Electron Transfer between Guanosine Radical and Amino Acids in

J. Phys. Chem. B 2007, 111, 7439-7448

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Electron Transfer between Guanosine Radical and Amino Acids in Aqueous Solution. 1. Reduction of Guanosine Radical by Tyrosine Olga B. Morozova,† Alexey S. Kiryutin,†,‡ Renad Z. Sagdeev,†,§ and Alexandra V. Yurkovskaya*,†,| International Tomography Center of SB RAS, Institutskaya 3a, 630090 NoVosibirsk, Russia, NoVosibirsk State UniVersity, PirogoVa 2, 630090 NoVosibirsk, Russia, Center of Magnetic Tomography and Spectroscopy, Moscow State UniVersity, Leninskie Gory 1-73, 119992 Moscow, Russia, and Institute of Experimental Physics, Free UniVersity of Berlin, Arnimallee 14, 14195 Berlin, Germany ReceiVed: NoVember 21, 2006; In Final Form: April 4, 2007

As a model of chemical DNA repair, the reductive electron transfer from the aromatic amino acid tyrosine to the radical of the purine base guanosine monophosphate (GMP) was studied by time-resolved chemically induced dynamic nuclear polarization (CIDNP). The guanosyl radicals were photochemically generated in the quenching reaction of the triplet excited dye 2,2′-dipyridyl. Depending on the pH of the aqueous solution, four different guanosyl radicals were observed. The identification of the radicals was possible because of the high sensitivity of CIDNP to distinguish them through their ability or disability of participating in the degenerate electron hopping reaction with the diamagnetic molecules of guanosine monophosphate in the ground state. The CIDNP kinetics in this three-component system containing the dye, GMP, and N-acetyl tyrosine is strongly dependent on the efficiency of the electron-transfer reaction from tyrosine to the nucleotide radical. Quantitative analysis of the CIDNP kinetics obtained at different concentrations of the amino acid, together with the comparison with the CIDNP kinetics of the two-component systems (dipyridyl/tyrosine and dipyridyl/GMP) allowed for the determination of the rate constant ke of the reductive electron-transfer reaction for five pairs of reactants, with different protonation states depending on the pH: GH++•/TyrOH (pH 1.3), G+•/TyrOH (pH 2.9), G(-H)•/TyrOH (pH 7.5), G(-H)•/TyrO- (pH 11.3), and G(-2H)-•/TyrO- (pH 13.3). The rate constant ke varies from (7.1 ( 3.0) × 108 M-1 s-1 (pH 1.3, 2.9) to less than 6 × 106 M-1 s-1 (pH 13.3).

Introduction Electron-transfer reactions often occur in living biological systems giving rise to many important functional processes. They are also involved in the pathological damage of DNA caused by electron removal from DNA bases under ionizing radiation,1 photoininization,2-4 chemical oxidation,5 and photosensitization.2 Guanine is the most easily oxidized DNA component.6 In liquid aqueous solution, the principal oneelectron-oxidized intermediate is the guanyl radical leading to formation of stable products or crosslinking in DNA. Guanyl radicals are produced not only in oxidative damage of DNA,7 but also in double stranded oligonucleotides,8,9 and in aqueous solution of the free bases.10 There are several enzymatic DNA repair pathways that have evolved in nature to prevent the accumulation of damages that block a number of critical processes, such as transcription and replication.11-15 Besides the relatively slow enzymatic processes of DNA repair for protecting genetic information, the electronic vacancies in DNA produced by oxidizing agents or by ionizing radiation on the guanyl base may be refilled rather fast via electron transfer from the surrounding protein pool (in particular, from histon proteins) thus preventing the radical chemistry to evolve into pathological DNA damage.7,16-20 This “chemical way” of * Author for correspondence. Tel. +7 383 3331333. Fax +7 383 3331399. E-mail: [email protected]. † International Tomography Center of SB RAS. ‡ Novosibirsk State University. § Moscow State University. | Free University of Berlin.

DNA reductive repair7,18,19-21 efficiently competes with the formation of modified sites that are targets for enzymatic repair. Recently it was shown that the yield of enzyme sensitive sites, formed in γ-irradiated plasmid DNA after its incubation with the base excision repair enzyme endonuclease that converts damaged guanosine sites into the strand breaks, was reduced on addition of single substituted phenols,7 aniline derivatives,19 the amino acids tyrosine and tryptophan,20 or their peptide derivatives.18 Among the six relatively easily oxidized amino acids studied,20 tryptophan and tyrosine residues were found to be the most efficient reducing agents for fast chemical repair of guanyl radicals formed in plasmid DNA under γ-irradiation. The bimolecular rate constant for reductive repair was estimated using an indirect method based on the analysis of the number of strand breaks formed in the irradiated plasmid DNA after its incubation with endonuclease.7,16,17,19-21 Both the process of electron removal from guanine and the repair of DNA guanyl radicals in vitro by electron transfer from mild reducing agents at physiological conditions were found to be coupled to proton transfer7,16-18,22 and therefore expected to be pH dependent, but this dependence has not been exhaustivly investigated so far. In part a of Chart 1, the structures of four possible guanyl radicals are shown along the pH axis. Since the concentration of the radical intermediates formed in the above-mentioned processes is very low, their in situ detection by conventional electron paramagnetic resonance spectroscopy is not feasible. An alternative approach is to use indirect detection by dynamic nuclear spin polarization that provides a much higher sensitivity. Chemically

10.1021/jp067722i CCC: $37.00 © 2007 American Chemical Society Published on Web 05/25/2007

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Morozova et al.

