Pourbaix diagram, proton-coupled electron transfer and decay kinetics of a protein tryptophan radical: Comparing the redox properties of W32\xc2\xb7 and Y32\xc2\xb7 generated inside the structurally characterized \xce\xb13W and \xce\xb13Y proteins
Protein \xe2\x80\x9chole\xe2\x80\x9d hopping typically involves spatially arranged redox-active tryptophan and/or tyrosine residues. Thermodynamic information is scarce for this type of process. The well-structured \xce\xb13W model protein was studied by protein film square wave voltammetry and transient absorption spectroscopy to obtain a comprehensive thermodynamic and kinetic description of a buried tryptophan residue. A Pourbaix diagram, correlating thermodynamic potentials (E\xc2\xb0\xe2\x80\xb2) with pH, is reported for W32 in \xce\xb13W and compared to equivalent data recently presented for Y32 in \xce\xb13Y (Ravichandran, K. R.; Zong, A. B.; Taguchi, A. T.; Nocera, D. G.; Stubbe, J.; Tommos, C. J. Am. Chem. Soc. 2017, 139, 2994\xe2\x80\x933004). The \xce\xb13W Pourbaix diagram displays a pKOX of 3.4, a E\xc2\xb0\xe2\x80\xb2(W32(N\xc2\xb7+/NH) of 1293 mV and a E\xc2\xb0\xe2\x80\xb2(W32(N\xc2\xb7/NH); pH 7.0) of 1095 \xc2\xb1 4 mV versus the normal hydrogen electrode. W32(N\xc2\xb7/NH) is 109 \xc2\xb1 4 mV more oxidizing than Y32(O\xc2\xb7/OH)) at pH 5.4\xe2\x80\x9310. In the voltammetry measurements, W32 oxidation-reduction occurs on a timescale of about 4 ms and is coupled to the release/uptake of one full proton to and from bulk. Kinetic analysis further shows that W32 oxidation likely involves pre-equilibrium electron transfer followed by proton transfer to a water or small water cluster as the primary acceptor. A well-resolved absorption spectrum of W32\xc2\xb7 is presented and analysis of decay kinetics show that W32\xc2\xb7 persists ~104 longer than aqueous W\xc2\xb7 due to significant stabilization by the protein. The redox characteristics of W32 and Y32 are discussed relative to global and local protein properties.
INTRODUCTION<!>Protein film SWV characterization of \xce\xb13W<!>E\xc2\xb0\xe2\x80\xb2 vs pH diagrams of \xce\xb13W and \xce\xb13Y constructed from protein film voltammetry and solution pH titration data<!>Redox kinetics of \xce\xb13W<!>Correlating data obtained from protein films and solution samples<!>Global and local protein effects on E\xc2\xb0\xe2\x80\xb2 of W and Y<!>Global and local protein effects on the stability of W\xc2\xb7 and Y\xc2\xb7<!>Radical formation in solvated \xce\xb13W<!>Summary and future perspectives
<p>Tryptophan (W) and tyrosine (Y) can serve as one-electron mediators in redox catalysis and in multistep "hole" hopping or proton coupled electron transfer (PCET) processes.1,2 The frequency of functional W/Y hole hopping/PCET chains remains to be established experimentally but may be quite significant.3 The thermodynamic landscape of protein ET is determined by the reduction potentials (E°′s) of the redox couples involved and how the surrounding protein environment influences these potentials. The molecular basis for tuning E°′s of metallo-cofactors has been the focus of much research effort and a range of mechanisms has been identified and characterized.e.g.4 In contrast, little is known regarding the thermodynamic properties of the less common amino-acid radical cofactors. This knowledge gap is due to the highly oxidizing potentials involved and the difficulty of probing the potential of a single specific protein residue. The literature contains only a handful of single-point E°′ or time-dependent "operational" potentials estimated from kinetic data.1,5 Few, if any, general trends can be extracted from the scattered and limited information available. This issue is addressed here.</p><p>The α3X model protein system was designed to systematically map and compare the E°′s and PCET characteristics of W and Y radicals formed inside a structured protein. The 65-residue three-helix bundle (α3) proteins contain a buried radical site (position 32), which is occupied by W32 (α3W),6,7 Y32 (α3Y),6,8,9 fluoro-Y32 (α3FnY, n = 2, 3)10,11 or 2-, 3- or 4-mercaptophenol (MP) ligated via a disulfide bond to C32 (2-, 3- or 4MP-α3C).12,13 In this report we used the structurally characterized α3W protein7 and protein film square-wave voltammetry (SWV)11 to measure E°′(W32) as a function of pH. We show that W is a "hot" protein hole-transfer agent and that there is a significant difference in the E°′s of W32 and Y32 when compared under highly similar experimental conditions. Additionally, flash-quench transient absorption (TA) methods were used to obtain high-quality spectra of W32· and to investigate the PCET kinetics of W32 oxidation and the stability of the W32· state. Experimental and modeled redox properties for W32, Y32, and aqueous (aq) W and Y are summarized and discussed relative to structural and dynamic properties of the α3W and α3Y proteins.