Proton-Coupled Electron Transfer in the Oxidation of Guanosine

Subscriber access provided by University of Sunderland

C: Surfaces, Interfaces, Porous Materials, and Catalysis

Proton-Coupled Electron Transfer in the Oxidation of Guanosine Monophosphate by Ru(bpy) 33+

Christine Fecenko Murphy, Prateek Dongare, Stephanie Weatherly, Christopher J Gagliardi, H. Holden Thorp, and Thomas J. Meyer J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b08382 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 13, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Proton-Coupled Electron Transfer in the Oxidation of Guanosine Monophosphate by Ru(bpy)33+ Christine Fecenko Murphy#$, Prateek Dongare†$, Stephanie C. Weatherly†, Christopher J. Gagliardi†, H. Holden Thorp§, Thomas J. Meyer†* Department of Chemistry, the University of North Carolina-Chapel Hill, Chapel Hill, NC 27599-3290 [email protected] Supporting Information The supplemental contains detailed methods for electrochemical analysis and digital simulation. ABSTRACT: Oxidation of guanine by the outer-sphere metal complex oxidant Ru(bpy)33+ (bpy is 2,2’bipyridine) has been explored in deoxyguanosine-5´-monophosphate (GMPH) with the added buffers succinic acid/succinate Hsuc/suc‒● (pKa = 5.6), H2PO4-/HPO42- (pKa = 7.2), and tris [(HOCH2)3CNH3+/(HOCH2)3CNH2] (pKa = 8.1) at 232 C. Over an extended range of buffer concentrations and ratios, there is clear evidence for the mechanistic importance of pathways involving concerted electron-proton transfer (EPT). In this pathway, proton transfer to the base form of the buffer occurs in concert with electron transfer to the oxidant, Ru(bpy)33+.

Oxidation of guanine is a key event in the oxidation of the biological polymers DNA and RNA.1-3 Given the redox potentials for the key constituent couples, 1.29 V vs. NHE for the guanine+/0 couple, 1.42 for the adenine+/0 couple, 1.6 V for the cytosine +/0 couple, and 1.7 V for the thymine+/0 couple

_ 4-6

guanine is a redox trap7 and a key site for oxidative damage.8-11 External factors _ ionizing radiation with UV light, chemical oxidation,6 and photoexcitation

_

have all been shown to occur at guanine with the

appearance of multiple products.12-13 Perturbations in the redox background of cells can lead to radicals and peroxides and to cellular damage of DNA by base lesions or by oxidation of DNA strands. Cell damage at this level can be repaired by photo-reactivation with mediation by the enzyme DNA photolyase. Unrepaired lesions can lead to a variety neurodegenerative disorders with oxidation of guanine a key event in the oxidation of both DNA and RNA.1-3 As noted above, in these structures guanine is the most vulnerable site for oxidative damage.9-11

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a)

Page 2 of 16

(b)

3+

O N

HN H 2N

N

N

N

N

O

N

Ru

N

O HO

OH OH P O

N

E0 = 1.25 V vs NHE

E0 = 1.29 V vs NHE pKa = 9.3

(c)

N

O OH

HO

NH2

E0 = 1.42 V vs NHE pKa = 12

Figure 1. Structures of (a) deoxyguanosine-5´-monophosphate (GMP-H) (b) Ru(bpy) 33+ (c), and tyrosine (Tyr). The microscopic details by which DNA is oxidized are not well understood including an absence of mechanistic information about the fate of the N1 proton (Figure 1a) and its role in structure and reactivity. 10, 14-22

The pKa value of the latter is 9.2, decreases to 3.9 in the protonated radical (GMPH +•), to 3.9 in

the free nucleoside, and to 4.5 in double-stranded DNA.6, 23 Following oxidation in neutral solution, the deprotonated radical is observed as a transient but it is not clear at which site and when, during the reaction, that PT occurs. There are suggestions that PT occurs from the N1 proton in DNA to the hydrogen-bonded nitrogen on cytosine15, 24-25 but there are also reports that cytosine plays no role in long range ET in DNA. There are conflicting reports on the low activation energy for transfer of the proton to cytosine.19 We report here the results of a study on the electron transfer oxidation of guanine in the presence of a series of proton acceptor bases.8, 26-27 The reactions were studied under conditions where the added bases play a critical role by inducing concerted electron-proton transfer (EPT).28-31 Kinetic analysis of the data with an analysis of kinetic isotope effects (KIE) in H2O/D2O mixtures, have provided detailed insight into the role that the bases play in the oxidative reactivity of guanine.32

RESULTS


Page 3 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In the electrochemical analysis, the kinetically stable oxidant Ru(bpy)33+, with E0’ = 1.25 V vs NHE22 for the Ru(bpy)33+/2+ couple, was used to explore mechanistic details in the outer sphere oxidation of deoxyguanosine-5’-monophosphate (GMPH, Figure 1a). The Ru(bpy)33+/2+ couple undergoes rapid electron transfer with a self-exchange rate constant near the diffusion-controlled limit.8,

