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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.

SUBMITTER: Connor HD

PROVIDER: S-EPMC2644638 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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

Connor Henry D HD Sturgeon Bradley E BE Mottley Carolyn C Sipe Herbert J HJ Mason Ronald P RP

Journal of the American Chemical Society 20080424 20

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 expe ...[more]

PMID: 18433127

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Project description:Cytochrome P450 enzymes are heme based monoxygenases that catalyse a range of oxygen atom transfer reactions with various substrates, including aliphatic and aromatic hydroxylation as well as epoxidation reactions. The active species is short-lived and difficult to trap and characterize experimentally, moreover, it reacts in a regioselective manner with substrates leading to aliphatic hydroxylation and epoxidation products, but the origin of this regioselectivity is poorly understood. We have synthesized a model complex and studied it with low-pressure Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry (MS). A novel approach was devised using the reaction of [FeIII(TPFPP)]+ (TPFPP = meso-tetrakis(pentafluorophenyl)porphinato dianion) with iodosylbenzene as a terminal oxidant which leads to the production of ions corresponding to [FeIV(O)(TPFPP+?)]+. This species was isolated in the gas-phase and studied in its reactivity with a variety of olefins. Product patterns and rate constants under Ideal Gas conditions were determined by FT-ICR MS. All substrates react with [FeIV(O)(TPFPP+?)]+ by a more or less efficient oxygen atom transfer process. In addition, substrates with low ionization energies react by a charge-transfer channel, which enabled us to determine the electron affinity of [FeIV(O)(TPFPP+?)]+ for the first time. Interestingly, no hydrogen atom abstraction pathways are observed for the reaction of [FeIV(O)(TPFPP+?)]+ with prototypical olefins such as propene, cyclohexene and cyclohexadiene and also no kinetic isotope effect in the reaction rate is found, which suggests that the competition between epoxidation and hydroxylation - in the gas-phase - is in favour of substrate epoxidation. This notion further implies that P450 enzymes will need to adapt their substrate binding pocket, in order to enable favourable aliphatic hydroxylation over double bond epoxidation pathways. The MS studies yield a large test-set of experimental reaction rates of iron(iv)-oxo porphyrin cation radical complexes, so far unprecedented in the gas-phase, providing a benchmark for calibration studies using computational techniques. Preliminary computational results presented here confirm the observed trends excellently and rationalize the reactivities within the framework of thermochemical considerations and valence bond schemes.

Dataset Information

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

Publications

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

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