{'fraction_non_alphanumeric': 0.04499370976097092, 'fraction_numerical': 0.04010952416191815, 'mean_word_length': 4.205701078582434, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 3, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Photochemical Tyrosine Oxidation in the Structurally Well-Defined α3Y Protein: Proton-Coupled Electron Transfer and a Long-Lived Tyrosine Radical

Tyrosine oxidation–reduction involves proton-coupled electron transfer (PCET) and a reactive radical state. These properties are effectively controlled in enzymes that use tyrosine as a high-potential, one-electron redox cofactor. The α3Y model protein contains Y32, which can be reversibly oxidized and reduced in voltammetry measurements. Structural and kinetic properties of α3Y are presented. A solution NMR structural analysis reveals that Y32 is the most deeply buried residue in α3Y. Time-resolved spectroscopy using a soluble flash-quench generated [Ru(2,2′-bipyridine)3]3+ oxidant provides high-quality Y32–O• absorption spectra. The rate constant of Y32 oxidation (kPCET) is pH dependent: 1.4 × 104 M–1 s–1 (pH 5.5), 1.8 × 105 M–1 s–1 (pH 8.5), 5.4 × 103 M–1 s–1 (pD 5.5), and 4.0 × 104 M–1 s–1 (pD 8.5). kH/kD of Y32 oxidation is 2.5 ± 0.5 and 4.5 ± 0.9 at pH(D) 5.5 and 8.5, respectively. These pH and isotope characteristics suggest a concerted or stepwise, proton-first Y32 oxidation mechanism. The photochemical yield of Y32–O• is 28–58% versus the concentration of [Ru(2,2′-bipyridine)3]3+. Y32–O• decays slowly, t1/2 in the range of 2–10 s, at both pH 5.5 and 8.5, via radical–radical dimerization as shown by second-order kinetics and fluorescence data. The high stability of Y32–O• is discussed relative to the structural properties of the Y32 site. Finally, the static α3Y NMR structure cannot explain (i) how the phenolic proton released upon oxidation is removed or (ii) how two Y32–O• come together to form dityrosine. These observations suggest that the dynamic properties of the protein ensemble may play an essential role in controlling the PCET and radical decay characteristics of α3Y.


Chemical denaturation of α 3 Y
The global stability of α 3 Y was determined by urea denaturation at pH 4.5, 8.5 and 9.9 ( Figure S1). Experimental details are provided in the Materials and Methods section in the main manuscript. The data in Figure S1 complement earlier reports on the stability of α 3 Y measured at pH 5.0, 5.5 and 8.2. 2,3 The global stability of α 3 Y remains unchanged between pH 5.0 and 8.5 (-3.8 ± 0.1 kcal mol -1 ) and is slightly lower at pH 4.5 (-3.3 ± 0.1 kcal mol -1 ) and 9.9 (-3.5 ± 0.1 kcal mol -1 ). 9.9. The grey lines represent nonlinear fits to determine the stability of the protein in the absence of urea. 4 The average standard error in the reported ΔG values is ± 0.1 kcal mol -1 .