</p><!><p>A SWV study of α3Y revealed that the Y32 redox cycle is fully reversible and that true thermodynamic potentials can be determined for the Y32(O·/OH) redox couple.14 Similar results were obtained for 2MP-α3C and α3(3,5)F2Y.10,13 In subsequent work the α3X/SWV approach was further refined to involve protein film samples. This method improved the Faradaic response significantly and facilitated studies of several very high potential α3FnY proteins.11 Here we apply protein film SWV to α3W and, for the first time, obtain thermodynamic E°′s and a Pourbaix diagram for a protein tryptophan residue.</p><p>A SWV measurement consists of a train of forward (here oxidative) and reverse (reductive) potential pulses superimposed on a staircase potential ramp. The induced current is sampled at the end of each applied pulse and plotted against the staircase potential. The measurement generates a forward (Ifor; Figure 1 shows oxidative currents in orange), a reverse (Irev; reductive currents in purple) and a net (Inet = Ifor − Irev; blue) voltammogram. All voltammograms are displayed in units of μA. When using SWV to investigate electrode mechanisms and to determine thermodynamic and kinetic parameters, it is essential to evaluate the lineshapes and positions of all three components as a function of experimental settings.15 The Faradaic response of a strongly adsorbed redox pair is at its maximum when the frequency of the applied pulses (f; s−1) is close to the surface standard ET rate constant (ksur; s−1).16 Under these conditions, Ifor and Irev are approximately equal in width and height, the maxima/minimum of the three individual currents (Ifor, Irev and Inet) are nearly coincident, and the peak potential of Inet (Enet) equals E°′.17 To find these conditions for α3W, protein film SW voltammograms were collected as a function of f. The observed α3W response was poor below 120 Hz, reached a maximum in the 200–300 Hz region, and rapidly declined above 300 Hz. These results show that 200 s−1 < ksur < 300 s−1 and a more detailed analysis was therefore conducted for this SW frequency range.</p><p>Figures 1 displays protein film voltammograms of α3W (in color) collected at 210, 240 and 270 Hz at pH 9.9 (panels A–C)), 8.4 (D–F) and 6.9 (G and H). There is no significant change in Enet as a function of f. Erev − Efor and Ifor/Irev values of − 8 ± 4 mV and 1.2 ± 0.2, respectively, were observed across the whole data set. The average Enet(210–270 Hz) value shifts by 55 ± 2 mV/pH unit between pH 6.93 ± 0.09 and 9.92 ± 0.06. (panel I). The pH dependence implies that there is a net of approximately one proton released upon W32 oxidation and taken up upon subsequent reduction of the radical. In these experiments, the W32 redox cycle occurs within 4.8 (210 Hz) to 3.7 (270 Hz) ms. We conclude that α3W is reversibly oxidized and reduced in an overall charge-neutral manner and that Enet(210–270 Hz) represents the true thermodynamic potential of the W32(N·/NH) redox couple.</p><p>Inet obtained from α3Y protein films was consistently ~ 2–4 times greater than Inet obtained from α3W protein film samples. This observation suggests a difference in the effective surface coverage, i.e. in the number of protein molecules electrically connected to the electrode. Y32 is centrally placed in the α3 scaffold9 making the Y32/electrode distance insensitive to the orientation of the protein in the film. In contrast, W32 is asymmetrically placed relative to the central axis of the protein.7 This is likely to result in a greater distribution in the W32/electrode distance and making some protein orientations electrochemically inactive.</p><!