CHART 1

induced dynamic nuclear polarization (CIDNP) is produced in the singlet-triplet transition of spin-correlated radical pairs in solution and manifests itself as anomalous intensity, emission or enhanced absorption, of NMR spectral lines of the nuclei that have hyperfine interaction with the unpaired electron in transient radicals.23 Strong enhancement factors of the nuclear polarization make the method very sensitive.24,25 Analysis of the intensities in the CIDNP spectrum allows one to get information about the spin density distribution in the intermediate paramagnetic particles. The first detection of purine radicals by CIDNP was done in 1979.26 A detailed description of photo CIDNP of tyrosine at different pH was given by Stob and Kaptein.27 Here we report studies of the short-lived intermediates in photo initiated free radical reactions of the RNA base guanosine monophosphate (GMP) with amino acids by timeresolved chemically induced dynamic nuclear polarization (TRCIDNP).28-30 The technique is based on the application of two synchronized pulses with variable delay τ between as it is shown in Chart 2. The first is a single laser pulse with a duration of a few nanoseconds that initiates photochemical reactions in the probe of the NMR spectrometer, and the second pulse is a radio frequency pulse that serves for the NMR detection of diamagnetic products formed during the time τ between these two pulses.

The time-resolved version of the CIDNP technique opens new possibilities for combining the structural information available from high-resolution NMR spectroscopy with the temporal resolution of laser flash photolysis. In this way one can monitor the chemical and structural changes associated with particular groups of atoms during a radical reaction on a microsecond timescale. By NMR detection of the nuclear polarization that is formed during the time between the short laser pulse and the RF-detection pulse, one can separate the contribution to the polarization formed in geminate recombination of radicals from the polarization formed in radical reactions, monitor the reaction kinetics, and study the nuclear paramagnetic relaxation of the radicals.28-30 The more conventional cw-CIDNP technique based on cw-irradiation can be used to monitor only processes on the millisecond time scale when it is combined with mechanical shutter.31 It has been shown that detection of shortlived paramagnetic particles by using TR-CIDNP provides detailed kinetic and structural information about radical intermediates. In our previous study, the technique was applied to reactions of the amino acids tyrosine,32 tryptophan,33 histidine,34 methionine,35 and the purine nuclear base guanosine monophosphate (GMP)36 in aqueous solution over a wide pH range. In particular, for the reactions of small peptides,37-39 and proteins40,41 containing two types of reactive amino acid residues, we found that information about the direction, reversibility and

Reduction of Guanosine Radical by Amino Acids

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CHART 2

the rate constants of bidirectional reductive electron transfer (RET) between one of the residues and the radical counterpart can be obtained by time-resolved CIDNP. Armed with this knowledge, we decided to apply this method to gaining understanding of the repair mechanism by a systematic investigation of the phochemical reactions of guanosine radicals at variable pH. For this purpose we extended our CIDNP study to a model system containing three components: the substrate (GMP) together with the photooxidative dye 2,2′-dipyridyl and the amino acid tyrosine acting as reductant of the GMP radical. Our investigation comprises kinetic measurements on the reversible photocycle that includes both the oxidation and reduction of the RNA base GMP under pulsed UV irradiation. The aim of our study is to explore the influence of pH on the mechanism of radical reduction, to determine the structure of participating intermediates, and to get detailed information about their reactivity in aqueous solution in the pH range from 1.3 to 13.3. The present paper describes the general approach for the analysis of the CIDNP kinetics in presence of intermolecular electron-transfer reaction between GMP and tyrosine. The scheme describing the structure of reactants, the mechanism of formation of the four GMP radicals from dicationic G++• to the anionic form G(-2H)-• together with the pKa values of the radicals and the corresponding ground state molecules, and the species participating in the RET reaction at different pH are shown in Chart 1, where GMP radicals at equilibrium conditions (a) and GMP radicals right after the quenching reaction in CIDNP experiment (b) are shown, together with the structure of Tyr molecules (c). Tyrosine molecules with different protonation states of the carboxyl group (pKa 2.2) are subsumed as TyrOH, because the protonation state of the carboxyl group does not play any role in the electron-transfer photoreaction. The protons of GMP that are sequentially released upon increasing pH are marked by an increasing number of apostrophes. GMP, Tyr are used in the text as general abbreviations. When the exact protonation state is mentioned, the notations from the Chart 1 such as TyrOH, TyrO-, GH+, G, and G(H)- are used. Experimental Section A detailed description of our TR-CIDNP setup was given previously.33 The samples, sealed in a standard 5 mm NMR Pyrex ampule, were irradiated by a COMPEX Lambda Physik XeCl excimer laser (wavelength 308 nm, pulse energy up to 150 mJ) in the probe of a 200 MHz Bruker DPX-200 NMR spectrometer. Light was guided to the sample using an optical system containing a quartz lens, a prism, and a cylindrical lightguide (5 mm diameter). The TR-CIDNP experiments were carried out applying the following pulse sequence: radiofrequency (RF) saturation pulses - laser pulse - evolution time

Figure 1. Aromatic region of 1H CIDNP spectra obtained in photoreactions of 2,2′-dipyridyl with 2.3 × 10-2 M guanosine-5′-monophosphate (left), 3.5 × 10-2 M N-acetyl tyrosine (right), and 2.0 × 10-2 M guanosine-5′-monophosphate and 1.0 × 10-2 M N-acetyl tyrosine (middle), pH 7.5. Upper spectra were taken immediately after the laser pulse; lower spectra, with a delay of 100 µs after the laser pulse.

