Use of 2,3,5-F(3)Y-ß2 and 3-NH(2)Y-α2 to study proton-coupled electron transfer in Escherichia coli ribonucleotide reductase.

Escherichia coli ribonucleotide reductase is an α2ß2 complex that catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in α2, and the essential diferric-tyrosyl radical (Y(122)(•)) cofactor that initiates transfer of the radical to the active site cysteine in α2 (C(439)), 35 Å removed, is in ß2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y(122) → W(48) → Y(356) in ß2 to Y(731) → Y(730) → C(439) in α2) and reversible proton-coupled electron transfer (PCET). Recently, 2,3,5-F(3)Y (F(3)Y) was site-specifically incorporated in place of Y(356) in ß2 and 3-NH(2)Y (NH(2)Y) in place of Y(731) and Y(730) in α2. A pH-rate profile with F(3)Y(356)-ß2 suggested that as the pH is elevated, the rate-determining step of RNR can be altered from a conformational change to PCET and that the altered driving force for F(3)Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH(2)Y(731(730))-α2, ß2, CDP, and ATP resulted in detection of NH(2)Y radical (NH(2)Y(•)) intermediates capable of dNDP formation. In this study, the reaction of F(3)Y(356)-ß2, α2, CDP, and ATP has been examined by stopped-flow (SF) absorption and rapid freeze quench electron paramagnetic resonance spectroscopy and has failed to reveal any radical intermediates. The reaction of F(3)Y(356)-ß2, CDP, and ATP has also been examined with NH(2)Y(731)-α2 (or NH(2)Y(730)-α2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH(2)Y(•) formation that support a change in the rate-limiting step at elevated pH. The results together with kinetic simulations provide a guide for future studies to detect radical intermediates in the pathway.

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation.

Expressed protein ligation (EPL) allows semisynthesis of a target protein with site-specific incorporation of probes or unnatural amino acids at its N or C termini. Here, we describe the protocol that our lab has developed for incorporating fluorotyrosines (F(n)Ys) at residue 356 of the small subunit of Escherichia coli ribonucleotide reductase using EPL. In this procedure, the majority of the protein (residues 1-353 out of 375) is fused to an intein domain and prepared by recombinant expression, yielding the protein in a thioester-activated, truncated form. The remainder of the protein, a 22-mer peptide, is prepared by solid-phase peptide synthesis and contains the F(n)Y at the desired position. Ligation of the 22-mer peptide to the thioester-activated R2 and subsequent purification yield full-length R2 with the F(n)Y at residue 356. The procedure to generate 100 mg quantities of Y356F(n)Y-R2 takes 3-4 months.

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.

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.

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.

Turning on ribonucleotide reductase by light-initiated amino acid radical generation.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms, providing the monomeric precursors required for DNA replication and repair. The class I RNRs are composed of two subunits; the R1 subunit contains the active site for nucleotide reduction and allosteric effector binding sites, whereas the R2 subunit houses the essential diirontyrosyl (Y.) radical cofactor. A major unresolved issue is the mechanism by which the tyrosyl radical on R2 (Y122, Escherichia coli numbering) reversibly generates the transient thiyl radical (S.) on R1 that initiates nucleotide reduction. This intersubunit radical initiation is postulated to occur through a defined pathway involving conserved aromatic amino acids (R2: Y122, W48, Y356; R1: Y731, Y730) over a long distance of 35 A. A 20-mer peptide identical to the C-terminal tail of R2 (356-375) and containing Y356 is a competitive inhibitor with respect to R2, and it effectively blocks nucleotide reduction. We now report that a 21-mer peptide, in which a tryptophan has been incorporated at the N terminus of the 20th mer, can replace the R2 subunit and initiate nucleotide reduction by photoinitiated radical generation. The deoxynucleotide generated depends on the presence of allosteric effector and is pathway-dependent. Replacement of Y731 of R2 with phenylalanine prevents deoxynucleotide formation. These results provide direct evidence for the chemical competence of aromatic amino acid radicals and the importance of Y356 in R2 in the radical initiation process of the class I RNRs.

Generation of the R2 subunit of ribonucleotide reductase by intein chemistry: insertion of 3-nitrotyrosine at residue 356 as a probe of the radical initiation process.

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. The enzyme is composed of two subunits: R1 and R2. R1 contains the active site for nucleotide reduction and the allosteric effector sites that regulate the specificity and turnover rate. R2 contains the diferric-tyrosyl (Y(*)) radical cofactor that initiates nucleotide reduction by a putative long-range proton-coupled electron transfer (PCET) pathway over 35 A. This pathway is thought to involve specific amino acid radical intermediates (Y122 to W48 to Y356 within R2 to Y731 to Y730 to C439 within R1). In an effort to study radical initiation, R2 (375 residues) has been synthesized semisynthetically. R2 (residues 1-353), attached to an intein and a chitin binding domain, was constructed, and the protein was expressed (construct 1). This construct was then incubated with Fe(2+) and O(2) to generate the diferric-Y(*) cofactor, and the resulting protein was purified using a chitin affinity column. Incubation of construct 1 with 2-mercaptoethanesulfonic acid (MESNA) resulted in the MESNA thioester of R2 (1-353) (construct 2). A peptide containing residues 354-375 of R2 was generated using solid-phase peptide synthesis where 354, a serine in the wild-type (wt) R2, was replaced by a cysteine. Construct 2 and this peptide were ligated, and the resulting full-length R2 was separated from truncated R2 by anion-exchange chromatography. The purified protein had a specific activity of 350 nmol min(-1) mg(-1), identical to the same protein generated by site-directed mutagenesis when normalized for Y(*). As a first step in studying the radical initiation by PCET, R2 was synthesized with Y356 replaced by 3-nitrotyrosine (NO(2)Y). The protein is inactive (specific activity 1 x 10(-4) that of wt-R2), which permitted a determination of the pK(a) of the NO(2)Y in the R1/R2 complex in the presence of substrate and effectors. Under all conditions, the pK(a) was minimally perturbed. This has important mechanistic implications for the radical initiation process.

2,3-difluorotyrosine at position 356 of ribonucleotide reductase R2: a probe of long-range proton-coupled electron transfer.

Escherichia coli class I ribonucleotide reductase catalyzes the conversion of ribonucleotides to deoxyribonucleotides and consists of two subunits: R1 and R2. R1 possesses the active site, while R2 harbors the essential diferric-tyrosyl radical (Y*) cofactor. The Y* on R2 is proposed to generate a transient thiyl radical on R1, 35 A distant, through amino acid radical intermediates. To study the putative long-range proton-coupled electron transfer (PCET), R2 (375 residues) was prepared semisynthetically using intein technology. Y356, a putative intermediate in the pathway, was replaced with 2,3-difluorotyrosine (F2Y, pKa = 7.8). pH rate profiles (pH 6.5-9.0) of wild-type and F2Y-R2 were very similar. Thus, a proton can be lost from the putative PCET pathway without affecting nucleotide reduction. The current model involving H* transfer is thus unlikely.