><p>Figure 2 was made as follows: (i) E°′(W32(N·/NH)) was determined at pH 6.9, 8.4 and 9.9. Replica voltammograms (n = 2–4) were collected at three different SW frequencies (210, 240 and 270 Hz, Figure 1) for each pH point. This entire data set was then reproduced with independently prepared samples. The average E°′ (1097 ± 4, 1023 ± 2 and 933 ± 3 mV) vs pH (6.93 ± 0.09, 8.39 ± 0.09 and 9.92 ± 0.06) values from these two data sets are shown as red circles. (ii) E°′s for Y32(O·/OH) were reproduced from ref 11 (blue circles). (iii) All E°′s were obtained from protein films prepared from solution samples containing stable and structurally well defined α3W and α3Y molecules (Figures S1 and S2).6–9 (iv) The pH dependence of E°′ (α3W 55 ± 2 mV/pH unit; α3Y 55 ± 1 mV/pH unit; Figure 1I) and the pK of oxidized W32 (pKOX 3.4; vide infra) and reduced Y32 (pKRED 11.3)6,18 were used to predict E°′s (α3W green lines; α3Y orange lines) outside the pH range investigated electrochemically (see Table S1 for details). (v) W (aq) has a pKOX of 4.2–4.3 ± 0.1.19,20 Incorporating a series of FnYs at site 32 increased their pKRED's by 0.83 ± 0.06 (presumably by destabilizing the FnY–O− state).11,18 A protein-induced shift of similar magnitude is likely for α3W (by destabilizing the W32·+ state) and a pKOX of 4.25 – 0.83 = 3.4 was predicted for W32.</p><!><p>The evolution and decay kinetics of oxidized W32 were followed by optical TA spectroscopy. In these experiments, a laser flash was supplied to α3W samples containing a photosensitizer, [Ru(L)3]2+ (L = 4,4′-R2-2,2′-bipyridine, R = H or CH3) and a sacrificial oxidative quencher, [Co(NH3)5Cl]2+. The laser flash-quench method generates the [Ru(L)3]3+ oxidant in situ, which subsequently oxidizes W32. Figure S3 summarizes the chemical reactions that occur during the flash-quench photolysis process. Details regarding sample preparations and experimental settings are also provided in the SI.</p><p>As described above, the W32 redox cycle is coupled to proton release and uptake to/from bulk (at pH > pKOX) and W32· is stable on the experimental timescale of the SWV measurements (≤ 4.8 ms as determined by the SW frequency). TA difference spectra obtained at pH 5.5 and 8.5 (Figures 3A and D) confirm the formation of a long-lived deprotonated W32· state. W· and W·+ (where W·+ is the protonated form of the radical) display absorption maxima at ~ 510 and 560 nm, respectively.19,20 The absorption spectra of oxidized α3W show a well-resolved peak with a maximum at 510 nm consistent with the formation of deprotonated W32· at both pH 5.5 and 8.5. TA kinetic traces were collected at 450, 510 and 560 nm (Figures 3B and E) to monitor the decay of [Ru(bpy)33+, the formation and yield of W32·, and the possible formation of W32·+, respectively. PCET rate constants (kPCET) associated with W32· formation were extracted from kinetic measurements as described in the SI (pages S3–4).</p><p>The amplitude of the positive signal at 510 nm was consistently ~ 4 times greater than at 560 nm in kinetic traces collected at pL 5.5 and 8.5, and in W32· spectra collected at pH 5.5 and 8.5. This observation demonstrates that the positive intensity at 560 nm at short timescales (Figures 3B and E) is due to absorption in the red edge of the W32· band and not from the accumulation of W32·+. The positive amplitude at 510 nm was used to calculate W32· and W· (aq) yields, which were ~ 1.0 for both systems. We note that the yields were obtained using an ε510(W·) of 230020 rather than 185019 M−1 cm−1. The latter value resulted in radical yields that were unrealistically high (i.e. significantly larger than 1.0). We conclude that the 2300 M−1 cm−1 value reported in ref 20 more accurately decribes ε510 for a deprotonated W· radical.</p><p>Decay of the long-lived W32· state was monitored at 510 nm (Figures 3C and F). W32· and W· (aq) decay was dominated by a second-order process attributed to radical-radical dimerization (kdim) at both pH 5.5 and 8.5. The predominance of a radical-radical coupling mechanism for decay was also observed for Y32· and Y· (aq).9 A second minor decay pathway was observed for W32·, which we assign to W32· reacting with a reduced α3W substrate molecule (ksub). Details regarding the curve fitting routines to determine rate constants kdim and ksub are given in the SI. Table 1 summarizes rate constants (kPCET, kdim, ksub), W32· yield and kinetic isotope effects (KIE) obtained from kinetic studies of W32 oxidation. Reference data were also collected on W (aq) (see Figure S7) and kPCET, kdim and W· yield are reported in Table 1.</p><!