τ - RF detection pulse - free induction decay (FID) as it is shown in Chart 2. Since the background signals in the spectrum originating from Boltzmann polarization are suppressed, only resonances from the polarized products formed during the variable delay τ appear in the 1H CIDNP spectra. In all kinetic measurements, an RF pulse with duration of 1 µs was used for detection. To account for the finite length of the RF-pulse, the time scale was shifted by one-half of the RF-pulse duration; that is, in all plots in Figures 2-7, the time of 0.5 µs corresponds to τ ) 0 µs, 1.5 µs stands for τ ) 1 µs, etc. (cf. Chart 2). Each of the kinetic data sets recorded for the mixture of guanosine-5′-monophosphate and N-acetyl tyrosine was recorded using 16 samples: each sample was used to acquire four scans for each of the 13 τ-values, so that every data point in Figures 2-7 represents 64 signal accumulations. Only 32 accumulations were used to acquire the kinetic data sets for the solutions containing the amino acid and the nucleotide separately. The sample depletion after the 52 light flashes was never more than 25%. To avoid any distortion of the kinetics from this source, ascending and descending order of the time delays was used alternately. Guanosine-5′-monophosphate, N-acetyl-L-tyrosine, 2,2′-dipyridyl, solvent D2O, DCl, and NaOD were used as received from Sigma-Aldrich. All 1H NMR measurements were performed in D2O. The pH of the NMR samples was adjusted by addition of DCl or NaOD. No correction was made for the deuterium isotope effect on the pH.42 In aqueous solution, the identities of the reactive species are pH-dependent (cf. Chart 1). Depending on the pH value of the solution, the guanine nucleotide can exist in three forms: positively charged (GH+, pKa ) 2.4), neutral (G, pKa ) 9.4),

7442 J. Phys. Chem. B, Vol. 111, No. 25, 2007

Figure 2. CIDNP kinetics for the proton H8 of guanosine-5′monophosphate obtained at pH 7.5 in the photoreactions of 2,2′dipyridyl with 1.7 × 10-2 M guanosine-5′-monophosphate (b), with 2.2 × 10-2 M guanosine-5′-monophosphate plus 2.5 × 10-3 M N-acetyl tyrosine (/), and with 2.0 × 10-2 M guanosine-5′-monophosphate plus 1.0 × 10-2 M N-acetyl tyrosine (4). Lines, simulations using eqs 1-5 according to the procedure described in the text with the parameters given in Tables 1 and 2.

Figure 3. CIDNP kinetics for the protons H3,5 of N-acetyl tyrosine obtained at pH 7.5 in the photoreactions of 2,2′-dipyridyl with 3.5 × 10-2 M N-acetyl tyrosine (O), with 1.7 × 10-2 M guanosine-5′monophosphate plus 2.0 × 10-2 M N-acetyl tyrosine (9), with 2.0 × 10-2 M guanosine-5′-monophosphate plus 1.0 × 10-2 M N-acetyl tyrosine (4), with 2.1 × 10-2 M guanosine-5′-monophosphate plus 5.0 × 10-3 M N-acetyl tyrosine ([), with 2.2 × 10-2 M guanosine-5′monophosphate plus 2.5 × 10-3 M N-acetyl tyrosine (*). Lines, simulations using eqs 1, 2, 6, and 7 according to the procedure described in the text with the parameters given in Tables 1 and 2.

and negatively charged (G(-H)-).10 Successive protonation of dipyridyl in aqueous solution yields the following species: neutral dipyridyl DP, protonated dipyridyl DPH+ (pKa ) 4.3), and doubly protonated dipyridyl DPH22+ (pKa ) -0.2).43,44 Irradiation of the dye solution leads to the formation of the triplet state of the dye. Triplet dipyridyl also can subsist in either protonated (TDPH+, pKa ) 5.8), or neutral (TDP) form.34 In our previous TR CIDNP and laser flash photolysis study, the influence of the pH on the quenching rate for the reactions of triplet dipyridyl with GMP36 and tyrosine,32 the primary step of radical formation via electron or hydrogen atom transfer, and the mechanism of CIDNP formation for the range of pH considered here were described in detail. In the present study, we chose the pH and the concentrations of GMP and Tyr according to the following considerations: (1) Since both Tyr and GMP compete for the triplet excited dye, the relative intensities of lines in the CIDNP spectra depend on the ratio of

Morozova et al.

Figure 4. CIDNP kinetics for the proton H8 of guanosine-5′monophosphate and H3,5 of N-acetyl tyrosine (insert) obtained at pH 1.3 in the photoreactions of 2,2′-dipyridyl with 6.4 × 10-3 M guanosine5′-monophosphate (b), with 2.5 × 10-3 M N-acetyl tyrosine (O in the insert), with 6.4 × 10-3 M guanosine-5′-monophosphate plus 2.5 × 10-3 M N-acetyl tyrosine (4), and with 6.4 × 10-3 M guanosine-5′monophosphate plus 1.3 × 10-3 M N-acetyl tyrosine (*). Lines, simulations using eqs 1-7 according to the procedure described in the text with the parameters given in Tables 1 and 2.