33

In the

electrochemical experiments reported here, a well-established procedure, based on cyclic voltammetry, was used to investigate the oxidation of GMPH by Ru(bpy)33+ following oxidation of Ru(bpy)32+ at planar indium tin dioxide electrodes.8, were

used36

26-27, 34-36,37

Analysis of voltammetric wave forms by digital simulation

to evaluate kinetic transfer rate constants for the oxidation of GMPH by Ru(bpy)33+ with the

added buffers succinic acid/succinate (Hsuc/suc‒; 5.6), H2PO4‒/HPO42‒ (pKa = 7.2), and tris [(HOCH2)3CNH3+/(HOCH2)3CNH2] (pKa = 8.1) phosphate in 0.8 M NaCl at 232 C.34 The electrochemical results reported here were consistent with the results obtained earlier by stopped-flow mixing.38 Evaluation of the data was based on Scheme 1, which was of the same form used to analyze data for the oxidation of tyrosine and cysteine.17-18 It incorporates proton coupled electron transfer (PCET) with baseinvolved pathways playing major role with added buffers. In the mechanism, PCET arises from two sources. In one, stepwise loss of a proton from GMPH is followed by oxidation of the anion by the oxidant in a PT-ET mechanism.20,21,34 In the second, proton transfer occurs to a base which is part of a multi-site electron proton transfer (MS-EPT) pathway39 with electron transfer from GMPH to the oxidant is integrated with proton loss to an added base.32 As with tyrosine and cysteine, the magnitudes of the current enhancements in the oxidation of GMPH varied systematically with the concentrations of GMPH, HPO42‒, and the oxidant, consistent with Scheme 1. Over an extensive range of buffer concentrations, the kinetic data were consistent with the mechanism in Scheme 1, analogous to related mechanisms for the oxidation of tyrosine by Ru(bpy)33+ and of cysteine by Os(bpy)33+.21-22, 38 Representative cyclic voltammograms with H2PO4‒/HPO42‒ as the buffer base pair are shown in Figure 2. The figure shows single CV scans of 20 μM in Ru(bpy)33+ and 0.1 mM GMPH solutions as a function of pH from pH 6.2 to 8.0 in 50 mM buffers at 232 C. The data clearly show the existence of a buffer base effect with limiting currents reached at high concentrations of added base. Under these conditions, GMPH is singly oxidized followed by dimerization to give the final dimeric GMPH product. Additional data are presented in the SI which provide additional experimental details including digital simulations and cyclic voltammograms with GMPH and Ru(bpy3)3+ in the succinate, phosphate, and with the Tris buffer.



Scheme 1. Mechanistic pathways for the oxidation of GMPH by Ru(bpy)33+ (RuIII) with H2PO4-/

HPO42- as the added buffer (B) in 0.8 M NaCl at 232 C.


Page 4 of 16

Page 5 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Experimental cyclic voltammograms (solid traces) in solutions 0.1 mM in GMPH and 20 µM in Ru(bpy)32+ showing simulated fits (dotted features) in pH regions where both phosphate pathways in Scheme 1 contribute mechanistically - 50 mM phosphate buffer, 0.8 M NaCl at 232 C and (A) pH 6.2, (B) pH 7.2, (C) and at the limit of PT-ET involvement at pH 8.2. (D) Overlay of CVs in all pH ranges

Based on the CV data, addition of the buffers, H2PO4-/HPO42- (pKa = 7.2), succinic acid/succinate (Hsuc/suc‒; 5.6), and tris [(HOCH2)3CNH3+/(HOCH2)3CNH2]; 8.1), all result in catalysis. Similar rate laws and buffer effects were observed for tyrosine21-22 and cysteine.38 The kinetic scheme is illustrated with the buffer H2PO4-/HPO42- with Scheme 1 and described by the rate constant expressions in eqs 1-3. In the mechanism there are two origins for the base effect, prior deprotonation of GMPH followed by oxidation of the anion, and PCET with sequential ET and PT from GMPH to the oxidant and buffer base, respectively.21-22 In the absence of an added buffer, the rate law is given by eq 1b with the reaction occurring by outer sphere electron transfer with kobs = kET. 𝐾𝐴[𝐺𝑀𝑃𝐻][𝐻𝑃𝑂24 ― ] 𝑘1𝑘2 𝑑[𝑅𝑢(𝐼𝐼𝐼)] ′𝐴𝑘𝑟𝑒𝑑 + =2 [𝑅𝑢(𝐼𝐼𝐼)] 𝐾 𝑑𝑡 1 + 𝐾𝐴[𝐻𝑃𝑂24 ― ] 𝑘 ―1[𝐻𝑃𝑂4― ] + 𝑘2[𝑅𝑢(𝐼𝐼𝐼)

[

]

(

)

𝑑[𝑅𝑢(𝐼𝐼𝐼)] = 𝑘𝑜𝑏𝑠[𝑅𝑢(𝐼𝐼𝐼)][𝐺𝑀𝑃𝐻] 𝑑𝑡

(

𝑘𝑜𝑏𝑠 = 𝐾′𝐴𝑘𝑟𝑒𝑑 +

𝑘1𝑘2

(1𝑎)

(1b)

)[

𝑘 ―1[𝐻𝑃𝑂4― ] + 𝑘2[𝑅𝑢(𝐼𝐼𝐼)

𝐻𝑃𝑂24 ― ]

(1c)

In the kinetic limit, k-1 >> k2, only the EPT pathway in Scheme 1 is significant. In this limit, the expression for kobs simplifies to Eq 2. 𝑘obs = 𝐾𝐴𝐾′𝐴𝑘red[HPO24 ― ]

(2)

In the limit, k-1 80 mM), saturation kinetics in added base were observed over the entire pH range. Under these conditions, the mechanism is rate-limited by the formation of an H-bonded adduct GMP-H---B dominating reactivity. A plot of

𝑘𝑜𝑏𝑠 [𝐺𝑀𝑃]

vs. HPO42- at pH 7.0 shown in Figure 3a.

