Generation of a Tryptophan Radical in High Quantum Yield from a Novel Amino Acid Analog Using Near-UV/Visible Light

Abstract

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides. An initial step in this process has been postulated to be a coupled proton and electron transfer between the essential tyrosyl radical (•Y122) on the R2 subunit and a cysteine residue (C439) on the R1 subunit, the site of nucleotide reduction. One of our long-term goals is to generate the cysteinyl radical on R1 in the absence of R2 using a photoreactive peptide that binds to the R2 binding site of R1. Toward this end, the synthesis of an N-hydroxypyridine-2-thione derivative of tryptophan, designed to generate a tryptophan radical with a quantum of near-UV/visible light, is described. Laser flash photolysis (λexc = 355 nm) of this derivative gave rise to a transient absorption spectrum which showed a ground state depletion centered at its absorption maximum near 370 nm and a broad absorption band at 490 nm. The latter band partially decayed with a lifetime of ∼3 μs to leave an underlying band at 510 nm with a much longer lifetime. We have assigned the transient at 490 nm to the 2-pyridylthiyl radical and the transient at 510 nm to the neutral tryptophan radical. The addition of methyl methacrylate, a known thiyl radical quencher, suppressed the transient at 490 nm while the addition of trifluoroacetic acid caused a shift in the tryptophan radical absorbance to 560 nm consistent with protonation to form the corresponding cation radical. Using comparative actinometry, the quantum yields for N−O bond cleavage and tryptophan radical formation were found to be 1.0 ± 0.1. This selective method for generating tryptophan radical, when incorporated into the appropriate peptide, may make this a useful probe for the study of electron transfer between the R1 and R2 subunits of RNR and may be generally applicable to other systems.