Figure 5. CIDNP kinetics for the proton H8 of guanosine-5′monophosphate and H3,5 of N-acetyl tyrosine (insert) obtained at pH 2.9 in the photoreactions of 2,2′-dipyridyl with 3.8 × 10-3 M guanosine5′-monophosphate (b), with 2.5 × 10-3 M N-acetyl tyrosine (O in the insert), with 3.8 × 10-3 M guanosine-5′-monophosphate and 2.5 × 10-3 M N-acetyl tyrosine (4), and with 3.8 × 10-3 M guanosine-5′monophosphate and 1.3 × 10-3 M N-acetyl tyrosine (*). Lines, simulations using eqs 1-7 according to the procedure described in the text with the parameters given in Tables 1 and 2.

concentrations of GMP and Tyr. The concentrations were chosen so that the signals for the two reactants of RET (Tyr and GMP) have analyzable intensities in their CIDNP spectra. (2) The highest concentration of Tyr was limited to a value ensuring that the resulting decay time of CIDNP of GMP was not shorter than the time resolution of our setup (1 µs) so that the CIDNP kinetics could be treated quantitatively. (3) The estimated initial radical concentration in our NMR ampule is below 10-4 M; hence, the lower limit for the concentration of Tyr is chosen to be 1 mM in order to be able to neglect the change of Tyr concentration due to the RET reaction and to consider it as a pseudo-first-order reaction. Results and Discussion The reaction that models chemical DNA repair consists of electron transfer from the protein residue tyrosine to the guanosyl radical that represents the oxidized nuclear acid base. Since in the nucleotide only the nucleobase is involved in

Reduction of Guanosine Radical by Amino Acids

Figure 6. CIDNP kinetics for the proton H8 of guanosine-5′monophosphate and H3,5 of N-acetyl tyrosine (insert) obtained at pH 11.3 in the photoreactions of 2,2′-dipyridyl with 4.5 × 10-3 M guanosine-5′-monophosphate (b), with 2.5 × 10-3 M N-acetyl tyrosine (O in the insert), with 4.5 × 10-3 M guanosine-5′-monophosphate plus 2.5 × 10-3 M N-acetyl tyrosine (4), and with 4.5 × 10-3 M guanosine5′-monophosphate plus 1.3 × 10-3 M N-acetyl tyrosine (*). Lines, simulations using eqs 1-7 according to the procedure described in the text with the parameters given in Tables 1 and 2.

Figure 7. CIDNP kinetics for the proton H8 of guanosine-5′monophosphate obtained at pH 13.3 in photoreactions of 2,2′-dipyridyl with 4.5 × 10-3 M guanosine-5′-monophosphate (b), with 4.5 × 10-3 M guanosine-5′-monophosphate plus 1.3 × 10-3 M N-acetyl tyrosine (4), and with 4.5 × 10-3 M guanosine-5′-monophosphate plus 2.5 × 10-3 M N-acetyl tyrosine (*). Lines, simulations using eqs 1-5 according to the procedure described in the text with the parameters given in Tables 1 and 2.

electron-transfer reactions, we chose guanosine monophosphate, because it was already studied by TR-CIDNP in our previous work.32 All of the reactants, the nucleobase, the photosensitizer, and the protein residue can exist in different protonation states. The investigation in the present paper represents the electrontransfer reaction from N-acetyl tyrosine to the radical of guanosine-5′-monophosphate, including the determination of the nature of the reactive species. Radicals were generated photochemically, via electron or hydrogen atom transfer from the nucleotide or the amino acid to the excited triplet dye 2,2′dipyridyl. At every pH value, the CIDNP formed in the photoreactions of DP with GMP, and of DP with Tyr, served as a reference, and was compared to that formed in the photoreaction of DP and GMP/Tyr mixture to reveal the

