Site-specific replacement of a conserved tyrosine in ribonucleotide reductase with an aniline amino acid: a mechanistic probe for a redox-active tyrosine.
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.
Chang
Michelle C Y
MC
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Yee
Cyril S
CS
Nocera
Daniel G
DG
Stubbe
JoAnne
J
D006860:Hydrogen Bonding; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D011522:Protons; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D011522:Protons; C009722:4-aminophenylalanine; D014443:Tyrosine; D010649:Phenylalanine; D012264:Ribonucleotide Reductases