Characterization of cobalt phosphate/oxide precipitates
As shown in Figure 2 in the main manuscript, kinetic traces collected on long timescales (>1 s) display a baseline offset. Transient absorption (TA) properties of the [Ru(bpy) 3 ]Cl 2 sensitizer and the [Co(NH 3 ) 5 Cl]Cl 2 quencher were assessed in the absence of the α 3 Y protein. These control experiments show that the observed baseline is due to light absorbing/scattering products formed in reactions between Co 2+ (from decomposed quencher) and buffer ions, phosphate as well as borate. Spectra of solutions containing [Ru(bpy) 3 ]Cl 2 and [Co(NH 3 ) 5 Cl]Cl 2 at pH 5.5 ( Figure S2A) and pH 8.5 ( Figure  S2B) were recorded at different time intervals on a diode array spectrometer. Kinetics traces of pH 5.5 and 8.5 solutions from laser flash photolysis experiments are shown in Figures 3a and 3c in the main text (grey traces). A FTIR spectrum of precipitates that were isolated after a flash photolysis experiment is shown in Figure S3. Infrared spectra were indistinguishable for precipitates collected from experiments performed with or without α 3 Y at both pH 5.5 and 8.5.
The difference spectra at pH 5.5 ( Figure S2A) were collected in an identical fashion to the Y32-O• spectra ( Figure 2, main manuscript). The displayed spectra correspond to the light-minus-dark difference observed at 2.5 s, 5 s and 26 minutes after the flash of light. The initial Ru(bpy) 2+ bleach appears at ~450 nm and does not fully recover over the course of the study due to the irreversibility of the quencher. The positive signal that covers the visible spectrum after 26 minutes is due to precipitated material and appears as broad and featureless from scattering of the probe light. The sample at pH 8.5 ( Figure S2B) was not subject to a pulse of excitation light, yet the formation of the S4 Figure S2. Control Figure S3. Representative infrared spectrum of precipitated products formed during a flash photolysis experiment. The spectrum shows peaks associated with water and phosphate stretches: intense bands due to water at ~3300 and 1630 cm -1 and medium bands due to PO 4 3at 1050, 630, 615 and 564 cm -1 . A medium band at 1340 cm -1 is consistent with borate and provides evidence that borate salt is also part of the precipitates. The small stretches at ~2900 cm -1 in the shoulder of the water band are consistent with small amounts of 2,2'-bipyridine from decomposed sensitizer. Sample Preparation: Spent flash photolysis solutions containing precipitates were centrifuged and the supernatant removed. The solids were washed and centrifuged 5 times with high purity water to remove soluble material and buffer salts. The solids were dried in vacuo for 12 h and then pressed into a KBr matrix. S5 precipitates was observed. The probe light of the diode array excites the sensitizer and drives the precipitate forming reactions. The initial and final spectra are the orange dotted and red solid line, respectively, while the difference spectrum is shown in grey. Again, the difference spectrum shows the absorption/light scattering profile of the precipitated material.
Flash photolysis experiments in the absence of α 3 Y (Figures 3a and 3c, main manuscript) show that transient absorption kinetic profile of these absorbing species is negligible on the ms timescale. Their formation is so slow that their impact on the α 3 Y and L-tyrosine kinetics (k obs ) would present only a minimal contribution.

Kinetic fits and simulations
Tyrosine radicals can be detected from their absorption at 410 nm (see Figure 2, main manuscript). At this wavelength, the Ru 2+ bleach is also apparent. Transient absorption traces collected at 410 nm thus report on Ru 2+ recovery as well as the formation of Y-O• (i.e. they provide information on k PCET associated with tyrosine oxidation and the Y-O• yield). Should Y-O• dimerize or be subject to other side reactions, the 410 nm signal will diminish in intensity. When the oxidation reaction is fast relative to the radical side reaction(s), the amplitude of the signal at 410 nm can reflect a Y-O• yield of 100%. Such is the case for the TA signal of L-tyrosine at pH 8.5, which displays a maximum positive amplitude at 410 nm that is ~2/3 of the Ru 2+ bleach at the same wavelength. The 2/3 factor comes from the ratio of ε 410 (Y-O•) and Δε 410 (Ru 2+ ), which are 3000 and 4500 M -1 cm -1 , respectively. 5,6 Figure 4B in the main manuscript shows the fitting result of the 410 nm TA signal for L-tyrosine at pH 8.5 applying a model of a pseudo-first order recovery ([Y-OH] >> [Ru 3+ ]) followed by a second order decay.
The concentrations of Ru 3+ , Y-O•, and Y dim as a function of time can be described by the following system of equations: The non-trivial analytical solution describing [Y-O•] as a function of time has been reported. 7,8 In this model [Y-O•] as a function of time depends on three parameters: the initial Ru 3+ concentration, the pseudo-first order rate constant, k 1 , and the second order rate constant, k 2 . The k 1 and k 2 rate constants are reported as k obs and k Y-O• in Table 2 of the main manuscript, respectively. The TA signal at 410 nm is reproduced by the sum of the contributions of the recovering Ru 2+ signal (solution to equation 1) and S6 the Y-O• signal (solution to equation 2). At 450 nm the Ru(bpy) 3 2+ bleach is stronger and the Y-O• absorption is weaker than at 410 nm. As pointed out in the main article, a single exponential fit to the 450 nm traces, reflecting (1) above, gave values of k obs that were the same as those derived at 410 nm. Figure 4A in the main manuscript shows the fitting result for L-tyrosine at pH 5.5 using the same model. In this case the rate of PCET that generates Y-O• is slower and the effect of the second order decay process is apparent. Thus the maximum positive amplitude of the 410 signal at pH 5.5 is significantly less than that of the maximum signal at pH 8.5. Despite the diminished 410 nm maximum amplitude at pH 5.5 the trace is consistent with 100% yield of Y-O• with respect to Ru 3+ generated per laser flash. At pH 5.5 conditions the lower amplitude of the 410 nm signal is simply due to concurrent tyrosine oxidation and radical-radical dimerization. The experimentally determined rates k obs and k Y-O• from Table 2   The fitting routine described above was applied to the 410 nm TA signals that were recorded for α 3 Y at pH(D) 5.5 and 8.5 in H 2 O and in D 2 O. Comparing the α 3 Y kinetics at these four different sample conditions revealed that Y32-O• yield is not 100% under any of the conditions studied. Figure  S4 displays simulated 410 nm TA traces (orange lines) that include the experimentally determined k obs and k Y-O• values in Table 2 (main manuscript) and a 100% Y32-O• yield at pH 5.5 and 8.5, S7 respectively. The radical yield is given relative to the flash-generated concentration of [Ru(bpy) 3 ] 3+ . These simulations show that the Y32-O• yield is less than unity. Figures S5A-F show TA traces at 410 nm and fits using the first order followed by second order kinetic model for α 3 Y at all pH(D) and protein concentration conditions that are not shown in the main manuscript. Specifics regarding each trace are given in the figure caption. The yield of Y32-O• was found to be ca. 40%, 35%, 58% and 50% for pH 8.5, pD 8.5, pH 5.5 and pD 5.5, respectively.