J. Phys. Chem. B, Vol. 111, No. 25, 2007 7443 presence of reductive electron transfer (RET) between the tyrosine and the guanosyl radical. Since the RET reaction affects the concentration of the involved radicals, it manifests itself in the CIDNP kinetics. Hence, the quantitative analysis of the CIDNP kinetics allowed for the determination of the RET rate constants. The quenching of the triplet dipyridyl by tyrosine proceeds via an electron transfer at acidic (pH < 5) and strongly basic (pH > 10.5) conditions and via a hydrogen transfer in neutral and moderately basic (6 < pH < 9.5) solution.32 The rate constant of the electron transfer is close to the diffusioncontrolled limit kq ) (2÷3.5) × 109 M-1 s-1, whereas the rate constant of the hydrogen transfer is significantly lower: kq ) 7 × 107 M-1 s-1 in nonbuffered aqueous solution.32 The pKa value of TyrOH, together with the mechanism of the GMP radical formation (electron or hydrogen transfer) and the three pKa values of the GMP radical, determines the pairs of species involved in the RET reaction at different pH values. This is summarized in Chart 1. The pKa of the phenolic tyrosine proton is 10.1. Charged guanosine-monophosphate with a pKa of 2.4 quenches triplet DPH+ (pKa 5.8) via electron transfer, resulting in a guanosine dication radical below this pKa. Neutral dipyridyl in its triplet state reacts with neutral G (pKa 9.4) via hydrogen transfer and with G(-H)- via electron transfer; in both reactions, the neutral radical G(-H)• is formed.36 This radical has a pKa of 10.8; however, at higher pH (11.8), this radical is stable on the microsecond time scale of the CIDNP experiment.36 Although the CIDNP kinetics at pH 13.3 was not studied in the previous paper,36 we found out that at a strongly basic pH (above 12.5) the interaction with OH- accelerates deprotonation (vide infra), resulting in the radical G(-2H)-•. Therefore, we extended our present study of the RET to that pH. An open point was whether CIDNP resulting from the cation radical G+• can be observed since the pKa of the cation radical is unknown, and the difference between the pKa values of the corresponding ground state molecule and the radical (2.4 and 3.9, respectively) is too low to ensure the pH condition for the ultimate predomination of G+• radicals in solution. From the values of the rate constants of quenching of triplet dipyridyl by G and by GH+,36 we chose pH 2.9 because the formation of monocationic G+• radicals resulting from quenching predominates over dicationic GH++• (vide infra). Also, at pH 2.9, the deprotonation of the cation radical (pKa 3.9) can be neglected. Thus, the following five pH values were chosen to represent all five possible pairs of reactants studied with respect to the efficiency of the RET reaction: GH++•/TyrOH (pH 1.3), G+•/ TyrOH (pH 2.9), G(-H)•/TyrOH (pH 7.5), G(-H)•/TyrO- (pH 11.3), and G(-2H)-•/ TyrO- (pH 13.3). CIDNP Spectra. Figure 1 shows the aromatic region of the CIDNP spectra obtained at pH 7.5 in the photoreaction of DP with GMP, DP with TyrOH, and DP with both species (left, right, and middle, respectively). The scaling of the three upper spectra was adjusted to have the same amplitude of the GMP H8 signal in the left pair of spectra and of the Tyr H3,5 signal in the right pair of spectra. The corresponding pairs of spectra taken at zero delay and 100 µs have the same scaling. Enhancements are observed for the nonexchangable protons having nonzero hyperfine interaction constants (HFI) in the intermediate radicals, the signs of the CIDNP signals are in accordance with Kaptein’s rule:45 Γ ) µ sgn(∆g) sgn(Α), where µ ) +1 for a triplet precursor and µ ) -1 for a singlet precursor,  ) +1 for polarization of geminate products and  ) -1 for products of radical recombination in the bulk, sgn-

7444 J. Phys. Chem. B, Vol. 111, No. 25, 2007 (∆g) and sgn(Α) are the signs of the difference in g factor and of the HFI constant of the protons under question. In our case, it is enhanced absorption for H3,5, H4 of DP, H2,6 of Tyr, and emission for H8 of GMP and H3,5 of Tyr (enhanced absorption is also observed for the β-CH2-protons of Tyr, but not shown). The CIDNP intensities of the protons belonging to the geminate products, when detected without delay between the laser and the rf-pulses, are usually proportional to the values of the hyperfine coupling (HFC) constants for these protons in the preceding radicals. At all other pH values studied, the CIDNP pattern is qualitatively the same as at neutral pH except for some variation of the chemical shifts of the diamagnetic molecules with pH, which does not play any role for the radical stage of the reactions. The mechanism of CIDNP formation in reversible photochemical reactions has been described in a number of papers27,33,41,46,47 and can be briefly summarized as follows. In the photochemical reaction with the triplet precursor molecule, a triplet spin-correlated radical pair is formed as the result of electron or hydrogen atom transfer. The recombination can only proceed from the singlet state of the pair. At the geminate stage of the reaction, the radical pair undergoes triplet-singlet conversion opening the recombination pathway for the radicals. Accordingly, the probability of in-cage recombination depends on the efficiency of the triplet-singlet transitions driven by magnetic interaction in the pair, namely by the difference in electronic Zeeman frequencies and by electron-nuclear hyperfine interactions. In this way, it depends on the nuclear spin configuration, giving rise to geminate CIDNP. Radicals that escape the geminate recombination carry nuclear polarization opposite in sign to the geminate one because the total polarization is conserved at times that are short compared to paramagnetic nuclear spin-lattice relaxation. The kinetic profile of CIDNP depends on the second-order termination rate (and thus on the initial radical concentration), the paramagnetic nuclear relaxation time, and the efficiency of the reaction of degenerate electron exchange. Termination of the radicals in the bulk leads to the transfer of polarization to the diamagnetic state, which is opposite in sign to the geminate one, and thus gives rise to the so-called CIDNP cancellation effect. In addition nuclear polarization is formed in diffusive collisions of noncorrelated radical pairs (F-pairs) in the bulk coinciding in sign with geminate CIDNP in the case of a triplet precursor. Summing this up, the CIDNP kinetics is determined in total by all of the above-mentioned processes: in reversible photochemical reactions, when initial compounds and products are the same species, fast rising geminate CIDNP is compensated by the slower transfer of polarization from the escaped radicals to the diamagnetic products in bulk reactions. The loss of polarization in the radicals caused by paramagnetic nuclear relaxation makes the cancellation of the geminate CIDNP incomplete. Therefore, the degree of cancellation depends on the nuclear relaxation time with respect to the radical lifetime. An additional CIDNP is created in F-pairs. Details of the CIDNP kinetics at each of the five pH values chosen to detect all five possible pairs of reactants, namely GH++•/TyrOH (pH 1.3), G+•/ TyrOH (pH 2.9), G(-H)•/TyrOH (pH 7.5), G(-H)•/TyrO- (pH 11.3), and G(-2H)-•/TyrO- (pH 13.3), are considered below. CIDNP Kinetics at pH 7.5. At neutral pH, the mechanism of quenching of the excited triplet of DP by both TyrOH and GMP is hydrogen atom transfer from the nuclear base and the amino acid to the dye resulting in the formation of DPH+•, neutral radicals of tyrosine TyrO•, and guanosine G(-H)• as it is shown in Chart 1.