><p>The redox properties of α3W and α3Y have been characterized by protein film voltammetry and by solution methods including CD, NMR, and steady-state and transient absorption spectroscopy. Protein films are not individual proteins in solution and the structure of the protein in the film may not be identical to the structure of the protein in solution. Nevertheless, earlier work has shown that the α3X proteins bind to the electrode surface in a reversible manner that is subject to the KCl concentration.13,14 Salt-dependent binding is consistent with a three-helix bundle fold6 where the apolar residues in the heptad a and d positions form the main part of the protein interior and the polar/charged residues in the remaining heptad positions form the exterior. The folded α3 scaffold extends the radical lifetime (evident from the reversible protein film voltammograms and measured directly by TA for α3W and α3Y in solution) and shields X32 from chemical groups present on the electrode surface. The latter is evident by the fact that E°′(X32·/X32) is independent of the material from which the working electrode is made.11,13,14</p><!><p>Figure 2 presents the absolute and relative E°′s of W32 and Y32 across a pH range that covers most biological redox processes. This data set provides a unique guide to predict how the thermodynamic profile of a radical/hole transfer chain will change when replacing a W with a Y, or vice versa. This statement is based on the following results: The Pourbaix diagrams are based on true thermodynamic potentials. This conclusion is evident from the SWV responses of α3W (Figure 1) and α3Y11,14 and consistent with the TA-monitored X32· decay kinetics presented here (Table 1) and in ref 9. A major effect of the protein matrix is to effectively suppress radical side reactions and extend the lifetime of the X32· state (seconds) well beyond the experimental timescale of the SWV measurements (ms). In contrast, there are large uncertainties in the potentials reported for the aqueous systems over the past few decades since irreversible radical side reactions compromise both pulse radiolysis and voltammetry measurements. Importantly, all SWV and TA studies are supported by extensive structural characterization. α3W (RCSB PDB ID 1LQ7)7 and α3Y (2MI7)9 form well-defined three-helix bundles at pH 5.5, as shown by solution NMR spectroscopy. W32 and Y32 are both buried (see Figure 4A for details).7,9 The two proteins remain stable (global stabilities of −3.7 to −4.0 kcal mol−1 at pH 5–8.5 and −3.2 to −3.5 kcal mol−1 at pH 8.5–10; Figure S1),8–10 helical (51–52 ± 1 residues of 65 in total; pH 5–10)6–9 and well-folded (Figure S2)8 across a broad pH range. While there are minor structural differences between the W32 and Y32 sites (vide infra), the overall environments of W32 and Y32 are highly similar.</p><p>Table S1 summarizes experimental and modeled pKOX, pKRED, E°′(X·+/X) and E°′(pH 7.0) values for W32, Y32, W (aq) and Y (aq). E°′(pH 7.0) of W32(N·/NH) and Y32(O·/OH) equal 1095 ± 4 mV and 986 ± 3 mV, respectively. ΔE°′(W32(N·/NH) − Y32(O·/OH)) is constant at 109 mV ± 4 mV between pH 5.4 and 10 (Figure 2). Table S1 lists recent literature consensus E°′(pH 7.0) values for aqueous W and Y.11,22 Comparing the E°′(pH 7.0) values of α3Y and α3W to those of aqueous Y and W gives a difference of ~45–65 mV11 and ~65 mV, respectively. Similarly, comparing the 1293 mV potential of W32(N·+/NH)) with the 1190 mV potential estimated for W(N·+/NH) (aq) yields a difference of ~100 mV. Despite the uncertainties in the aqueous potentials, it appears clear that the protein milieu increases E°′ for both the neutral (X·/X) and the cation (X·+/X) redox pair. The latter result was expected due the energetic penalty of solvating a charge in the low dielectric protein medium. The protein-induced increase in E°′(X32·/X32) for the overall charge-neutral α3X redox proteins represents a more surprising result.</p><p>There is a small difference in the pH dependence of the α3X potentials, 55 ± 1–2 mV/pH unit, relative to the theoretically predicted value of ln(10)RT/F = 59 mV/pH unit at 25 °C. This observation was originally made for α3Y and assigned to redox Bohr effects.14 Alternatively, it reflects a more global effect. The isoelectric point was determind to be ~8.0 for both α3W and α3Y by isoelectric gel electrophoresis (not shown). The minor deviation in the pH dependence relative to theory may result from a weak electrostratic influence on the W32(N·/NH) and Y32(O·/OH) redox pairs as the net charge of the α3 scaffold transition from positive in the acid-neutral range of the Pourbaix diagram to negative in the alkaline region.