CPD-PL, because both sq and ox contribute to the absorbance at 450 nm, the growth is fit to a double exponential expression to yield rate constants for sq (k₁) and ox (k₂) formation. The latter is an upper limit because oxidation is incomplete (33). f, UV-visible absorption spectra recorded during white light photoreduction of N386D CPD-PL in aqueous buffer containing 10 mm EDTA at pH 9 (solid line) and pH 5.4 (dashed line). The concentration of holoproteins was ∼15 μm. Spectra shown correspond to the photoreduction time at which sq formation was at its maximum at pH 9.

FIGURE 3.

Mechanistic model and energy profile for exchange of FAD redox states in wild type and N386D CPD-PL. a, model of PCET for interconversion between flavin sq/ox in PLs/CRYs with an N5-proximal Asn or Asp. Oxidation of CPD-PL (top) requires participation beyond the N5-proximal Asn, possibly by a bound/mobile water, as shown. The deprotonation is shown to follow ET. It could be concerted. A conformational change of the Asn side chain is also necessary to achieve H-bonding with the deprotonated flavin N5. When the N5-proximal residue is an Asp (bottom), the proton can be exchanged between the flavin and the carboxylate with no additional participant in the PT or a conformational change. The identity and properties of the N5-proximal residue are thus expected to regulate the activation barrier for PCET. R = ribitol diphosphoadenosine; R′ = polypeptide chain. b, qualitative reaction coordinate diagram for exchange among FAD redox states in wild type CPD-PL and its N386D mutant (normalized to the energy of ox).