L-tryptophan radical cation electron spin resonance studies: connecting solution-derived hyperfine coupling constants with protein spectral interpretations

Abstract

Fast-flow electron spin resonance (ESR) spectroscopy has been used to detect a free radical formed from the reaction of l-tryptophan with Ce (4+) in an acidic aqueous environment. Computer simulations of the ESR spectra from l-tryptophan and several isotopically modified forms strongly support the conclusion that the l-tryptophan radical cation has been detected by ESR for the first time. The hyperfine coupling constants (HFCs) determined from the well-resolved isotropic ESR spectra support experimental and computational efforts to understand l-tryptophan's role in protein catalysis of oxidation-reduction processes. l-Tryptophan HFCs facilitated the simulation of fast-flow ESR spectra of free radicals from two related compounds, tryptamine and 3-methylindole. Analysis of these three compounds' beta-methylene hydrogen HFC data along with equivalent l-tyrosine data has led to a new computational method that can distinguish between these two amino acid free radicals in proteins without dependence on isotope labeling, electron-nuclear double resonance, or high-field ESR. This approach also produces geometric parameters (dihedral angles for the beta-methylene hydrogens) that should facilitate protein site assignment of observed l-tryptophan radicals as has been done for l-tyrosine radicals.

Fig. 1. l-Tryptophan radical cation ESR spectrum

ESR fast-flow spectrum of l-tryptophan radical cation produced in a system of l- tryptophan, Ce⁴⁺, and H₂SO₄ having concentrations of 0.5 mM, 0.25 mM, and 0.225 M, respectively. Equal volumes of aqueous solutions of l-tryptophan/H₂SO₄ and Ce⁴⁺/ H₂SO₄ were mixed milliseconds before entering the ESR flat cell at a total flow rate of 60 mL/min. ESR spectra were recorded at 20 mW microwave power, 0.1mT field modulation, 6.00 mT field sweep width, 163 ms time constant, 81 ms conversion time and 86 scans of 1024 data points (Red). Also shown is the l-tryptophan radical cation simulation spectrum with coupling constants given in Table 1 (Green).

Fig. 2. Tryptamine radical cation ESR spectrum

ESR fast-flow spectrum of tryptamine radical cation produced in a system of tryptamine, Ce⁴⁺, and H₂SO₄ having concentrations of 0.5 mM, 0.25 mM, and 0.225 M, respectively. Equal volumes of aqueous solutions of tryptamine/H₂SO₄ and Ce⁴⁺/H₂SO₄ were mixed milliseconds before entering the ESR flat cell at a total flow rate of 30 mL/min. ESR spectra were recorded at 8 mW microwave power, 0.025 mT field modulation, 6.00 mT field sweep width, 163 ms time constant, 81 ms conversion time, and 230 scans of 2048 data points (Red). Also shown is the tryptamine radical cation simulation spectrum with coupling constants given in Table 1 (Green).

Fig. 3. 3-methylindole radical cation ESR spectrum

ESR fast-flow spectrum of 3-methyl indole radical cation produced in a system of 3-methyl indole, Ce⁴⁺, acetic acid, and H₂SO₄ having concentrations of 0.5 mM, 0.25 mM, 1.75 M, and 0.113 M, respectively. Equal volumes of aqueous solutions of 3-methyl indole/acetic acid and Ce⁴⁺/H₂SO₄ were mixed milliseconds before entering the ESR flat cell at a total flow rate of 50 mL/min. ESR spectra were recorded at 20 mW microwave power, 0.10 mT field modulation, 8.00 mT field sweep width, 163 ms time constant, 81 ms conversion time, and 79 scans of 1024 data points (Red). Also shown is the 3-methyl- indole radical cation simulation spectrum with coupling constants given in Table 1 (Green).

Fig. 4

l- Tryptophan molecular model (A) and corresponding β-methylene hydrogen representation (B).

Fig. 5

l-Tryptophan conformers that contribute to β-methylene a_β^H inequivalency.