At moderate concentrations of acid with [H2PO4-]/[HPO42-] > 10, the EPT pathway in Scheme 1 dominates. Under these conditions, kobs is given by eq 4a. GMPH exists predominantly as the H-bonded adduct (GMP-H--B) with B the buffer base, HPO42-. The overall reaction is dominated by EPT within the association complex between Ru(bpy)33+ and GMP-H--B, Ru(bpy)33+,GMPH--B

𝒌𝒓𝒆𝒅

Ru(bpy)32+,GMP●--

H-B. Reactions run in phosphate buffer ratios < 1:5 (base:acid) show a significant and increased dependence on the oxidation potential of the metal mediator as exhibited in Figure 3a. In this limit, the rate law simplifies for Eq 4a and the EPT reactivity constant (KAKA’kred) can be calculated directly from simulation of cyclic voltammograms. 𝑘𝑜𝑏𝑠 [𝐺𝑀𝑃]

= 𝐾𝐴𝐾′𝐴𝑘𝑟𝑒𝑑[𝐻𝑃𝑂24 ― ]

(4a)

Similarly, at low buffer ratios, with [HPO4-]/[HPO42-] < 10, the pathway labeled PT-ET dominates reactivity. In the limit, k2[Ru(bpy)33+] >> k-1[H2PO4-], the general rate law in eq 4a reduces to eq 4b with kobs independent of [Ru(bpy)33+] as shown in Fig 3 Saturation kinetics are observed at high concentrations of [HPO42-] consistent with a simplified form of the rate law, reported in eq 4b. Under these conditions, k1 = 5.0±0.3×10 s-1, with k1 the rate constant for proton transfer within the association complex. This kinetic limit is illustrated in figure 4b where the concentration of metal complex is increased with no impact on reaction rate. Digital simulations using a proton transfer mechanism use the pre-established pKa values for reactants and result in a k-1 value of 5.0x106M-1s-1. The influence of the acid component of the phosphate buffer was also investigated by simulation of voltammograms under conditions where [HPO42-] was held constant, with variations in [H2PO4-] between 0.002M-1 – 48M-1 (fig 3b), with a slope of 1.9x107 and an intercept of 5.1x106. These values represent the presence of two kinetically distinct ranges, the acid dependent PT pathway (the slope) and the acid independent MS-EPT pathway (the intercept). The intercept value for the reactivity constant is in good agreement with values determined from digital simulation of cyclic voltammograms. 𝑘𝑜𝑏𝑠 [𝐺𝑀𝑃]

= 𝐾𝐴𝑘1[𝐻𝑃𝑂24 ― ]

(4b)


Page 7 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a)

(b)

Figure 3. Plot of (a) kobs/[GMPH] vs [HPO42‒] and its reciprocal (inset) illustrating saturation kinetics over an extended range of concentrations for HPO42‒ in 0.1 mM GMPH and (b) kobs vs [H2PO4‒]-1 illustrating two distinct kinetic regimes one dependent on the presence of H2PO4‒ (the proton transfer pathway) and one independent of H2PO4- (the EPT pathway) in 0.1 mM GMPH with 20 μM Ru(bpy)33+ at 23±2ºC.

(a)

(b)

Figure 4. A) A plot of kobs/[GMPH] vs [Ru(bpy)33+] illustrating the onset of saturation kinetics for Ru(bpy)33+ at high concentrations of Ru(bpy)33+: at pH 6.2 in a 0.5 M phosphate buffer, at 232 C, 0.1 mM in GMPH. B) A plot of kobs/[GMPH] vs [Ru(bpy)33+] illustrating the lack of metal dependence at



increased metal concentrations a pH 8.0 of 0.5 M phosphate buffer, at 232 C, 0.1 mM in GMPH and 2.0x10-5M dGMP The rate constant, k2 = 1.1(±0.2)×107 M-1s-1, was determined by digital simulation of fig 2C, acquired in 0.5 M phosphate buffer (pH 8.5at 232 C), assuming that GMP● was generated and dominates reactivity at higher pH. PCET Kinetic Isotope Effects. The oxidation of GMPH by Ru(bpy)33+ was also investigated in D2O to explore the role of H/D kinetic isotope effects on the PCET pathways. A representative data set used to extract the kobs (k) in H2O or D2O phosphate buffer is provided in fig 5. Because of overlapping contributions from both the MS-EPT and PT-ET pathways over a wide range of buffer concentrations and ratios, the isotope effects were investigated under limiting conditions where one of the pathways dominates, at pH 6.0 for MS-EPT with KAKA’kred = 3.3(±0.4)×106 M-2s-1, and at pH 8.2, where PT-ET dominates, with k2 = 1.1×107 M-1 s-1 in phosphate buffered solution. Under the same buffer conditions, but in D2O as solvent, KAKA’kred = 1.3(±0.2)×106 s-1 and k1 = 8.4(±0.3)×106 M-1 s-1. Based on the data, the isotope effects under the two sets of conditions were relatively small with k(H2O)/k(D2O) = 2.4±0.4 at pH = 6.0 with MS EPT dominating and k(H2O)/k(D2O) = 1.3±0.2 at pH = 8.2 with PT-ET dominating. Related kinetic isotope effects have been obtained for the PCET oxidation of tyrosine by Os(bpy)33+ in phosphate buffers. For the MS-EPT pathway the rate constant ratio was, k(H2O)/k(D2O) = 2.1±0.6. With PT-ET dominating, k(H2O)/k(D2O) = 1.2±0.4 has been reported.21-22


