Investigation of the pH-dependence of the oxidation of FAD in VcCRY-1, a member of the cryptochrome-DASH family.

Vibrio cholerae cryptochrome-1 (VcCRY-1) is a member of the cryptochrome DASH family. The flavoprotein appears to use blue light both for repair of cyclobutane pyrimidine dimers (CPDs) on DNA and signal transduction. Earlier, we found that it was almost impossible to oxidize the FADH· state upon binding to a CPD, and, in the absence of substrate, the rate of FADH· oxidation was much larger at high pH (Gindt et al. in Biochemistry 54:2802-2805, 2015). Here, we present the pH-dependence of the oxidation of FADH· by ferricyanide, which revealed a switch between slow and fast oxidation with a pKa ≈ 7.0. Stopped-flow mixing was used to measure the oxidation of FADH- to FADH· at pH 6.7 and 7.5. Substrate binding was required to slow down this oxidation such that it could be measured with stopped flow, but there was only a small effect of pH. In addition, resonance Raman measurements of FADH· in VcCRY-1 at pH 6.5 and 7.5 were performed to probe for structural changes near the FAD cofactor related to the observed changes in rate of FADH· oxidation. Only substrate binding seemed to induce a change near the FAD cofactor that may relate to the change in oxidation kinetics. The pH-effect on the FADH· oxidation rate, which is rate-limited by the proton acceptor, does not seem to be due to a protein structural change near the FAD cofactor. Instead, a conserved glutamate in CRY-DASH may control the deprotonation of FADH· and give rise to the pH-effect.

A Review of Spectroscopic and Biophysical-Chemical Studies of the Complex of Cyclobutane Pyrimidine Dimer Photolyase and Cryptochrome DASH with Substrate DNA.

Cyclobutane pyrimidine dimer (CPD) photolyase (PL) is a structure-specific DNA repair enzyme that uses blue light to repair CPD on DNA. Cryptochrome (CRY) DASH enzymes use blue light for the repair of CPD lesions on single-stranded (ss) DNA, although some may also repair these lesions on double-stranded (ds) DNA. In addition, CRY DASH may be involved in blue light signaling, similar to cryptochromes. The focus of this review is on spectroscopic and biophysical-chemical experiments of the enzyme-substrate complex that have contributed to a more detailed understanding of all the aspects of the CPD repair mechanism of CPD photolyase and CRY DASH. This will be performed in the backdrop of the available X-ray crystal structures of these enzymes bound to a CPD-like lesion. These structures helped to confirm conclusions that were drawn earlier from spectroscopic and biophysical-chemical experiments, and they have a critical function as a framework to design new experiments and to interpret new experimental data. This review will show the important synergy between X-ray crystallography and spectroscopic/biophysical-chemical investigations that is essential to obtain a sufficiently detailed picture of the overall mechanism of CPD photolyases and CRY DASH proteins.

The Missing Electrostatic Interactions Between DNA Substrate and Sulfolobus solfataricus DNA Photolyase: What is the Role of Charged Amino Acids in Thermophilic DNA Binding Proteins?

DNA photolyase can be used to study how a protein with its required cofactor has adapted over a large temperature range. The enzymatic activity and thermodynamics of substrate binding for protein from Sulfolobus solfataricus were directly compared to protein from Escherichia coli. Turnover numbers and catalytic activity were virtually identical, but organic cosolvents may be necessary to maintain activity of the thermophilic protein at higher temperatures. UV-damaged DNA binding to the thermophilic protein is less favorable by ∼2 kJ/mol. The enthalpy of binding is ∼10 kJ/mol less exothermic for the thermophile, but the amount and type of surface area buried upon DNA binding appears to be somewhat similar. The most important finding was observed when ionic strength studies were used to separate binding interactions into electrostatic and nonelectrostatic contributions; DNA binding to the thermophilic protein appears to lack the electrostatic contributions observed with the mesophilic protein.

Enzyme-Substrate Binding Kinetics Indicate That Photolyase Recognizes an Extrahelical Cyclobutane Thymidine Dimer.