</p><!><p>The use of a sacrificial quencher in the TA measurements permitted the observation of W32· and W· decay kinetics. Kinetic analysis shows that W32· decays with intermolecular radical-radical and radical-substrate reactions as the major and minor pathways, respectively. Similarly, Y32· decays via radical-radical dimerization.9 No evidence of intramolecular W32· or Y32· decay was observed. These observations show that the native state of the protein protects X32· from reacting (Figure 4B and Figure 6 in ref 9). Furthermore, it is clear that protein dynamics, i.e. local, sub-global or global unfolding events, are involved in the decay of W32· and Y32·. In other words, the protein transiently opens and exposes W32·, which then reacts irreversibly with a second transiently exposed W32·/α3 species (major decay pathway) or with a reduced α3W molecule (minor decay pathway). For α3Y, Y32·–Y32· dimerization was the only detected radical decay pathway.9 These equilibrium structural fluctuations of the protein result in an apparent reduction in kdim of W32· relative to W· (aq) and is similar to that for Y32· relative to Y· (aq) (both on the order of 104 M−1 s−1). Additionally, kdim(W32·)/kdim(Y32·) and kdim(W·)/kdim(Y·) both equal 1 and 0.4 at the pH 5.5 and 8.5, respectively (Table 1). These results suggest that the decays of W32· and Y32· are both subject to the same dynamic event(s) in the protein. NMR studies to investigate these issues further are in progress (Glover, Valentine, Liang, Wand, Tommos, on-going).</p><!><p>W32 is oxidized to a neutral radical with water as the primary proton acceptor. This conclusion is based on the constant kPCET between pH 5.5 and 8.5 (i.e. OH− or phosphate buffer ions are not significant) and on the structural features of the W32 site. The very minor solvent exposure of W32 (with an average solvent accessible surface area (SASA) of < 4%) is localized to the indole edge of the aromatic ring and centered at the indole hydrogen (white labeles display atom-level SASAs in Figure 4A). There are no basic side chains in close vicinity to the W32 indole hydrogen. Nonetheless, the SWV data described above show that redox-drived proton release and uptake occur within a few ms. Likewise, formation of the neutral W32· radical was observed by TA spectroscopy to occur on a ms time scale (Figure S5). Thus, the indole proton is most likely connected to the bulk phase either by water penetration or by protein fluctuations.</p><p>Setting aside the precise mechanism by which the indole group contacts water, the oxidation mechanism is either (a) pre-equilibrium ET followed by proton transfer (ETPT) or (b) pre-equilibrium concerted electron-proton transfer (CEPT) followed by escape of the proton from the primary water cluster to the bulk. A pre-equilibrium model predicts, for both case (a) and (b), a tenfold increase in kPCET for each 59 mV increase in oxidant strength. This prediction is in very good agreement with the observed 660 times increase in kPCET when using [Ru(bpy)3]3+ relative to [Ru(dmb)3]3+ as the flash-quench oxidant (160 meV difference in driving force; Table S1). Additionally, ΔG°ET is predicted to be 35 and 195 meV uphill for α3W/[Ru(bpy)3]3+ and α3W/[Ru(dmb)3]3+, respectively (Table S1). The observed KIE (Table 1) arises from the PT step in case (a) or from the CEPT step itself in case (b).</p><p>We favor case (a) the ETPT mechanism. Following Krishtalik's argument,26 CEPT to a small water cluster is much more uphill than ET, because of the unfavorable transfer of a proton from W32·+ (pKOX = 3.4) to a small water cluster (pKa(H3O+/H2O) = 0). With "small water cluster" we are referring to the molecular description of a proton released into bulk water. Here the primary PT is always to a small water cluster and is followed by thermal transfer to new water clusters. These clusters break up and reform on a 10's of fs time scale.27 Modeling predicts a ΔG°ET of 35 meV, ΔG°PT of 200 meV and a ΔG°CEPT of 235 meV for the α3W/[Ru(bpy)3]3+ system (Table S1). One caveat to this argument is results obtained on linked Ru(bpy)3-tryptophan model systems that show that CEPT nevertheless can be competitive.28 The moderate KIE's observed for W32 oxidation are not evidence for a CEPT reaction. They may be due to an isotope effect on the position of the initial ET equilibrium or the follow-up PT step from