Pulse Radiolysis Studies of Intramolecular Electron Transfer in Model Peptides and Proteins. 8. Trp[NH^•+] → Tyr[O^•] Radical Transformation in H-Trp-(Pro)_n-Tyr-OH, n = 3−5, Series of Peptides

Abstract

The kinetics of intramolecular long-range electron transfer (LRET) between the protonated tryptophan indolyl radical cation Trp[NH •+ ], remaining in equilibrium with its neutral Trp[N • ] form, and tyrosine in aqueous H-Trp-(Pro) n -Tyr-OH, n ) 3-5, peptides (abbreviated 3, 4, and 5) has been studied by pulse radiolysis at pH 2-8 at 298 K and at pH 4 over the temperature range 283-328 K. LRET has been found to occur down to pH 2, contrary to expectations based on the electrochemical redox potentials for Trp and Tyr in the form of free amino acids. The first-order rate constants of LRET, k obs , varied sigmoidally with pH, allowing evaluation of pK a1 's for deprotonation of Trp[NH •+ ]: 3.7, 4.1, and 4.3 for 3, 4, and 5, respectively, as well as the intrinsic rate constants, k 2 , of LRET involving solely Trp[NH •+ ] radicals. Variation of pK a1 was attributed to electrostatic interactions between the indolyl radical and the COOH group of terminal Tyr, expected to decrease in the shown order, and taken as an indication of variation, in the same order, of the electrochemical driving force ∆G°of LRET. In 4 and 5, k 2 proved to be about 60-fold larger than the corresponding k 1 values characteristic for Trp[N • ], which indicates that the kinetics of LRET involving Trp[NH •+ ] exhibits similar through-bond distance dependence as that found earlier for the reaction with Trp[N • ]. For 3 the ratio of k 2 /k 1 was found to be about 3-fold lower than for its two longer-bridged analogues, 4 and 5. Arrhenius activation energies, E a2 , of LRET involving Trp[NH •+ ] proved to be rather low for 4 and 5, 8.6 and 7.2 kJ mol -1 , respectively. In the case of 3, however, k obs varied nonlinearly with temperature. A nonlinear fit of Arrhenius type function to k obs (T) data under the assumption δE a2 /δT ) 0 and δpK a1 /δT * 0 gave E a2 of 31.6 kJ mol -1 and indicated a decrease in pK a1 of Trp[NH •+ ] by about one unit in the studied temperature range. Conformational preferences of 3 were thus studied by means of molecular dynamics modeling to understand why the parameters of LRET in this peptide were so different from those of 4 and 5. It has been shown that a f R transition at the central ψ(Pro 3 ) dihedral angle, which could bring NH (indole) and COOH groups into a close contact compatible with formation of a hydrogen bond, may occur in 3 on the time scale of the observed electron-transfer reaction. In longer-bridged analogues two such concerted transitions would be necessary to perturb LRET measurably, an event of extremely low probability. It is thus argued that this transition may explain the kinetic and energetic peculiarities of LRET in 3. Analysis of thermodynamic parameters indicated that in reactions involving both Trp[NH •+ ] as well as Trp[N • ] radicals the free energy barrier of activation of LRET, ∆G # , is for the most part entropic in nature. A more detailed analysis of thermodynamics of these reactions must await experimental determination of ∆G°for both reactions in the peptides studied.

Molecular basis of intramolecular electron transfer in proteins during radical-mediated oxidations: Computer simulation studies in model tyrosine–cysteine peptides in solution

“…For this subpopulation the rate is directly the ET rate, and final reprotonation of TyrO- is expected to be very fast as mentioned previously. For the protonated Cys species the estimated rates for Step 1D (based on pKa value) are similar to the electron migration rates in the peptide unit as reported by (9, 15, 52). On the other hand, the rate of this step is expected to be similar in the studied peptides of different length.…”

“…In this case, using Eyring equation (51) 2 , the proton transfer rate constant from Cys to water (k Cys-) is estimated to be 2.4 × 10 4 s −1 , which is similar or even faster than the estimates for the overall rate reaction in tyrosine-containing peptides k =10 3 -10 4 s −1 (9, 15, 52) suggesting that ET (Step 2D) being at least partially rate-limiting. On the other hand, tyrosine protonation is spontaneous, and so, it is expected to be diffusion-controlled.…”

“…Our results show that tyrosine radical (Tyr-O ● ) to cysteine transfer is most likely to be mediated by an acid/base equilibrium that involves deprotonation of cysteine residue to form thiolate, followed by a fast ( e.g. k ≅10 3 -10 4 s −1 in tyrosine-containing peptides (9, 15, 52)), yet rate limiting, internal electron transfer ( i.e . charge redistribution) resulting in cysteine radical and tyrosine phenolate.…”

Thioredoxin Ch1 of Chlamydomonas reinhardtii displays an unusual resistance toward one‐electron oxidation

“…These values are compared to those proposed for other peptides or proteins. Data are an average of values given in [29,30,43]. The distance is taken from Figs 4 and 5 of [43].…”

“…(0.46 ± 0.01) · 10 9 Hen egg white lysozyme (7.9 ± 0.8) · 10 8 120 ± 10 [6] 14 (Trp62/63-Tyr53) Trp-(Pro) 3 [43] a Authors gave only the order of magnitude of this rate constant. between Trp35 and Tyr (53 and 85) were calculated at 29.8 and 18.4 Å , respectively ( Table 1).…”