Spectroscopy and Reactivity of a Photogenerated Tryptophan Radical in a Structurally Defined Protein Environment

Abstract

The mutations W48F, Y72F, H83Q, Q107H, Y108W were carried out stepwise using Invitrogene primers and a QuickChange® kit (Stratagene). The modified DNA plasmid (verified sequencing) was incorporated into BL21(DE3) E. Coli (Novagene). Bacteria were grown in a standard LB broth to an optical density of ~2. After harvesting, the cells were subjected to lysis by the osmotic shock method. The periplasmic extrudate was buffered with 25 mM NaOAc pH 4.5, 5 mM of either Zn(II) or Cu(II) acetate, and allowed to sit for 3 days at 4 o C. The precipitate was removed by centrifugation and the supernatant liquid concentrated by an Amicon Ultrafiltration (Millipore) device using an YM-10 membrane (Millipore). The concentrated periplasmic extrudate was then washed several times with 25 mM NaOAc pH 4.5 to remove remaining DNA. Azurin was purified and labeled as previously reported (Di Bilio et al., JACS 1998, 120,

A Tryptophan Neutral Radical in the Oxidized State of Versatile Peroxidase from Pleurotus eryngii

“…It is very possible that the true catalytic intermediate in VP is the tryptophan cation radical, which becomes (in the absence of substrate), during the time needed for freezing, the protein deprotonated at the indole nitrogen to form the probably more stable observed tryptophan neutral radical. Formation of a relatively stable deprotonated neutral radical was also observed for a tryptophan radical generated by photoinduced electron transfer in azurin (54). Moreover, the existence of exposed tyrosine residues may lead to enzyme inactivation due to intermolecular cross-linking (between the phenoxy radicals formed), as reported during spontaneous decay of cytochrome c peroxidase Compound I (74).…”

“…For a tryptophan radical, the field corresponding to 94 GHz EPR (3.3 tesla) does still not represent the so-called high-field limit, where the g-tensor shifts exceed the hyperfine splitting and all three g-tensor components are separated in the spectrum. This situation was nearly met at 285 GHz EPR in a study of a tryptophan radical in azurin (54). However, the hf-tensor resolution in the spectrum was lost at 285 GHz.…”

Characterization of NocL Involved in Thiopeptide Nocathiacin I Biosynthesis

“…The EPR spectra of glycyl radicals characterized so far are characteristic of a 2-fold splitting caused by the nuclear hyperfine coupling of the hydrogen with the unpaired electron on C␣ (32,33). The spectra of Trp radicals, on the other hand, are anisotropic and variable in different cases (34,35). We examined the glycyl radical and/or possibly the Trp radical during the steady state of NocL catalysis by EPR spectroscopy under conditions that focus on and enhance the free radical signal.…”

l-Tryptophan Radical Cation Electron Spin Resonance Studies: Connecting Solution-Derived Hyperfine Coupling Constants with Protein Spectral Interpretations

“…2) 25 . (2) In this equation, ρ C π is the unpaired electron spin density on the ring carbon adjacent to the methylene group, B′ is a small constant which is usually set equal to 0, B″ is a proportionality constant which varies with the type of free radical (neutral, anion, or cation 26 ) and θ is the dihedral angle between the α-carbon p z axis and the projected C β H β bond (Fig. 4) 27 .…”