Page 8 of 16

Page 9 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5: Cyclic voltammograms of solutions of GMPH (0.1 mM) and Ru(bpy)32+ (20 μM) in phosphate buffer (red) and deuterated phosphate buffer (blue) solutions at pH 6.0, 0.8 M NaCl at 232 C.

EPT Oxidation of GMPH. Buffer Base Catalysis: The mechanism for oxidation of GMPH by Ru(bpy)32+ was further investigated by varying the buffer base from succinate (pKa = 5.6 ) to tris (pKa =8.1) (Fig S4). Catalytic currents were responsive to base strength, showing a large increase in peak current as the base strength increased from succinate to tris. Digital simulation of cyclic voltammograms, 0.1 mM in GMPH with 20 μM Ru(bpy)33+ in 0.5 M buffer (10:1 Base:Acid) showed a ~10 fold increase in rate from succinate to tris (see Table 1). Table-1. List of EPT reactivity constants for MS-EPT oxidation of GMPH, tyrosine (0.1 mM), and cysteine (0.1 mM) by Ru(bpy)33+ (20 μM) in 0.5 M buffer at 23±2 0C. Values derived from simulation of cyclic voltammograms of GMP under stated conditions. Buffer Base KAKA’kred, M-2s-1 KAKA’kred, M-2s-1 KAKA’kred M-2s-1 (GMPH)[a]

(Tyrosine)[b]

(Cysteine)[b]

Succinate (H+Suc/Suc−)

1.1×106

8.5x105

1.5x106

Phosphate (H2PO4-/HPO42-)

3.3×106

1.2x106

4.3x106

Tris

4.8×106

2.1x106

-

[(HOCH2)3CNH3+/(HOCH2)3CNH2] asee

previous section for details bFrom ref 21, 38, 40

DICUSSION Rate and equilibrium constants for PCET oxidation of GMPH by Ru(bpy)33+ with H2PO4‒/HPO42‒ as the added buffer are summarized in Table 2. Comparable data for the Ru(bpy)33+ oxidation of tyrosine21, 40 and Os(bpy)33+ oxidation of cysteine38 are shown as a comparison. Table 2. Parameters for EPT oxidation of GMPH, TyrOH by Ru(bpy)33+ with H2PO4‒/HPO42‒ as the added buffer at 23±0.2°C. Values are also reported for CySH with Os(bpy)33+ as oxidant under the same conditions. Parameter KA, M-1

k1/k-1

GMPH[a] 1.1x101

TyrOH[b] 3.0x101

CySH[c] 5.0x101

9.8x10-3

4.2x10-5

7.5x10-2



k2 (M-1 s-1) k(H2O)/k(D2O)[c] kET, M-1s-1

Page 10 of 16

1.1×107

1.7×107

1.7x107

2.40.4

2.10.6

1.52

3.6×102

1.7x102

3.5x102

aAverage

rate constants were acquired by voltammetry in phosphate buffers from 0.05 – 1.3 M at 23±2 ºC reported with the error as the standard deviation. bFrom ref 21 c from ref 38 and 40; dsee text for details.

The similarity in mechanism between the two is striking, as is the shared use of PCET pathways that dominate reactivity at high concentrations of added buffers. As shown by the data, even at relatively moderate concentrations of added buffers, over a range in buffer pKa values from 5.6 to 8.1, reactivity is dominated by PCET with prior loss of a proton and oxidation of GMP− by Ru(bpy)33+ by PT-ET, eq 5, 6, or oxidation of GMPH---B by MS-EPT, Scheme 2.

RuII + GMPH

RuIII + GMPH GMPH

(5)

GMP + H+

(6)

The comparison of rate constants in Table 2 provides clear kinetic evidence for significant rate enhancements for EPT in the oxidation of both GMPH and TyrOH when compared to electron transfer. Outer-sphere electron transfer for GMPH occurs with kET = 3.6×102 M-1 s-1 (Table 2). The origin of the PCET effect arises from the redox potentials for the 1-electron couples with Eo’ = 1.34 V vs. NHE for the GMPH/GMPH+● couple and 1.05 V for the GMP−/GMP● couple at neutral pH. Oxidation of the association complex, GMP-H--HPO42-, by Ru(bpy)33+ by MS-EPT occurs with KAKA’kEPTred = 3.3×106 M2s-1,

Table 1, enhanced by a factor of ~104 compared to oxidation of GMPH, Scheme 2. For prior proton

dissociation from GMP-H---HPO42‒ to give GMP−, followed by its outer sphere oxidation by Ru(bpy)33+, the rate constant for oxidation of GMP− was 2.1×109 s-1. Experiments, under conditions where H/D kinetic isotope effects were measured, gave k(H2O)/k(D2O) = 2.4±0.4 at pH = 6.0 with MS EPT dominating and at pH = 8 with k(H2O)/k(D2O) = 1.3±0.2 with PT-ET dominating. The values are relatively small but consistent with H/D isotope effects observed in other PCET reactions.41-44 For example, transient absorption measurements on GMPH oxidation by purine radicals show that the reactions occur with kinetic isotope effects of 1.5-2.20, 45

46

The small KIE effects

for proton transfer suggest that the extent of PT in both reactions is relatively small compared to the overall distance for proton transfer in both proton transfer reactions.47-50 Scheme 2. Illustration of proton transfer in the oxidation of GMPH by MS-EPT with the added base, B.