Escherichia coli DNA photolyase is a DNA-repair enzyme that repairs cyclobutane pyrimidine dimers (CPDs) that are formed on DNA upon exposure of cells to ultraviolet light. The light-driven electron-transfer mechanism by which photolyase catalyzes the CPD monomerization after the enzyme-substrate complex has formed has been studied extensively. However, much less is understood about how photolyase recognizes CPDs on DNA. It has been clearly established that photolyase, like many other DNA-repair proteins, requires flipping of the CPD site into an extrahelical position. Photolyase is unique in that it requires the two dimerized pyrimidine bases to flip rather than just a single damaged base. In this paper, we perform direct measurements of photolyase binding to CPD-containing undecamer DNA that has been labeled with a fluorophore. We find that the association constant of ∼2 × 10(6) M(-1) is independent of the location of the CPD on the undecamer DNA. The binding kinetics of photolyase are best described by two rate constants. The slower rate constant is ∼10(4) M(-1) s(-1) and is most likely due to steric interference of the fluorophore during the binding process. The faster rate constant is on the order of 2.5 × 10(5) M(-1) s(-1) and reflects the binding of photolyase to the CPD on the DNA. This result indicates that photolyase finds and binds to a CPD lesion 100-4000 times slower than other DNA-repair proteins. In light of the existing literature, we propose a mechanism in which photolyase recognizes a CPD that is flipped into an extrahelical position via a three-dimensional search.

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

VcCry1, a member of the CRY-DASH family, may serve two diverse roles in vivo, including blue-light signaling and repair of UV-damaged DNA. We have discovered that the electrochemistry of the flavin adenine dinucleotide cofactor of VcCry1 is locked to cycle only between the hydroquinone and neutral semiquinone states when UV-damaged DNA is present. Other potential substrates, including undamaged DNA and ATP, have no discernible effect on the electrochemistry, and the kinetics of the reduction is unaffected by damaged DNA. Binding of the damaged DNA substrate determines the role of the protein and prevents the presumed photochemistry required for blue-light signaling.

Evidence from thermodynamics that DNA photolyase recognizes a solvent-exposed CPD lesion.

Binding of a cis,syn-cyclobutane pyrimidine dimer (CPD) to Escherichia coli DNA photolyase was examined as a function of temperature, enzyme oxidation state, salt, and substrate conformation using isothermal titration calorimetry. While the overall ΔG° of binding was relatively insensitive to most of the conditions examined, the enthalpic and entropic terms that make up the free energy of binding are sensitive to the conditions of the experiment. Substrate binding to DNA photolyase is generally driven by a negative change in enthalpy. Electrostatic interactions and protonation are affected by the oxidation state of the required FAD cofactor and substrate conformation. The fully reduced enzyme appears to bind approximately two additional water molecules as part of substrate binding. More significantly, the experimental change in heat capacity strongly suggests that the CPD lesion must be flipped out of the intrahelical base stacking prior to binding to the protein; the DNA repair enzyme appears to recognize a solvent-exposed CPD as part of its damage recognition mechanism.

Evidence for concerted electron proton transfer in charge recombination between FADH- and 306Trp• in Escherichia coli photolyase.

Proton-coupled electron-transfer (PCET) is a mechanism of great importance in protein electron transfer and enzyme catalysis, and the involvement of aromatic amino acids in this process is of much interest. The DNA repair enzyme photolyase provides a natural system that allows for the study of PCET using a neutral radical tryptophan (Trp(•)). In Escherichia coli photolyase, photoreduction of the flavin adenine dinucleotide (FAD) cofactor in its neutral radical semiquinone form (FADH(•)) results in the formation of FADH(-) and (306)Trp(•). Charge recombination between these two intermediates requires the uptake of a proton by (306)Trp(•). The rate constant of charge recombination has been measured as a function of temperature in the pH range from 5.5 to 10.0, and the data are analyzed with both classical Marcus and semi-classical Hopfield electron transfer theory. The reorganization energy associated with the charge recombination process shows a pH dependence ranging from 2.3 eV at pH ≤ 7 and 1.2 eV at pH(D) 10.0. These findings indicate that at least two mechanisms are involved in the charge recombination reaction. Global analysis of the data supports the hypothesis that PCET during charge recombination can follow two different mechanisms with an apparent switch around pH 6.5. At lower pH, concerted electron proton transfer (CEPT) is the favorable mechanism with a reorganization energy of 2.1-2.3 eV. At higher pH, a sequential mechanism becomes dominant with rate-limiting electron-transfer followed by proton uptake which has a reorganization energy of 1.0-1.3 eV. The observed 'inverse' deuterium isotope effect at pH < 8 can be explained by a solvent isotope effect that affects the free energy change of the reaction and masks the normal, mass-related kinetic isotope effect that is expected for a CEPT mechanism. To the best of our knowledge, this is the first time that a switch in PCET mechanism has been observed in a protein.

Spectroscopic and thermodynamic comparisons of Escherichia coli DNA photolyase and Vibrio cholerae cryptochrome 1.

Escherichia coli DNA photolyase and cryptochrome 1 isolated from Vibrio cholerae, a member of the CRY-DASH family, are directly compared using a variety of experimental methods including UV-vis and Raman spectroscopy, reduction potential measurements, and isothermal titration calorimetry. The semiquinone form of the cryptochrome has an absorption spectrum that is red-shifted from that of the photolyase, but the Raman spectrum indicates that the FAD binding pocket is similar to that of photolyase. The FADH(-)/FADH* reduction potential of the cryptochrome is significantly higher than that of the photolyase at 164 mV vs NHE, but it also increases upon substrate binding (to 195 mV vs NHE), an increase similar to what is observed in photolyase. The FADH(-)/FADH* reduction potential for both proteins was found to be insensitive to ATP binding. Isothermal titration calorimetry found that photolyase binds tighter to substrate (K(A) approximately 10(5) M(-1) for photolyase and approximately 10(4) M(-1) for cryptochrome 1), and the binding constants for both proteins were slightly sensitive to oxidation state. Based upon this work, it appears that this cryptochrome has significant spectroscopic and electrochemical similarities to CPD photolyase. The thermodynamic cycle of the enzymatic repair in the context of this work is discussed.

Effect of the cyclobutane cytidine dimer on the properties of Escherichia coli DNA photolyase.

Cyclobutane pyrimidine dimer (CPD) photolyases are structure specific DNA-repair enzymes that specialize in the repair of CPDs, the major photoproducts that are formed upon irradiation of DNA with ultraviolet light. The purified enzyme binds a flavin adenine dinucleotide (FAD), which is in the neutral radical semiquinone (FADH(*)) form. The CPDs are repaired by a light-driven, electron transfer from the anionic hydroquinone (FADH(-)) singlet excited state to the CPD, which is followed by reductive cleavage of the cyclobutane ring and subsequent monomerization of the pyrimidine bases. CPDs formed between two adjacent thymidine bases (T< >T) are repaired with greater efficiency than those formed between two adjacent cytidine bases (C< >C). In this paper, we investigate the changes in Escherichia coli photolyase that are induced upon binding to DNA containing C< >C lesions using resonance Raman, UV-vis absorption, and transient absorption spectroscopies, spectroelectrochemistry, and computational chemistry. The binding of photolyase to a C< >C lesion modifies the energy levels of FADH(*), the rate of charge recombination between FADH(-) and Trp(306)(*), and protein-FADH(*) interactions differently than binding to a T< >T lesion. However, the reduction potential of the FADH(-)/FADH(*) couple is modified in the same way with both substrates. Our calculations show that the permanent electric dipole moment of C< >C is stronger (12.1 D) and oriented differently than that of T< >T (8.7 D). The possible role of the electric dipole moment of the CPD in modifying the physicochemical properties of photolyase as well as in affecting CPD repair will be discussed.

Resonance Raman spectroscopic investigation of the light-harvesting chromophore in escherichia coli photolyase and Vibrio cholerae cryptochrome-1.

