title: pH Rate profiles of FnY356-R2s (n = 2, 3, 4) in Escherichia coli ribonucleotide reductase: evidence that Y356 is a redox-active amino acid along the radical propagation pathway.
abstract: The Escherichia coli ribonucleotide reductase (RNR), composed of two subunits (R1 and R2), catalyzes the conversion of nucleotides to deoxynucleotides. Substrate reduction requires that a tyrosyl radical (Y(122)*) in R2 generate a transient cysteinyl radical (C(439)*) in R1 through a pathway thought to involve amino acid radical intermediates [Y(122)* --> W(48) --> Y(356) within R2 to Y(731) --> Y(730) --> C(439) within R1]. To study this radical propagation process, we have synthesized R2 semisynthetically using intein technology and replaced Y(356) with a variety of fluorinated tyrosine analogues (2,3-F(2)Y, 3,5-F(2)Y, 2,3,5-F(3)Y, 2,3,6-F(3)Y, and F(4)Y) that have been described and characterized in the accompanying paper. These fluorinated tyrosine derivatives have potentials that vary from -50 to +270 mV relative to tyrosine over the accessible pH range for RNR and pK(a)s that range from 5.6 to 7.8. The pH rate profiles of deoxynucleotide production by these F(n)()Y(356)-R2s are reported. The results suggest that the rate-determining step can be changed from a physical step to the radical propagation step by altering the reduction potential of Y(356)* using these analogues. As the difference in potential of the F(n)()Y* relative to Y* becomes >80 mV, the activity of RNR becomes inhibited, and by 200 mV, RNR activity is no longer detectable. These studies support the model that Y(356) is a redox-active amino acid on the radical-propagation pathway. On the basis of our previous studies with 3-NO(2)Y(356)-R2, we assume that 2,3,5-F(3)Y(356), 2,3,6-F(3)Y(356), and F(4)Y(356)-R2s are all deprotonated at pH > 7.5. We show that they all efficiently initiate nucleotide reduction. If this assumption is correct, then a hydrogen-bonding pathway between W(48) and Y(356) of R2 and Y(731) of R1 does not play a central role in triggering radical initiation nor is hydrogen-atom transfer between these residues obligatory for radical propagation.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139-4307, USA.
authors.forename: Mohammad R
authors.initials: MR
authors.lastname: Seyedsayamdost
authors.forename: Cyril S
authors.initials: CS
authors.lastname: Yee
authors.forename: Steven Y
authors.initials: SY
authors.lastname: Reece
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Nocera
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: C066447:tyrosyl radical Y(D); D014443:Tyrosine; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit