Olga B. Morozova

Time resolved CIDNP study of electron transfer reactions in proteins and model compounds

Intramolecular electron transfer (IET) from tyrosine to tryptophan cation radicals is investigated using time resolved chemically induced dynamic nuclear polarization (CIDNP) spectroscopy in combination with laser flash photolysis. In both the tryptophan-tyrosine dipeptide and the denatured state of hen lysozyme in aqueous solution, the transformation TrpH+ → TyrO by IET leads to an increase in the tyrosine radical concentration, growth in the tyrosine CIDNP signal, fast decay of the tryptophan CIDNP, and inversion of the phase of the CIDNP of the photosensitizing dye, 2,2′-dipyridyl. IET effects are not observed for mixtures of the amino acid or for the native state of lysozyme. The steady state CIDNP effects seen for denatured lysozyme thus depend not only on the accessibility of the amino acid residues on the surface of the protein but also on the reactivity of the radical intermediates.

Photo-CIDNP Study of Transient Radicals of Met-Gly and Gly-Met Peptides in Aqueous Solution at Variable pH

Time-resolved chemically induced dynamic nuclear polarization (CIDNP) was applied to the investigation of the photo-oxidation of two sulfur containing peptides, glycylmethionine (Gly-Met) and methionylglycine (Met-Gly). It was established that the reaction of Gly-Met with a photosensitizer, triplet 4-carboxybenzophenone, occurs via electron transfer from the sulfur atom and also from the terminal amino group in its uncharged state. The latter process leads to the formation of nuclear polarization of the alpha-protons of the glycine residue. The sulfur-centered cation radical of Gly-Met formed as a result of triplet quenching participates in the degenerate electron exchange reaction with the parent molecule. The rate constant of this reaction obtained from a simulation of the CIDNP kinetics is 2x10(8) M(-1) s(-1). Two channels of triplet quenching were also found for the Met-Gly peptide at pH values above the pKa of the terminal amino group: electron transfer from the amino group and from the sulfur atom. On the basis of the analysis of the CIDNP spectra and kinetics, it was found that at pH below pKa of the terminal amino group photo-oxidation of Met-Gly leads to the formation of an open-chain S-centered cation radical, which releases a proton from its N-terminal amino group to form a five-membered cyclic radical structure with a three electron bond between the S and N atoms. The rate constant of deprotonation obtained to be 1.8x10(5) s(-1) is in agreement with the pKa=4.7 of the S-centered radical of Met-Gly determined from the pH dependence of nuclear polarization. At pH>pKa, the aminium radicals formed in both peptides as a result of electron transfer from the lone pair of N-terminal amino group undergo deprotonation to the neutral aminyl radical on the submicrosecond time scale. The involvement of the different radicals was confirmed by the dependence of CIDNP on the external magnetic field ranging from 0.1 T to 7 T.

Intramolecular Electron Transfer in the Photooxidized Peptides Tyrosine–Histidine and Histidine–Tyrosine: A Time‐Resolved CIDNP Study

The long-range electron-transfer (ET) reaction with the participation of tyrosyl radicals is known to be of great importance in proteins. A binding partner of tyrosyl radicals at the active sites of several enzymes and in model peptides is histidine. [1] For example, the formation of the remarkably stable tyrosine radical in photosystem II is attributed to proton-coupled electron transfer, during which a proton is relocated to the imidazole group of an adjacent histidine residue. [2] The efficiency of this kind of ET was measured in model peptides containing a photochemically generated electron-acceptor group, N-terminal tyrosine as a donor, and a relay amino acid in between. [3] Indirect evidence for the proton-coupled electron transfer from tyrosine to the hystidyl radical was drawn from the optical detection of tyrosyl radicals when histidine was used as the relay amino acid instead of alanine. However, kinetic information about electron transfer from tyrosine to the histidyl radical has not been obtained so far. To study the kinetics of intramolecular electron transfer between tyrosine and the histidyl radical, we chose two dipeptides, histidine–tyrosine (His-Tyr) and tyrosine–histidine (Tyr-His) as model systems (Scheme 1). The method of time-resolved chemically induced dynamic nuclear polarization [4, 5] (TR CIDNP) used in the present study has the following advantage over conventional pulse radiolysis: the histidine radical is a weak chromophore, and transient absorption measurements restrict observation to the tyrosyl radical, [6] whereas the CIDNP technique enables the reactions of transient histidyl and tyrosyl radicals to be followed by NMR spectroscopic detection of signals of both residues. [7, 8] TR CIDNP has proved to be a good tool for establishing reaction mechanisms and for the determination of intra- and intermolecular electron-transfer rate constants. [9–11] The nonequilibrium population of nuclear spin states, known as CIDNP, arises from the dependence of the rate of intersystem crossing in a radical pair on the configuration of nuclear spins that have a nonzero hyperfine interaction (HFI) with an unpaired electron and results in anomalous NMR intensities for the nuclei involved. [5, 12] The essence of the TR CIDNP method in the investigation of reductive electron-transfer reactions is as follows: The radicals whose reduction is the subject of study are generated by quenching of a photoexcited dye molecule. At the geminate stage of the reaction, which is not resolved in our experiment, the formation of CIDNP is detected as NMR enhancement with no delay after the laser pulse. According to the spin-sorting nature of the S–T0 mechanism of CIDNP formation, the radicals that escape geminate termination are “marked” by nuclear polarization of the opposite sign to that of the geminate polarization. Upon the reduction of radicals in the bulk, this CIDNP is canceled out by the geminate polarization, and the decay of CIDNP is observed (the so-called CIDNP cancellation effect). The high sensitivity of the observed CIDNP kinetics to the rate of reductive electron transfer permits a quantitative study of this reaction. [9]

