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Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the de novo transformation of nucleoside 5- di(tri)phosphates [ND(T)Ps, where N is A, U, C, or G] to their corresponding deoxynucleotides. Despite the diversity of factors required for function and the low sequence conservation across RNRs, a unifying apparatus consolidating RNR activity is explored. We combine aspects of the protein subunit simplicity of class II RNR with a modified version of Escherichia coli class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme. The design of this RNR involves fusing a truncated form of the active site containing α subunit with the functionally important C- terminal tail of the radical- generating β subunit to render a chimeric RNR. Inspired by a recent cryo- EM structure, a [Re] photooxidant is located adjacent to Y356[β], which is an essential component of the radical transport pathway in class I RNRs. Combination of this RNR photochimera with cytidine diphosphate (CDP), adenosine triphosphate (ATP), and light resulted in the generation of Y356• along with production of deoxycytidine diphosphate (dCDP) and cytosine. The photoproducts reflect an active site chemistry consistent with both the consensus mechanism of RNR and chemistry observed when RNR is inactivated by mechanism- based inhibitors in the active site. The enzymatic activity of the RNR photochimera in the absence of any β metallocofactor highlights the adaptability of the 10- stranded αβ barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs. [ABSTRACT FROM AUTHOR]

The authors review the publications on ribonucleotide reductases. The topics of interest include the electron transfer principles, proton-coupled electron transfer, and electron transfer in biological systems.

Ribonucleotide reductases (RNRs) convert nucleotides to deoxynucleotides in all organisms. Activity of the class Ia and Ib RNRs requires a stable tyrosyl radical (Ycan be generated by the reaction of O2 with a diferrous cluster on the β subunit to form active diferric-Y. Recent experiments have demonstrated, however, that in vivo the class Ib RNR contains an active dimanganese(III)-Y. The similar metal binding sites of the class Ia and Ib RNRs, their ability to bind both MnII and FeII, and the activity of the class Ib RNR with both diferric-Ynganese(III)-Ys raise the intriguing question of how the cell prevents mismetallation of these essential enzymes. The presence of the class Ib RNR in numerous pathogenic bacteria also highlights the importance of manganese for these organisms’ growth and virulence. [ABSTRACT FROM AUTHOR]

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 Å. 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. [ABSTRACT FROM AUTHOR]

New insights have been gained into the formation of the di-iron tyrosyl radical cofactor, which is essential for nucleotide reduction in the class I ribonucleotide reductases. Recent advances include Insight into the function of the tyrosyl radical in initiation of nucleotide reduction. [Copyright &y& Elsevier]

Studies the principles that govern the formation of protein radicals and their roles in catalysis. Involvement of non-amino acid in enzymatic systems; Chemical properties of enzymes; Conversion of ribonucleotide reductases; Detection of the thiyl radical.

Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2′-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide reduction reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, a and b. Because of the transient subunit association, an atomic resolution structure of an active a2b2 RNR complex has been elusive. We used a doubly substituted b2, E52Q/(2,3,5)-trifluorotyrosine122-b2, to trap wild-type a2 in a long-lived a2b2 complex. We report the structure of this complex by means of cryo–electron microscopy to 3.6-angstrom resolution, allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity. [ABSTRACT FROM AUTHOR]

The mechanism of radical transport in the α2 (R1) subunit of class I E. coli ribonucleotide reductase (RNR) has been investigated by the phototriggered generation of a tyrosyl radical, •Y356, on a 20-mer peptide bound to α2. This peptide, Y-R2C19, is identical to the C-terminal peptide tail of the β2 (R2) subunit and is a known competitive inhibitor of binding of the native β2 protein to α2. •Y356 radical initiation is prompted by excitation (λ ≥ 300 nm) of a proximal anthraquinone, Anq, or benzophenone, BPA, chromophore on the peptide. Transient absorption spectroscopy has been employed to kinetically characterize the radical-producing step by time resolving the semiquinone anion (Anq•-), ketyl radical (•BPA), and Y• photoproducts on (i) BPA-Y and Anq-Y dipeptides and (ii) BPNAnq-Y-R2C19 peptides. Light- initiated, single-turnover assays have been carried out with the peptide/α2 complex in the presence of [14C]-labeled cytidine 5′-diphosphate substrate and ATP allosteric effector. We show that both the Anq- and BPA-containing peptides are competent in deoxycytidine diphosphate formation and turnover occurs via Y731 to Y730 to C439 pathway-dependent radical transport in α2. Experiments with the Y730F mutant exclude a direct superexchange mechanism between C439 and Y731 and are consistent with a PCET model for radical transport in which there is a unidirectional transport of the electron and proton transport among residues of α2. [ABSTRACT FROM AUTHOR]

The article discusses a study which observes a transient tyrosine radical competent for initiating turnover in a photochemical ribonucleotide reductase. Proton-coupled electron transfer is vital to the generation and transport of amino acid radicals within class I Escherichia coli ribonucleotide reductase (RNR). RNR catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. It also plays a vital role in deoxyribonucleic acid replication and repair.