Morozova et al. At pH 7.5, the CIDNP kinetics obtained for solutions containing either GMP (Figure 2) or Tyr (Figure 3) plus the triplet dye are shown by circles. For both cases, the CIDNP signal increases within the first few microseconds due to polarization formation in F-pairs, and then drops to its stationary value, determined by the paramagnetic nuclear relaxation of the corresponding protons. The kinetics are in full agreement with the CIDNP data obtained previously. Degenerate electron exchange is not operative for Tyr nor for GMP in the pairs TyrO•/TyrOH and G(-H)•/G, respectively, whereas hydrogen atom exchange seems to be too slow to compete with the radical termination reactions. The RET reaction occurs disregarding the spin state of reactants and converts GMP radicals in the bulk into ground state molecules. Since escaped GMP radicals carry nuclear polarization that has a sign opposite to the polarization of geminate products, this reaction leads to cancellation of CIDNP manifesting itself in the decay of nuclear polarization of GMP which becomes faster with increasing concentration of the electron donor tyrosine. Concerning tyrosine, RET serves as a source of nonpolarized Tyr radicals, which acquire nuclear polarization later in second-order termination reactions with DP radicals. That is why the stationary CIDNP of Tyr with respect to the geminate one is higher in the presence of GMP. This is illustrated in Figure 1 (lower spectra), as well as in Figures 2 and 3, where the kinetic curves are shown. The increase of tyrosine concentration accelerates the decay of CIDNP of GMP, which under our experimental conditions can be treated as a pseudo-first-order process. For convenient presentation, the CIDNP kinetics of GMP in Figure 2 is scaled to normalized units: here the initial value in the simulation is set to unity as it represents the geminate CIDNP better than the first experimental point obtained immediately after the laser flash with a rf-pulse of 1 µs, since its effective delay corresponds to 0.5 µs. For tyrosine, the variation of the CIDNP with respect to the geminate polarization is determined not only by the RET rate but also by the initial number of tyrosyl with respect to guanosyl radicals. The larger the share of GMP radicals is, the higher the concentration of tyrosine radicals can be generated via RET. The data set at pH 7.5 was obtained at a variety of initial concentrations of GMP and Tyr. The CIDNP kinetic data for Tyr were scaled in the following way: the first point in the kinetics with the lowest concentration of Tyr was taken as unity, the other points were rescaled accordingly. It is seen that, although the concentrations differ by more than a factor of 5, for the resulting CIDNP a change by less than a factor of 2 is observed. For the quantitative data treatment with the aim to obtain the RET rate constant, the CIDNP kinetics was simulated using the model that was developed previously by us for the description of the CIDNP kinetics in the dipeptide tryptophantyrosine where intramolecular electron transfer from the tyrosine residue to the oxidized tryptophanyl moiety occurs.39 This model is based on the approach suggested by Fischer for the description of the CIDNP kinetics30 and includes the solution of a system of coupled differential equations for (i) the radical concentrations, (ii) the CIDNP in the radicals and (iii) the CIDNP in the ground state molecules of the reacting species. The radical concentrations RDP, RGMP, and RTyr are described by the following equations:

dRDP ) - k1RDPRGMP - k2RDPRTyr dt

(1)

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dRGMP ) - k1RDPRGMP - k′eRGMP dt

(2)

dRTyr ) - k1RDPRTyr + k′eRGMP dt

(3)

where k1 and k2 are the second-order termination rate constants of the pairs of radicals of dipyridyl/guanosine and dipyridyl/ tyrosine, respectively, and k′e ) keCTyr describes the RET proceeding with the monomolecular rate constant proportional to the concentration of tyrosine, CTyr. In eqs 1-3, we neglected the small fraction of geminate recombination, which is a very good approximation for tripletborn radical pairs in a solvent with low viscosity,30 and considered the formation of radicals to be instantaneous. The initial concentration of radicals is R0 for DP radicals, RR0 for GMP radicals, and (1 - R)R0 for Tyr radicals with R ) kq(GMP)/ R and [kq(GMP)+kq(Tyr)]. Nuclear polarization in the radicals, PGMP R PTyr, and in the ground state molecules, PGMP andPTyr, is described by the following equations:

PRGMP dPRGMP R ) - k1PGMPRDP - k1βRGMPRDP dt T1(GMP) k′ex(GMP)PRGMP - k′ePRGMP (4) dPGMP ) k1PRGMPRDP + k1βRGMPRDP + k′ex(GMP)PRGMP + dt k′ePRGMP (5) PRTyr dPRTyr ) - k2PRTyrRDP - k2βRTyrRDP dt T1(Tyr) k′ex(Tyr)PRTyr (6) dPTyr ) k2PRTyrRDP + k2βRTyrRDP + k′ex(Tyr)PRTyr dt

(7)

