Site-Specific Replacement of a Conserved Tyrosine in Ribonucleotide Reductase with an Aniline Amino Acid: A Mechanistic Probe for a Redox-Active Tyrosine

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Abstract
An aniline-based amino acid provides a powerful mechanistic probe for redox-active tyrosines, affording a general method for elucidating the sequence of proton and electron transfer events during side-chain oxidation in biological systems. Intein technology allows Y356 to be site-specifically replaced with p-aminophenylalanine (PheNH2) on the R2 subunit of the class I ribonucleotide reductase. Analysis of the pH rate profile of Y356PheNH2−R2 strongly suggests that the mechanism of long-distance intrasubunit radical transfer through position 356 proceeds with electron transfer prior to proton transfer. In addition, we propose that radical transfer through position 356 only becomes rate-limiting upon raising the reduction potential of the residue at that location and is not affected by protonation state of either the ground state or oxidized amino acid.