Page 11 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


e3+ N N N

N

Ru

III

N

N

,

N

N

N

N

2+

B

O

H

N N

NH2

B O

N

N

RuII

N

N

,

N

N

H N

N

NH2

Free Energy Dependence. The availability of data for added bases for the MS-EPT pathway provides an additional experimental probe into the microscopic details of the integrated, electron-proton pathway. As shown in Scheme 2, in this pathway, electron transfer occurs from the GMPH---B base pair to Ru(bpy)33+. From Marcus-Hush theory for outer sphere electron transfer, the barrier to electron transfer is given by eq 5.22, 52 The free energy of activation for electron transfer, ∆G*, is given by eq 7, ∆Go is the driving force for the reaction and λ is the sum of the reorganizational energies for both intramolecular structural changes, λi, and the medium, λo. A slope of 0.5 in the variation of RT ln kred with -∆G in eV is predicted by the classical Marcus-Hush expression for electron transfer with ∆Go’ < λ.53-55 2

∆𝐺 ∗ = (𝜆 + ∆𝐺0′) 4𝜆 ≈ 𝜆 4 + ∆𝐺0′ 2 (𝜆 ≫ ∆𝐺0 ) ′

(7)

A plot of RTln(kEPT) vs. ΔGEPTo' is shown in Figure 6 with ΔGEPTo' varied with Ru(bpy)33+ as the common oxidant for the series of bases in Table 1. Values of ΔGEPTo' were calculated by using eqs 8 and S12. The data show that there is a variation in rate constant with the base strength of the acceptor bases consistent with an important role for proton transfer in the overall reaction that is independent of the proton acceptor base over a pKa range from 5.6 to 8.1. There are insufficient data to discern a detailed role for the proton transfer step in the overall reaction in Figure 6A. A full analysis of oxidants and bases is reported in Figure 6B. However, based on the linear correlation that does exist, the variations in driving force with added base over the pKa range 5.6 to 8.1 are consistent with d(lnk(obs))/d(dG0) = 1/(50) with parallel contributions from both electron and proton transfer. There was no evidence in the data for contributions from vibrational levels above v = 0 in the H-B+ proton acceptor mode in the proton transfer step. 22,56,57-58

0 ′ +• ― E ′ 3 + 2 + ― 59 mV(p𝐾a ∆𝐺0𝐸𝑃𝑇 = E0GMPH HB + ― p𝐾aGMPH +•) Ru


(8)


(b)

Figure 6. (a) Variation of RTlnkEPT with the base strength, -RTlnKEPT, for succinate, HPO4-, and tris. The slope of the drawn line is 0.55 in 0.1 mM GMPH with 20 μM Ru(bpy)33+ in 0.5 M buffers at 23±2°C. (b) The free energy dependence was examined over the entire range of buffer and metal oxidant concentrations indicating a free energy dependence as a function of base pH and oxidation potential of the metal mediator over the entire kinetic range. Conclusions We have shown that oxidation of guanine, as GMPH, is dependent on the base form of the added succinate (H+Suc/Suc−), phosphate (H2PO4-/HPO42-), and Tris [(HOCH2)3CNH3+/(HOCH2)3CNH2] buffers with the base acting as the proton acceptor. At high concentrations of added buffer, oxidation of the preassociated GMPH-buffer complex occurs by the parallel pathways as shown in Scheme 1. In one, a GMPH-buffer complex undergoes MS-EPT with Ru(bpy)33+ as the electron acceptor. In the other, initial PT is followed by rapid ET, GMPH + buffer → GMP- + BH+. Considering the pKa values, pKa(H3O+) = 0 (for the standard state, with activity = 1) and pKa(H2PO4-) = 7.1, addition of the buffer base creates a thermodynamic advantage of -0.5 eV (12 kcal/mol) for HPO42- over H2O, for example, as the acceptor base. The kinetics for the MS-EPT step also illustrate the importance of the base-catalyzed step in the oxidation of guanine by Ru(bpy)33+. The mechanistic details are analogous to those for the oxidation of TyrOH. In both cases, with high concentrations of added bases, reactivity is dominated by pathways involving prior dissociation of GMPH to GMP- or TyrOH to TyO- followed by oxidation by Ru(bpy)33+ (PT-ET) or by concerted electron-proton transfer by MS EPT with simultaneous transfer of an electron to Ru(bpy)33+ and a proton to the added base.32 Based on the free energy dependence in the oxidation of GMPH with variations as the base was varied, the barrier to electron transfer for the MS-EPT step includes a MarcusHush component arising from electron transfer and a proton transfer component from proton transfer. The overall variation with driving force was d(lnk(obs))/d(dG0) = 1/(50).