The first term in each equation describes the polarization transfer from radicals to ground state molecules in the termination reaction. The second term refers to polarization formation in radical pairs in the bulk, i.e., F-pairs. Also, in eqs 4 and 6, GMP and Tyr paramagnetic nuclear relaxation with the time T1 is taken into account. The polarization created in F-pairs isproportional to the value of geminate polarization PG with the factor β: β ) γPG/R0. The quantity γ is the ratio of CIDNP created in geminate and in F-pairs and is usually taken to be equal to 3 in the case of a triplet precursor. The terms describing polarization transfer in the degenerate electron exchange reaction with the pseudo-first-order rate constants k′ex(Tyr) and k′ < remark > ex(GMP) are also included in eqs 4-7 using a monomolecular rate constant that is proportional to the concentration of the diamagnetic molecules, Tyr and GMP, respectively. The RET between tyrosine and GMP radicals is also considered as a pseudo-first-order reaction. Used as fitting parameters were ke, R0k1, k1/k2, and the scaling factor. The paramagnetic nuclear relaxation times are known from previous studies. As we demonstrated previously in the similar quenching reaction of DP by a dipeptide with two reactive residues,39 the parameter R can be directly determined from the relative CIDNP intensities in the geminate spectra obtained at different reactant concentrations. The same approach for determination of R we use here because reliable data concerning the quenching rate constant of GMP are not available

TABLE 1: Species Participating in the Reductive Electron Transfer and Rate Constants of the RET Reactions, ke pH

guanosine radical

tyrosine

ke, M-1 s-1

1.3 2.9 7.5 11.3 13.3

GH++• G+• G(-H)• G(-H)• G(-2H)-•

TyrOH

(7.1 ( 3.0) × 108

TyrO-

(6.0 ( 1.0) × 107 (1.6 ( .0.4) × 108 14,49 thus the protonation reaction DP-• + H2O f DPH• + OH- occurs very fast with changing the spin density distribution in one of the partners in the primary radical pair. The changing of the electron spin density distribution and of the g-factor in radical G(-2H)-• as compared to G(-H)• occurs upon deprotonation and may lead to the changing of the CIDNP enhancement factors and of the rate of paramagnetic relaxation. The former reveals itself in the deviation of the parameter γ from the value of 3. And indeed, the best fit was obtained using γ ) 1.6 and a relaxation time T1(GMP) ) 38 µs of the anionic radical G(-2H)-•, almost twice long as that of the G(-H)• radical. In the presence of TyrO-, the CIDNP signal of H8 decreases and the decay rate of the whole kinetics accelerates with increasing concentration of Tyr. Qualitatively, this fact is in accordance with the decrease of the concentration of purine radicals in the solution. However, we failed to describe all the data sets obtained at different concentrations of Tyr by changing ′ only the parameter kex(GMP) ; a noticeable change in the initial second-order termination rate was also required (for the parameters, see figure caption and Table 2). A plausible explanation is the photoionization of tyrosine at this pH resulting in a neutral radical and a solvated electron:50

TyrO- + hV f Tyr• + e-solv The solvated electron can participate in the reduction of the GMP radical. At these conditions, the value of the RET rate constant can be estimated as follows: ke < 6 × 106 M-1 s-1. As it seen in Table 1, where the structures of the five possible pairs reactive species studied in the present work and the rate constants of RET are shown, it is the lowest rate constant for reductive electron transfer among all of the data obtained for reactive pairs of GMP radicals and tyrosine. This result is in agreement with the reported very weak oxidation ability of G(2H)-•: only for the strong reducing agent as N,N,N′,N′tetrametyl-p-phenylendiamine the oxidation by the deprotonated anion radical G(-2H)-• was detected but not for thiolates, ascorbate, or phenolates.10 Conclusion The application of time-resolved CIDNP to the quantitative investigation of the reductive electron transfer between photochemically generated GMP radicals and N-acetyl tyrosine, a system modeling the chemical repair of nucleic acid damage by proteins, revealed that the reaction rate constants are dependent on the protonation state of the reacting species. Successive protonation of the initial compounds (DP and GMP) at changing pH causes a change of the primary photochemical step: starting from electron transfer under strongly basic conditions, it changes to hydrogen transfer at neutral and

J. Phys. Chem. B, Vol. 111, No. 25, 2007 7447 moderately basic pH and back again to electron transfer in acidic solutions with the formation of four different forms of guanosyl radicals: GH++• (pH 1.3), G+• (pH 2.9), G(-H)• (pH 7.5), G(H)• (pH 11.3), and G(-2H)-• (pH 13.3). Detailed kinetic investigations of the reversible photocycle that includes both the oxidation and reduction of the RNA base GMP under pulsed UV irradiation were possible due to strong spin polarization enhancement as it was found in the reaction of the triplet dye DP with both GMP and Tyr in that pH range (1.3-13.3). The photoreaction was carried out at different concentrations of the amino acid at five selected pH values with the aim to determine the rate constant of the reductive electron transfer to all four forms of GMP radicals from tyrosine, with the neutral and the anionic form of the phenolic ring depending on the pH (TyrOH and TyrO-, respectively). Unambiguous identification of the different elusive GMP radicals was possible because of the high sensitivity of CIDNP to distinguish them through their ability or disability of participating in the degenerate electron hopping reaction with diamagnetic GMP molecules in the ground state. This is not feasible by transient optical absorption, because this reaction is not accompanied by a change of the optical density. Accordingly, in the kinetic study of the interaction of deprotonated purine radicals with amino acids and peptides,48 the contribution of oxidation of tyrosine by the cation dGMP radical and the neutral radical could not be distinguished. Our data however show that the different forms of GMP radicals are characterized by their remarkably different oxidative property with the rate constant changing from (7.1 ( 3.0) × 108 M-1 s-1 as obtained for G+• and GH++• to a value less than 6.0 × 106 M-1s-1 for G(-2H)-•. Another drawback of optical detection is the spectral overlap with the absorption spectrum of the tyrosyl radical, making the kinetic analysis rather tangled.48 Last but not least, the transient radicals of the purine base have broad and weak lines in the optical absorption spectrum. By contrast, from our study using photochemical generation of radicals at selected pH values and high-resolution NMR detection of the CIDNP kinetics the rate constants of electron transfer were obtained for all five possible combinations of GMP radicals and tyrosine in the pH range from 1.3 to 13.3. Therefore, the values of rate constants obtained by use of the monomeric nuclear base GMP and the amino acid tyrosine can serve as reference for further understanding the electron transfer in complex DNA-protein systems and the driving forces for repair of guanyl radicals in DNA. The results are summarized in Table 1. From this detailed study, it becomes clear that the most efficient reductive electron transfer occurs in acid solution between the protonated phenol ring and the cationic and dicationic radicals of GMP with ke ) (7.1 ( 3.0) × 108 M-1 s-1. The electron transfer from the anionic phenol ring to the neutral GMP radical proceeds with a rate constant that is by a factor of 4-5 smaller than that of the reaction in acidic solutions. In neutral solution, the reductive electron transfer is less efficient by 1 order of magnitude than in acid. In strongly basic solution (pH 13.3), the direct photoionization of tyrosine leading to formation of solvated electrons precludes the quantitative study of interaction between the anionic radical and the amino acid, therefore only the upper limit of the rate constant (ke < 6 × 106 M-1 s-1) was obtained. Currently, we are investigating the nucleotide polarization in the presence of reductive electron transfer from tryptophan using the same approach. First results show, that depending on the pH of aqueous solution, the reaction proceeds with an efficiency which in acids is comparable to that for tyrosine but much higher in neutral and basic conditions. The other amino