The class III ribonucleotide reductases (RNRs) are glycyl radical (G•) enzymes that provide the balanced pool of deoxynucleotides required for DNA synthesis and repair in many facultative and obligate anaerobic bacteria and archaea. Unlike the class I and II RNRs, where reducing equivalents for the reaction are delivered by a redoxin (thioredoxin, glutaredoxin, or NrdH) via a pair of conserved active site cysteines, the class III RNRs examined to date use formate as the reductant. Here, we report that reaction of the Escherichia coli class III RNR with CTP (substrate) and ATP (allosteric effector) in the absence of formate leads to loss of the G• concomitant with stoichiometric formation of a new radical species and a “trapped” cytidine derivative that can break down to cytosine. Addition of formate to the new species results in recovery of 80% of the G• and reduction of the cytidine derivative, proposed to be 3’-keto-deoxycytidine, to dCTP and a small amount of cytosine. The structure of the new radical has been identified by 9.5 and 140 GHz EPR spectroscopy on isotopically labeled varieties of the protein to be a thiosulfuranyl radical [RSSR2]•, composed of a cysteine thiyl radical stabilized by an interaction with a methionine residue. The presence of a stable radical species on the reaction pathway rationalizes the previously reported [3H]-(kcat/KM) isotope effect of 2.3 with [3H]-formate, requiring formate to exchange between the active site and solution during nucleotide reduction. Analogies with the disulfide anion radical proposed to provide the reducing equivalent to the 3’-keto-deoxycytidine intermediate by the class I and II RNRs provide further evidence for the involvement of thiyl radicals in the reductive half-reaction catalyzed by all RNRs. [ABSTRACT FROM AUTHOR]

The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or "hole" over a distance of 35 Å from the stable diferric/tyrosyl-radical (Y122•) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep "hopping" mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122• by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y122• Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme's essential Y122•, but also directly in catalysis. [ABSTRACT FROM AUTHOR]

The Escherichia co/i ribonucleotide reductase (RNR), composed of two subunits (R1 and R2), catalyzes the conversion of nucleotides to deoxynucleotides. Substrate reduction requires that a tyrosyl radical (Y122·) in R2 generate a transient cysteinyl radical (C439·) in Al through a pathway thought to involve amino acid radical intermediates [Y122· → W48 → Y356 within R2 to Y731 → V730 → C439 within R1]. To study this radical propagation process, we have synthesized R2 semisynthetically using intein technology and replaced V356 with a variety of fluorinated tyrosine analogues (2,3-F2Y, 3,5-F2Y, 2,3,5-F3Y, 2,3,6-F3Y, and F4Y) 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 PKaS that range from 5.6 to 7.8. The pH rate profiles of deoxynucleotide production by these FnY356-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 V356· using these analogues. As the difference in potential of the FnY· 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 Y356 is a redox-active amino acid on the radical-propagation pathway. On the basis of our previous studies with 3-NO2Y356-R2, we assume that 2,3,5-F3Y356, 2,3,6-F3Y356, and F4Y356-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 W48 and Y356 of R2 and Y731 of R1 does not play a central role in triggering radical initiation nor is hydrogen-atom transfer between these residues obligatory for radical propagation. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides providing the monomeric precursors required for DNA replication and repair. The class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 has the active site where nucleotide reduction occurs, and R2 contains the diiron tyrosyl radical (Y•) cofactor essential for radical initiation on R1. Mechanism-based inhibitors, such as 2'-azido-2'-deoxyuridine-5'-diphosphate (N³UDP), have provided much insight into the reduction mechanism. N³UDP is a stoichiometric inactivator that, upon interaction with RNR, results in loss of the Y• in R2 and formation of a nitrogen-centered radical (N•) covalently attached to C225 (R-S-N•-X) in the active site of R1. N2; is lost prior to N• formation, and after its formation, stoichiometric amounts of 2-methylene-3-furanone, pyrophosphate, and uracil are also generated. On the basis of the hyperfine interactions associated with N•, it was proposed that N• is also covalently attached to the nucleotide through either the oxygen of the 3'-OH (R-S-N•-O-R') or the 3'-C (R-S-N•-C-OH). To distinguish between the proposed structures, the inactivation was carried out with 3'-[17O]-N3UDP and N• was examined by 9 and 140 GHz EPR spectroscopy. Broadening of the N• signal was detected and the spectrum simulated to obtain the [17O] hypertine tensor. DFT calculations were employed to determine which structures are in best agreement with the simulated hypertine tensor and our previous ESEEM data. The results are most consistent with the R-S-N•-C-OH structure and provide evidence for the trapping of a 3'-ketonucleotide in the reduction process. [ABSTRACT FROM AUTHOR]

Describes the generation of a tryptophan radical in high quantum yield from a novel amino acid analog. Role of ribonucleotide reductase in catalyzing the conversion of nucleotides to deoxynucleotides; Assignment of transients to the radicals; Usefulness of the method for generating tryptophan radical in the study of electron transfer.

We describe a general method for the unimolecular photochemical generation of tyrosyl radicals from a diaryl oxalate ester platform on the nanosecond time scale, Symmetric and asymmetric tyrosine oxalate esters have been prepared in gram quantities, Direct photocleavage of the oxalate linkage by laser flash photolysis affords tyrosyl radicals within 50 ns. This approach provides unnatural caged amino acids that may be incorporated into model and biological systems for the study of proton-coupled electron transfer in enzymatic catalysis. [ABSTRACT FROM AUTHOR]

The article reports that Escherichia coli ribonucleotide reductase catalyzes the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates. The active protein is composed of two homodimeric subunits (R1 and R2) thought to form a 1:1 complex. R1 binds the NDP substrates, and houses the essential cysteines required for catalysis and the binding sites for the allosteric effectors that govern substrate specificity and turnover rates. R2 harbors the essential diiron tyrosyl radical cofactor on residue.