Page 12 of 16

Page 13 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGMENT: This work was funded by the National Science Foundation under Grant CHE-0957215. $authors

contributed equally. Current address: #Office of the Dean of the Graduate School, Princeton University, Princeton NJ, 08540, §Office of the Provost, University of Washington, St. Louis, St. Louis, MO, †Department of Chemistry, the University of North Carolina-Chapel Hill, Chapel Hill, NC 275993290 REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Kumar, A.; Sevilla, M. D., Proton-Coupled Electron Transfer in DNA on Formation of Radiation-Produced Ion Radicals. Chem. Rev. 2010, 110, 7002-7023. Swiderek, P., Fundamental Processes in Radiation Damage of DNA. Angew. Chem. Int. Ed. 2006, 45, 4056-4059. Lukin, M.; de los Santos, C., Nmr Structures of Damaged DNA. Chem. Rev. 2006, 106, 607-686. Steenken, S.; Jovanovic, S. V., How Easily Oxidizable Is DNA? One-Electron Reduction Potentials of Adenosine and Guanosine Radicals in Aqueous Solution. J. Am. Chem. Soc. 1997, 119, 617-618. Prat, F.; Houk, K. N.; Foote, C. S., Effect of Guanine Stacking on the Oxidation of 8-Oxoguanine in B-DNA. J. Am. Chem. Soc. 1998, 120, 845-846. Thapa, B.; Schlegel, H. B., Calculations of Pka’s and Redox Potentials of Nucleobases with Explicit Waters and Polarizable Continuum Solvation. J. Phys. Chem. A 2015, 119, 5134-5144. Choi, J.; Park, J.; Tanaka, A.; Park, M. J.; Jang, Y. J.; Fujitsuka, M.; Kim, S. K.; Majima, T., Hole Trapping of G-Quartets in a G-Quadruplex. Angew. Chem. Int. Ed. 2013, 52, 1134-1138. Thorp, H. H., Electrocatalytic DNA Oxidation. In Long-Range Charge Transfer in DNA Ii, Schuster, G. B., Ed. Springer Berlin Heidelberg: Berlin, Heidelberg, 2004; pp 159-182. Cadet, J.; Douki, T.; Ravanat, J.-L., Oxidatively Generated Damage to the Guanine Moiety of DNA: Mechanistic Aspects and Formation in Cells. Acc. Chem. Res. 2008, 41, 1075-1083. Burrows, C. J.; Muller, J. G., Oxidative Nucleobase Modifications Leading to Strand Scission. Chem. Rev. 1998, 98, 1109-1152. Wu, L.; Liu, K.; Jie, J.; Song, D.; Su, H., Direct Observation of Guanine Radical Cation Deprotonation in G-Quadruplex DNA. J. Am. Chem. Soc. 2015, 137, 259-266. Fleming, A. M.; Alshykhly, O.; Zhu, J.; Muller, J. G.; Burrows, C. J., Rates of Chemical Cleavage of DNA and Rna Oligomers Containing Guanine Oxidation Products. Chem. Res. Toxicol. 2015, 28, 1292-1300. Nguyen, K. V.; Burrows, C. J., Whence Flavins? Redox-Active Ribonucleotides Link Metabolism and Genome Repair to the Rna World. Acc. Chem. Res. 2012, 45, 2151-2159. Steenken, S.; Jovanovic, S. V.; Candeias, L. P.; Reynisson, J., Is “Frank” DNA-Strand Breakage Via the Guanine Radical Thermodynamically and Sterically Possible? Chem. Eur. J. 2001, 7, 2829-2833. Steenken, S., Electron Transfer in DNA? Competition by Ultra-Fast Proton Transfer? Biol. Chem. 1997, 378, 1293-7. Pratviel, G.; Meunier, B., Guanine Oxidation: One- and Two-Electron Reactions. Chem. Eur. J. 2006, 12, 6018-6030. Sistare, M. F.; Codden, S. J.; Heimlich, G.; Thorp, H. H., Effects of Base Stacking on Guanine Electron Transfer: Rate Constants for G and Gg Sequences of Oligonucleotides from Catalytic Electrochemistry. J. Am. Chem. Soc. 2000, 122, 4742-4749. Duarte, V.; Muller, J. G.; Burrows, C. J., Insertion of Dgmp and Damp During in Vitro DNA Synthesis Opposite an Oxidized Form of 7,8Dihydro-8-Oxoguanine. Nucleic Acids Res. 1999, 27, 496-502. Gasper, S. M.; Schuster, G. B., Intramolecular Photoinduced Electron Transfer to Anthraquinones Linked to Duplex DNA: The Effect of Gaps and Traps on Long-Range Radical Cation Migration. J. Am. Chem. Soc. 1997, 119, 12762-12771.



20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

34. 35. 36. 37. 38. 39. 40. 41.