Aminium Cation Radical of Glycylglycine and its Deprotonation to Aminyl Radical in Aqueous Solution

The photochemical reaction between glycylglycine and triplet 4-carboxybenzophenone has been investigated using time-resolved chemically induced dynamic nuclear polarization (CIDNP). It is shown that the mechanism of the peptide reaction with triplet excited carboxybenzophenone is electron transfer from the amino group of the peptide, leading to the formation of an aminium cation radical that deprotonates to a neutral aminyl radical. Simulation of the CIDNP kinetics leads to an estimation of the paramagnetic relaxation time for the alpha-protons at the N-terminus at 20 to 40 mus with the best-fit value of 25 mus.

Time-resolved CIDNP and laser flash photolysis study of the photoreaction between triplet 2,2′-dipyridyl and guanosine-5′-monophosphate in water

Laser flash photolysis and time-resolved CIDNP have been applied to the investigation of the kinetics and the mechanism of the photoreaction between triplet 2,2′-dipyridyl (DP) and guanosine-5′-monophosphate (GMP) over a wide pH range in aqueous solution. The pH dependence of the rate constant kq of quenching the triplet dipyridyl by the nucleotide has been measured. Upon pH titration, four pairs of the reacting species contribute to the observed value of kq: pH 9.4, TDP and G(−H)−, with the corresponding quenching rate constants k1=1.3×109 M−1 s−1, k2=2.7×109 M−1 s−1, k3=1.6×108 M−1 s−1, k4=1.1×109 M−1 s−1. Based on LFP and CIDNP data, the established mechanism of the quenching reaction is hydrogen atom transfer in neutral solution (5.8<pH<9.4), and electron transfer in all other pH regions. Kinetic CIDNP measurements reveal that in acidic and basic solutions the CIDNP kinetics for GMP is determined by the degenerate electron exchange between the GMP radical and its parent molecule with the rate constants 1.3×108 M−1 s−1 (acidic conditions) and 4.0×107 M−1 s−1 (basic conditions). The nuclear paramagnetic relaxation time for the proton H8 of GMP, T1=20±5 μs, obtained from the simulations of the CIDNP kinetics, is found to be independent of the protonation state of the radical.

Effects of Surfactants on the Photosensitized Production of Tyrosine Radicals Studied by Photo-CIDNP¶

The influence of the surfactants sodium dodecyl sulphate, cetyltrimethyl‐ammonium bromide and triton X‐100 on the photochemically induced dynamic nuclear polarization (CIDNP) of N‐acetyl tyrosine has been investigated. Three photosensitizers were used to generate polarization: thionin, eosin Y and flavin mononucleotide. 600 MHz 1H photo‐CIDNP experiments, supported by laser flash photolysis transient absorption measurements, indicate that the neutral triton surfactant has no influence on the nuclear polarization, but that the other two, charged, amphiphiles affect the photochemistry in a variety of ways, depending on the surfactant concentration and the identity of the sensitizer.

Changing the Direction of Intramolecular Electron Transfer in Oxidized Dipeptides Containing Tryptophan and Tyrosine

Intramolecular electron transfer (IET) in the oxidized dipeptide Tyr-Trp was investigated in the pH range from 1.0 to 3.1 by the method of time-resolved chemically induced dynamic nuclear polarization. The results were compared with data obtained earlier for Trp-Tyr. Surprisingly, it was found that the direction of IET changes with the order of the amino acid residues in the peptide. For Tyr-Trp, the rate constant of electron transfer from tyrosine residue to tryptophanyl cation radical is below 1.2 × 10(4) s(-1), whereas for Trp-Tyr, the value of this rate constant is 5.5 × 10(5) s(-1). Conversely, for oxidized Tyr-Trp at pH range 2.1 and lower, electron transfer from tryptophan residue to tyrosyl radical is observed. The rate constant of this reaction is proportional to the concentration of protons in aqueous solution, and at pH 1.0 is equal to 6.5 × 10(5) s(-1). The change in direction of IET observed for oxidized Tyr-Trp dipeptide is presumably due to the positive charge at the N-terminal amino group of the peptide, which promotes electron transfer in the direction of the N-terminus.