the W32·/(small water cluster) intermediate. A study on Ru(bpy)3-linked bromo-W reported KIE of 2 for a reaction attributed to ETPT.29</p><p>Most likely, W32 and W (aq) oxidation occur via ETPT while Y32 and Y (aq) oxidation occur via a CEPT or PTET mechanism.9 The kPCET(W/W32) ratio is much larger than kPCET(Y/Y32): 3000 vs 200 at pH 5.5 (Table 1). The protein has a more pronounced effect on the kinetics of W oxidation relative to the kinetics of Y oxidation. Modeling suggests significant differences in the ΔG°ET step of W32 and W (aq) oxidation (30 vs −65 meV; Table S1) in contrast to the ΔG°CEPT step of Y32 and Y (aq) oxidation (100 vs 85 meV; Table S1). The decrease in pKOX and increase in E°′(W(N·+/NH)) induced by the low dielectric protein milieu slows down ETPT. Nevertheless, E°′(W32(N·+/NH)) is still much lower than E°′(Y32(O·+/OH)) (1293 vs 1510 mV; Table S1). This situation favors an ETPT mechanism via the protonated radical intermediate in α3W, instead of CEPT or PTET as in α3Y. Thus, while E°′(pH 7.0) is clearly greater for α3W than α3Y, the much greater pKOX values of W32 (3.4 vs −2.5; Table S1) stabilizes the W32·+ intermediate relative to Y32·+, and enables a different oxidation mechanism.</p><!><p>The α3X model protein system was made to gain a better understanding of how natural proteins effectively manage amino-acid based redox reactions and long-range radical/hole transfer. These types of reactions are integral to many important processes in biology.1–3 The α3W data presented here, combined with other recent α3X studies,9,11,14 allowed us to start formulating a detailed description of key similarities and differences in the redox properties of W and Y when residing in a highly similar protein milieu. These are the main observations: α3W and α3Y each contain a redox-active aromatic residue at position 32. W32 and Y32 exhibit an average SASA of <4 and 0% and a residue depth of 5.9 ± 0.3 and 7.7 ± 0.3 Å, respectively. SWV studies showed that W32 and Y32 can be oxidized and reduced in a reversible manner. Their redox cycles are charge neutral with ET (between X32 and the working electrode) and coupled protonic reactions (between α3X and the bulk phase) occurring on the time-scale of 2–4 ms. The combination of redox reversibility and broad structural pH stability allowed Pourbaix diagrams to be obtained for W32 and Y32. This unique data set revealed that the protein matrix increases E°′s for the (X32·/X32) and (X32·+/X32) redox couples by about 45–65 and 100 mV, respectively, relative to the corresponding redox couples of aqueous W and Y. A small but statistically significant change in the slope of the α3X Pourbaix plots relative to theory indicates that the net charge of the α3X proteins has a weak influence on the E°′s of the (X32·/X32) redox couples. Overall, W32 and Y32 are very hot protein oxidants with E°′(pH 7.0) of 1095 ± 4 mV and 986 ± 3 mV, respectively. W32(N·/NH) is 109 ± 4 mV more oxidizing than Y32(O·/OH) at pH 5.4–10. This difference gradually decreases as the pH approaches either the pKOX of W32 (3.4) or the pKRED of Y32 (11.3). TA spectroscopy, using a flash-quench generated [Ru(bpy)3]3+ oxidant, was employed to monitor the kinetics of W32· and Y32· formation resulting in radical yields of ~ 1.0 and 0.3–0.6, respectively. High-quality absorption spectra of W32· and Y32· have been obtained. Kinetic analyses suggested that W32 is oxidized via a pre-equilibrium ETPT path while Y32 oxidation follows a CEPT, or possibly PTET, mechanism. Water or a small water cluster serves as the primary proton acceptor for W32. Water and/or buffer serve as the primary proton acceptor for Y32. In light of these latter results, it is interesting that the predicted distances between the indole N and the phenol O to the closest bulk water are 4.1 ± 0.4 and 6.3 ± 0.4 Å, respectively. The protein matrix stabilizes the X32· state by effectively blocking radical migration (E°′ of X32 ≪ E°′ of all other residues in the α3 scaffold) and radical-radical recombination (major decay pathway of aqueous W· and Y·). Equilibrium structural fluctuations in the protein matrix are likely to be involved in both the PCET of radical formation (connecting X32 to the primary proton acceptor) and decay (connecting two X32· residues). These observations set the stage for future investigations aimed at delineating the role of the protein ensemble on PCET and radical-stabilization reactions.</p>