Shafirovich, V.; Dourandin, A.; Geacintov, N. E., Proton-Coupled Electron-Transfer Reactions at a Distance in DNA Duplexes: Kinetic Deuterium Isotope Effect. J. Phys. Chem. B 2001, 105, 8431-8435. Fecenko, C. J.; Meyer, T. J.; Thorp, H. H., Electrocatalytic Oxidation of Tyrosine by Parallel Rate-Limiting Proton Transfer and Multisite Electron−Proton Transfer. J. Am. Chem. Soc. 2006, 128, 11020-11021. Fecenko, C. J.; Thorp, H. H.; Meyer, T. J., The Role of Free Energy Change in Coupled Electron−Proton Transfer. J. Am. Chem. Soc. 2007, 129, 15098-15099. Kobayashi, K.; Yamagami, R.; Tagawa, S., Effect of Base Sequence and Deprotonation of Guanine Cation Radical in DNA. J. Phys. Chem. B 2008, 112, 10752-10757. Feeney, M. M.; Kelly, J. M.; Tossi, A. B.; Mesmaeker, A. K.-d.; Lecomte, J.-P., Photoaddition of Ruthenium(II)-Tris-1,4,5,8Tetraazaphenanthrene to DNA and Mononucleotides. J Photochem Photobiol 1994, 23, 69-78. Buchko, G. W.; Cadet, J., Identification of 2-Deoxy-D-Ribono-1,4-Lactone at the Site of Benzophenone Photosensitized Release of Guanine in 2′-Deoxyguanosine and Thymidylyl-(3′-5′)-2′-Deoxyguanosine. Can. J. Chem. 1992, 70, 1827-1832. Weatherly, S. C.; Yang, I. V.; Armistead, P. A.; Thorp, H. H., Proton-Coupled Electron Transfer in Guanine Oxidation: Effects of Isotope, Solvent, and Chemical Modification. J. Phys. Chem. B 2003, 107, 372-378. Weatherly, S. C.; Yang, I. V.; Thorp, H. H., Proton-Coupled Electron Transfer in Duplex DNA: Driving Force Dependence and Isotope Effects on Electrocatalytic Oxidation of Guanine. J. Am. Chem. Soc. 2001, 123, 1236-1237. Costentin, C.; Robert, M.; Savéant, J.-M., Concerted Proton−Electron Transfers: Electrochemical and Related Approaches. Acc. Chem. Res. 2010, 43, 1019-1029. Saveant, J. M., Concerted Proton-Electron Transfers: Fundamentals and Recent Developments. Annu Rev Anal Chem (Palo Alto Calif) 2014, 7, 537-60. Warren, J. J.; Tronic, T. A.; Mayer, J. M., Thermochemistry of Proton-Coupled Electron Transfer Reagents and Its Implications. Chem. Rev. 2010, 110, 6961-7001. Hammarstrom, L.; Styring, S., Coupled Electron Transfers in Artificial Photosynthesis. Philos Trans R Soc Lond B Biol Sci 2008, 363, 128391; discussion 1291. Huynh, M. H. V.; Meyer, T. J., Proton-Coupled Electron Transfer. Chem. Rev. 2007, 107, 5004-5064. Young, R. C.; Keene, F. R.; Meyer, T. J., Measurement of Rates of Electron Transfer between Tris(2,2'-Bipyridine)Ruthenium(3+) and Tris(1,10-Phenanthroline)Iron(2+) Ions and between Tris(1,10-Phenanthroline)Ruthenium(3+) and Tris(2,2'-Bipyridine)Ruthenium(2+) Ions by Differential Excitation Flash Photolysis. J. Am. Chem. Soc. 1977, 99, 2468-2473. Johnston, D. H.; Glasgow, K. C.; Thorp, H. H., Electrochemical Measurement of the Solvent Accessibility of Nucleobases Using Electron Transfer between DNA and Metal Complexes. J. Am. Chem. Soc. 1995, 117, 8933-8938. Elgrishi, N.; McCarthy, B. D.; Rountree, E. S.; Dempsey, J. L., Reaction Pathways of Hydrogen-Evolving Electrocatalysts: Electrochemical and Spectroscopic Studies of Proton-Coupled Electron Transfer Processes. ACS Catal. 2016, 6, 3644-3659. Holmberg, R. C.; Thorp, H. H., Electrochemical Determination of Triple Helices: Electrocatalytic Oxidation of Guanine in an Intramolecular Triplex. Inorg. Chem. 2004, 43, 5080-5085. Szalai, V. A.; Thorp, H. H., Electrocatalysis of Guanine Electron Transfer: New Insights from Submillimeter Carbon Electrodes. J. Phys. Chem. B 2000, 104, 6851-6859. Gagliardi, C. J.; Murphy, C. F.; Binstead, R. A.; Thorp, H. H.; Meyer, T. J., Concerted Electron–Proton Transfer (EPT) in the Oxidation of Cysteine. J. Phys. Chem. C 2015, 119, 7028-7038. Weinberg, D. R.; Gagliardi, C. J.; Hull, J. F.; Murphy, C. F.; Kent, C. A.; Westlake, B. C.; Paul, A.; Ess, D. H.; McCafferty, D. G.; Meyer, T. J., Proton-Coupled Electron Transfer. Chem. Rev. 2012, 112, 4016-4093. Murphy, C. F. Coupled Electron Proton Transfer Reactions in Biological Redox Active Substrates. University of North Carolina at Chapel Hill, Chapel Hill, 2009. Dongare, P.; Bonn, A. G.; Maji, S.; Hammarström, L., Analysis of Hydrogen Bonding Effects on Excited State Proton-Coupled Electron Transfer from a Series of Phenols to a Re(I) Polypyridyl Complex. J. Phys. Chem. C 2017, 121, 12569–12576.