7448 J. Phys. Chem. B, Vol. 111, No. 25, 2007 acids (methionine, cysteine, and histidine) that were found involved into chemical DNA repair20 also exhibit strong CIDNP effects. We envision that the TR CIDNP technique has a great potential for protein research and for exploration of nucleotideprotein interaction and the electron-transfer reaction responsible for the reductive protection of RNA and DNA from damage. Acknowledgment. The financial support of INTAS (Project No. 05-100000-8070) and RFBR (Projects 05-03-32370 and 0603-32993), RAS (Grant 5.2.3), the Russian President’s program of support of the leading scientific schools (Grant NSch4821.2006.3), and Presidium of SBRAS (Project No. 60) is gratefully acknowledged. O.B.M. is indebted to the Young Scientists Grant Program of the President of Russian Federation (Project No. MK-2547.2005.3) for financial support. A.V.Y. acknowledges support by the EU FP6 program of Marie Curie action (Project IIF 2208) for the fellowship at FU Berlin. References and Notes (1) Cai, Z.; Gu, Z.; Sevilla, M. D. J. Phys. Chem. B 2001, 105, 60316041. (2) Melvin, T.; Botchway, S. W.; Parker, A. W.; O’Neill, P. J. Am. Chem. Soc. 1996, 118 (42), 10031-10036. (3) Melvin, T.; Cunniffe, S. M.; O’Neill, P.; Parker, A. W.; RoldanArjona, T. Nucl. Acids Res. 1998, 26 (21), 4935-4942. (4) Douki, T.; Angelov, D.; Cadet, J. J. Am. Chem. Soc. 2001, 123 (46), 11360-11366. (5) Burrows, C. J.; Muller, J. G. Chem. ReV. 1998, 98 (3), 11091152. (6) Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119 (3), 617-618. (7) Milligan, J. R.; Aguilera, J. A.; Hoang, O.; Ly, A.; Tran, N. Q.; Ward, J. F. J. Am. Chem. Soc. 2004, 126 (6), 1682-1687. (8) Weatherly, S. C.; Yang, I. V.; Thorp, H. H. J. Am. Chem. Soc. 2001, 123 (6), 1236-1237. (9) Weatherly, S. C.; Yang, I. V.; Armistead, P. A.; Thorp, H. H. J. Phys. Chem. B 2003, 107 (1), 372-378. (10) Steenken, S. Chem. ReV. 1989, 89 (3), 503-20. (11) Showalter, A. K.; Lamarche, B. J.; Bakhtina, M.; Su, M.-I.; Tang, K.-H.; Tsai, M.-D. Chem. ReV. 2006, 106 (2), 340-360. (12) Rossi, M. L.; Purohit, V.; Brandt, P. D.; Bambara, R. A. Chem. ReV. 2006, 106 (2), 453-473. (13) Samaranayake, M.; Bujnicki, J. M.; Carpenter, M.; Bhagwat, A. S. Chem. ReV. 2006, 106 (2), 700-719. (14) O’Brien, P. J. Chem. ReV. 2006, 106 (2), 720-752. (15) Kimura, S.; Sakaguchi, K. Chem. ReV. 2006, 106 (2), 753-766. (16) Ly, A.; Tran, N. Q.; Ward, J. F.; Milligan, J. R. Biochemistry 2004, 43 (28), 9098-9104. (17) Ly, A.; Bandong, S. L.; Tran, N. Q.; Sullivan, K. J.; Milligan, J. R. J. Phys. Chem. B 2005, 109 (27), 13368-13374. (18) Milligan, J. R.; Tran, N. Q.; Ly, A.; Ward, J. F. Biochemistry 2004, 43 (17), 5102-5108. (19) Ly, A.; Tran, N. Q.; Silliavan, K.; Bandong, S. L.; Ward, J. F.; Milligan, J. R. Org. Biomol. Chem. 2005, 3, 917-923.

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