α 3 F/trifluoroacetic acid control studies
A ns flash photolysis control study was performed with α 3 F in lieu of α 3 Y to assess the reactivity of the protein scaffold. The F32 residue in α 3 F is expected to be redox inert under the conditions employed in this study, and any photochemical reactivity from the protein scaffold should be apparent. The experimental conditions used in this control experiment were identical to the conditions used for the α 3 Y flash photolysis experiments. Inspection of each plot in Figure S6 shows that after 4-5 laser shots the α 3 F samples give the same response as samples containing only [Ru(bpy) 3 ]Cl 2 and [Co(NH 3 ) 5 Cl]Cl 2 in buffered solution. Any reactivity from α 3 F would give many reproducible traces on account of the relatively high concentration of protein (300 µM) versus the concentration of [Ru(bpy) 3 ] 3+ generated via a laser flash (3-9 µM). The data shown in Figure S6 verifies that the α 3 protein scaffold is unreactive under the present experimental conditions.  Figure S7) is strikingly similar to the solutions containing α 3 F ( Figure  S6). As with α 3 F, any reactivity from TFA itself would give reproducible shots on account of the high concentration of TFA versus [Ru(bpy) 3 ] 3+ . Thus, a likely candidate for the competitive consumption of [Ru(bpy) 3 ] 3+ in flash photolysis experiments is a small impurity present in the TFA.  Table S2 contains the details for the transient absorption measurements where the initial concentration of Y32-O• was varied by the use of neutral density filters and a 500 ms excitation pulse. With the exception of the TA trace with the smallest initial concentration, the second order rate constants from this experiment are comparable to those from experiments where the initial concentration was varied by lengthening the pulse duration ( Figure S8). The pH 5.5 solution had the following concentrations: 70 µM α 3 Y, 30 µM [Ru(bpy) 3 ]Cl 2 and 2 mM [Co(NH 3 ) 5 Cl]Cl 2 .  Dityrosines exhibit a diagnostic emission spectrum with a maximum at ca. 400 nm when excited at 310-325 nm. [9][10][11] Fluorescence spectroscopy of both the isolated Y32-O• reaction products ( Figure S9B) and L-tyrosine reaction products ( Figure S9C) showed this characteristic emission while unreacted α 3 Y, [Ru(bpy) 3 ]Cl 2 , [Co(NH 3 ) 5 Cl]Cl 2 , [Ru(bpy) 3 ]Cl 2 and [Co(NH 3 ) 5 Cl]Cl 2 products, and buffer did not emit under the same conditions. The emission spectra for Y32-O• reaction products and L-tyrosine reaction products match previously reported spectra for dityrosine. Further, the absorbance ( Figure  S9A) and fluorescence excitation spectrum ( Figure S9D) of Y32-O• reaction products show the same structural features that have been reported previously for dityrosine at pH ~7. 12 The series of absorption and fluorescence spectra displayed in Figure S9 verify that Y32-O• flash photolysis products exhibit the characteristic spectral properties of dityrosine.   5 Cl]Cl 2 oxidative quencher. α 3 Y was dissolved in 20 mM potassium, 20 mM sodium borate, pH 7.0, and diluted to 60 µM in buffer only (Fig. S10A, red spectrum) and in buffer containing 40 µM [Ru(bpy) 3 ]Cl 2 (blue spectrum). The 222 nm ellipticity, which directly reflects the helicity of α 3 Y, is the same in the two spectra. Due to total light absorption and/or scattering, CD spectra could not be collected on samples containing [Co(NH 3 ) 5 Cl]Cl 2 . Gel filtration samples were prepared from a 600 µM α 3 Y solution diluted to 300 µM protein in buffer only (Fig. S10B)