CROSS-RELAXATION MECHANISM FOR THE FORMATION OF NUCLEAR POLARIZATION : A QUANTITATIVE TIME-RESOLVED CIDNP STUDY

Abstract The kinetics of the nuclear polarization formed during the photolysis of acetone in isopropanol- d 8 were analyzed quantitatively. Model calculations show that the spin-selective recombination of radicals gives rise to the electron polarization and, with regard to the electron-nuclear cross-relaxation, are adequate to describe the formation of the net nuclear polarization of the reaction products even if the solution contains only one type of radical. For the 2-hydroxy-2-propyl radicals at a magnetic field of 7 T, fitting the theoretical results to the experimental data gives the electron relaxation time T 1 e = 1.0 ± 0.2 μ s and the cross-relaxation time T x = 92 ± 18 μ s.

Modulation of the rate of reversible electron transfer in oxidized tryptophan and tyrosine containing peptides in acidic aqueous solution.

Time-resolved chemically induced dynamic nuclear polarization (CIDNP) was used to investigate reversible intramolecular electron transfer (IET) in short-lived oxidized peptides, which had different structures and contained tryptophan and tyrosine residues, in an acidic aqueous solution with a pH below the pKa of the tryptophanyl cation radical. The CIDNP kinetic data were obtained at the microsecond scale and were analyzed in detail to calculate the rate constants for electron transfer in both directions: from the tyrosine residue to the tryptophanyl cation radical, kf, and from the tryptophan residue to the neutral tyrosyl radical, kr. The charge of the terminal amino group and the presence of glycine and proline spacers were shown to strongly affect the rate constants of the reaction under study. Among these functional groups, the presence and the location of the positive charge on the amino group in close proximity to the cationic indolyl radical had the strongest effect on the rate constant of the forward IET from the tyrosine residue to the tryptophanyl radical cation, kf. This effect was manifested as an increase of 2 orders of magnitude in kf for a change in the linkage order between residues in the dipeptide: kf = 4 × 10(3) s(-1) for the oxidized Tyr-Trp increased to kf = 5.5 × 10(5) s(-1) in oxidized Trp-Tyr. The reverse rate constant for IET was less sensitive to the amino group charge. Moreover, the presence of glycine or proline spacers in the peptides with a tryptophan residue at the N-terminus not only reduced the IET rate constant but also shifted the equilibrium of the IET in the reaction under study toward the formation of tyrosyl radicals with respect to the peptide Trp-Tyr. That is, the glycine or proline spacers affected the difference in the reduction potential of the tryptophanyl and tyrosyl radicals.

Magnetic Resonance Characterization of One-Electron Oxidized Cyclic Dipeptides with Thioether Groups

Photo-oxidation of seven cyclic dipeptides containing methionine, Met, and/or S-methylcysteine, Cys(Me) by electron transfer from the sulfur atom was studied in aqueous solution by time-resolved and field dependent CIDNP (chemically induced dynamic nuclear polarization). Hyperpolarized high resolution NMR spectral patterns of the starting peptides detected immediately after pulsed laser excitation show signals of all protons that are bound to carbons neighboring the sulfur atom, thus proving the involvement of sulfur-centered cation radicals. The magnetic field dependence of CIDNP shows a pronounced maximum that is determined by the g-factors and hyperfine coupling constants of the transient radical species. From simulation of the experimental data obtained for the magnetic field dependences of CIDNP, three types of radical structures were characterized: (1) a linear sulfur-centered cation radical of the methionine (Met) residue (g = 2.0107 ± 0.0010) for cyclo-(d-Met-l-Met) (trans-configuration), cyclo-(d-Met-l-Cys(Me)) (trans-configuration), and cyclo-(Gly-Met); (2) a cyclic radical (S∴O)(+) (g = 2.0088 ± 0.0010) with a two-center three-electron bond (2c-3e) structure between the sulfur atom of the Cys(Me) residue and the oxygen atom of cyclo-(d-Met-l-Cys(Me)) and cyclo-(Gly-Cys(Me)); (3) a cyclic radical (S∴S)(+) (g = 2.013 ± 0.0020) with a two-center three-electron bond structure between the two sulfur atoms of the peptides cyclo-(l-Met-l-Met), cyclo-(l-Met-l-Cys(Me)), and cyclo-(l-Cys(Me)-l-Cys(Me)). In contrast, no indication of any type of cyclic radicals with a two-center three-electron bond between sulfur and nitrogen atoms was found. In addition, the hyperfine coupling constants (HFCCs) were determined.