Photolyases and cryptochromes are flavoproteins that belong to the class of blue-light photoreceptors. They usually bind two chromophores: flavin adenine dinucleotide (FAD), which forms the active site, and a light-harvesting pigment, which is a 5,10-methenyltetrahydrofolate polyglutamate (MTHF) in most cases. In Escherichia coli photolyase (EcPhr), the MTHF cofactor is present in substoichiometric amounts after purification, while in Vibrio cholerae cryptochrome-1 (VcCry1) the MTHF cofactor is bound more strongly and is present at stoichiometric levels after purification. In this paper, we have used resonance Raman spectroscopy to monitor the effect of loss of MTHF on the protein-FAD interactions in EcPhr and to probe the protein-MTHF interactions in both EcPhr and VcCry1. We find that removal of MTHF does not perturb protein-FAD interactions, suggesting that it may not affect the physicochemical properties of FAD in EcPhr. Our data demonstrate that the pteridine ring of MTHF in EcPhr has different interactions with the protein matrix than that of MTHF in VcCry1. Comparison to solution resonance Raman spectra of MTHF suggests that the carbonyl of its pteridine ring in EcPhr experiences stronger hydrogen bonding and a more polar environment than in VcCry1, but that hydrogen bonding to the pteridine ring amine hydrogens is stronger in VcCry-1. These differences in hydrogen bonding may account for the higher binding affinity of MTHF in VcCry1 compared to EcPhr.

The free energy of dissociation of oligomeric structure in phycocyanin is not linear with denaturant.

Using SEC HPLC and fluorescence anisotropy, absorption spectra were assigned to the specific oligomeric structures found with phycocyanin. The absorption spectra were used to quantify the population of each oligomeric form of the protein as a function of both urea concentration and temperature. Phycocyanin hexamers dissociate to trimers with equilibrium constants of 10(-6) to 10(-5). Phycocyanin trimers dissociate to monomers with equilibrium constants of 10(-15) to 10(-12). Both dissociation constants increase linearly with increasing urea concentration, and deltaG(o) values calculated from the equilibrium constants fit best with an exponential function. Our findings appear in contrast with the commonly used linear extrapolation model, deltaG(urea)(o) = deltaG(water)(o) + A[denaturant], in which a linear relationship exists between the free energy of protein unfolding or loss of quaternary structure and the denaturant concentration. Our data examines a smaller range of denaturant concentration than generally used, which might partially explain the inconsistency.

Substrate binding modulates the reduction potential of DNA photolyase.

The reduction potential of the (FADH-/FADH*) couple in DNA photolyase was measured, and the value was found to be significantly higher than the values estimated in the literature. In the absence of substrate, the enzyme has a reduction potential of 16 +/- 6 mV vs NHE. In the presence of excess substrate the reduction potential increases to 81 +/- 8 mV vs NHE. The increase in reduction potential has physiological relevance since it gives the catalytic state greater resistance to oxidation. This is the first measurement of a reduction potential for this class of DNA-repair enzymes and the larger family of blue-light photoreceptors.

Substrate electric dipole moment exerts a pH-dependent effect on electron transfer in Escherichia coli photolyase.

Transient absorption spectroscopy is used to demonstrate that the electric dipole moment of the substrate cyclobutane thymine dimer affects the charge recombination reaction between fully reduced flavin adenine dinucleotide (FADH-) and the neutral radical tryptophan 306 (Trp306*) in Escherichia coli DNA photolyase. At pH 7.4, the charge recombination is slowed by a factor of 1.75 in the presence of substrate, but not at pH 5.4. Photolyase does bind substrate at pH 5.4, and it seems that this pH effect originates from the conversion of FADH- to FADH2 at lower pH. The free-energy changes calculated from the electric field parameters and from the change in electron transfer rate are in good agreement and support the idea that the substrate electric dipole is responsible for the observed change in electron transfer rate. It is expected that the substrate electric field will also modify the physiologically important from excited 1FADH- to the substrate in the DNA repair reaction.