Page 14 of 16

Page 15 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

Gagliardi, C. J.; Wang, L.; Dongare, P.; Brennaman, M. K.; Papanikolas, J. M.; Meyer, T. J.; Thompson, D. W., Direct Observation of LightDriven, Concerted Electron–Proton Transfer. Proc. Natl. Acad. Sci. 2016, 113, 11106-11109. Irebo, T.; Zhang, M. T.; Markle, T. F.; Scott, A. M.; Hammarstrom, L., Spanning Four Mechanistic Regions of Intramolecular Proton-Coupled Electron Transfer in a Ru(bpy)32+-Tyrosine Complex. J. Am. Chem. Soc. 2012, 134, 16247-54. Costentin, C.; Louault, C.; Robert, M.; Saveant, J. M., The Electrochemical Approach to Concerted Proton--Electron Transfers in the Oxidation of Phenols in Water. Proc Natl Acad Sci 2009, 106, 18143-18148. Shafirovich, V.; Dourandin, A.; Luneva, N. P.; Geacintov, N. E., The Kinetic Deuterium Isotope Effect as a Probe of a Proton Coupled Electron Transfer Mechanism in the Oxidation of Guanine by 2-Aminopurine Radicals. J. Phys. Chem. B 2000, 104, 137-139. Krishtalik, L. I., The Mechanism of the Proton Transfer: An Outline. Biochim. Biophys. Acta 2000, 1458, 6-27. Lu, W.; Liu, J., Deprotonated Guanine•Cytosine and 9-Methylguanine•Cytosine Base Pairs and Their "Non-Statistical" Kinetics: A Combined Guided-Ion Beam and Computational Study. PCCP 2016, 18, 32222-32237. Venkateswarlu, D.; Lyngdoh, R. H. D., Proton Transfer Reactions of Nucleic Acid Bases: A Semiempirical Molecular Orbital Study. Proc Ind Acad Sci - Chem Sci 1995, 107, 221. Holcomb, D. R.; Ropp, P. A.; Theil, E. C.; Thorp, H. H., Nature of Guanine Oxidation in Rna Via the Flash-Quench Technique Versus Direct Oxidation by a Metal Oxo Complex. Inorg. Chem. 2010, 49, 786-795. Kobayashi, K.; Tagawa, S., Direct Observation of Guanine Radical Cation Deprotonation in Duplex DNA Using Pulse Radiolysis. J. Am. Chem. Soc. 2003, 125, 10213-10218. Hammarström, L.; Styring, S., Proton-Coupled Electron Transfer of Tyrosines in Photosystem II and Model Systems for Artificial Photosynthesis: The Role of a Redox-Active Link between Catalyst and Photosensitizer. Energy Environ. Sci. 2011, 4, 2379. Chen, P.; Meyer, T. J., Medium Effects on Charge Transfer in Metal Complexes. Chem. Rev. 1998, 98, 1439-1478. Marcus, R. A.; Sutin, N., Electron Transfers in Chemistry and Biology. Biochim. Biophys. Acta 1985, 811, 265-322. Sutin, N., Theory of Electron Transfer Reactions: Insights and Hindsights. In Prog. Inorg. Chem., John Wiley & Sons, Inc.: 2007; pp 441-498. Hush, N. S., Adiabatic Theory of Outer Sphere Electron-Transfer Reactions in Solution. J. Chem. Soc. Faraday Trans. 1961, 57, 557-580. Ram, M. S.; Hupp, J. T., Linear Free Energy Relations for Multielectron Transfer Kinetics: A Brief Look at the Broensted/Tafel Analogy. J. Phys. Chem. 1990, 94, 2378-2380. Iordanova, N.; Decornez, H.; Hammes-Schiffer, S., Theoretical Study of Electron, Proton, and Proton-Coupled Electron Transfer in Iron BiImidazoline Complexes. J. Am. Chem. Soc. 2001, 123, 3723-3733. Iordanova, N.; Hammes-Schiffer, S., Theoretical Investigation of Large Kinetic Isotope Effects for Proton-Coupled Electron Transfer in Ruthenium Polypyridyl Complexes. J. Am. Chem. Soc. 2002, 124, 4848-4856. Rhile, I. J.; Markle, T. F.; Nagao, H.; DiPasquale, A. G.; Lam, O. P.; Lockwood, M. A.; Rotter, K.; Mayer, J. M., Concerted Proton−Electron Transfer in the Oxidation of Hydrogen-Bonded Phenols. J. Am. Chem. Soc. 2006, 128, 6075-6088. Soetbeer, J.; Dongare, P.; Hammarstrom, L., Marcus-Type Driving Force Correlations Reveal the Mechanism of Proton-Coupled Electron Transfer for Phenols and [Ru(bpy)3]3+ in Water at Low pH. Chem. Sci. 2016, 7, 4607-4612.

TOC



Page 16 of 16

e3+ N N N

Ru N

N III

N

N

,

N

N N

2+

B

O

H

N N

NH2

B O

N

RuII

N N

N

,

N


N

H N

N

NH2

Proton-Coupled Electron Transfer in the Oxidation of Guanosine

Recommend Documents