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Photochemical radical initiation is a powerful tool for studying radical initiation and transport in biology. Ribonucleotide reductases (RNRs). which catalyze the conversion of nucleotides to deoxynucleotides in all organisms, are an exemplar of radical mediated transformations in biology. Class Ia RNRs are composed of two subunits: α2 and β2. As a method to initiate radical formation photochemicatly within β2, a single surface-exposed cysteine of the β2 subunit of Escherichia coli Class Ia RNR has, been labeled (98%) with a photooxidant ([Re] = tricarbonyl (1,10-phenanthroline)(methylpyridyl)rhenium(l)). The labeling was achieved by incubation of S355C-β2 with the 4-(bromomethyl) pyridyl derivative of [Re] to yield the labeled species, [Re]-S355C-β2. Steady-state and time-resolved emission experiments reveal that the metal-to-ligand charge transfer (MLCT) excited-state ³[Re] is not significantly perturbed after bioconjugation and is available as a phototrigger of tyrosine radical at position 356 in the β2 subunit; transient absorption spectroscopy reveals that the radical lives for microseconds. The work described herein provides a platform for photochemical radical initiation and study of proton-coupled electron transfer (PCET) in the β2 subunit of RNR, from which radical initiation and transport for this enzyme originates. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is essential in all organisms. Class I RNRs consist of two homodimeric subunits: α2 and β2. The α subunit contains the site of nucleotide reduction, and the β subunit contains the essential diferric-tyrosyl radical (Y•) cofactor. Escherichia coli contains genes encoding two class I RNRs (Ia and Ib) and a class III RNR, which is active only under anaerobic conditions. Its class Ia RNR, composed of NrdA (α) and NrdB (β), is expressed under normal aerobic growth conditions. The class lb RNR, composed of NrdE (α) and NrdF (β), is expressed under oxidative stress and iron-limited growth conditions. Our laboratory is interested in pathways of cofactor biosynthesis and maintenance in class I RNRs and modulation of Y• levels as a means of regulating RNR activity. Our recent studies have implicated a [2Fe2S]-ferredoxin, YfaE, in the NrdB diferric-Y• maintenance pathway and possibly in the biosynthetic and regulatory pathways. Here, we report that NrdI is a flavodoxin counterpart to YfaE for the class lb RNR. It possesses redox properties unprecedented for a flavodoxin (Eox/sq = 264 ± 17 mV and Esq/hq = -255 ± 17 my) that allow it to mediate a two-electron reduction of the diferric cluster of NrdF via two successive one-electron transfers. Data presented support the presence of a distinct maintenance pathway for NrdEF, orthogonal to that for NrdAB involving YfaE. [ABSTRACT FROM AUTHOR]

Discusses the mechanism of long-range electron transfer (ET) and proton-coupled electron transfer (PCET) in biology, using the radical initiation process of class I ribonucleotide reductases (RNR) as a focal point. Theoretical framework of ET and PCET; Principles of ET; Methods to study radical initiation in RNR.

The article focuses on the study about the forward and reverse electron transfer with the Y356dihydroxyphenylalanine (DOPA)-beta2 Heterodimer of Escherichia coli ribonucleotide reductases (RNRs). Results show that the amounts of DOPA suggest that the interaction of the alpha2/beta2 complex is asymmetric. In addition, results also constitutes the first direct observation of reverse electron transfer between residues 356 and 122 in the beta subunit of RNR.

Escherichia coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and β2. β2 contains a stable di-iron tyrosyl radical (Y122•) cofactor required to generate a thiyl radical (C439•) in α2 over a distance of 35 Å, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y122 W48[?] ⇆ Y356 in β2, across the subunit interface to Y731 ⇆ Y730 ⇆ C439 in α2. Within α2 a colinear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH2Y) replaced Y730 in α2, and this mutant was incubated with β2, cytidine 5′-diphosphate, and adenosine 5′-triphosphate to generate a NH2Y730• in D2O. [2H]-Electron-nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained, and together with DFT models of α2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C439 and Y731. A third proton was assigned to a water molecule in close proximity (2.2 Å O-H⋯O distance) to residue 730. The calculations also suggest that the unusual g-values measured for NH2Y730• are consistent with the combined effect of the hydrogen bonds to Cys439 and Tyr731, both nearly perpendicular to the ring plane of NH2Y730. The results provide the first experimental evidence for the hydrogen-bond network between the pathway residues in α2 of the active RNR complex, for which no structural data are available. [ABSTRACT FROM AUTHOR]

E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5′-diphosphates into 2′-deoxynucleotides and is composed of two subunits: α2 and β2. During turnover, a stable tyrosyl radical (Y•) at Y122-β2 reversibly oxidizes C439 in the active site of α2. This radical propagation step is proposed to occur over 35 Å, to use specific redox-active tyrosines (Y122 and Y356 in β2, Y731 and Yβ in α2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO2Y, pKa 7.1) has been incorporated in place of Y122, Y731, and Y730 to probe how the protein environment perturbs each pKa in the presence of the second subunit, substrate (S), and allosteric effector (E). The activity of each mutant is <4 × 10-3 that of the wild-type (wt) subunit. The [NO2Y730]-α2 and [NO2Y731]-α2 each exhibit a pKa of 7.8-8.0 with E and E/β2. The pKa of [NO2Y730]-α2 is elevated to 8.2-8.3 in the S/E/β2 complex, whereas no further perturbation is observed for [NO2Y731]-α2. Mutations in pathway residues adjacent to the NOαY that disrupt H-bonding minimally perturb its pKa. The pKa of NO2Y122-β2 alone or with α2/S/E is >9.6. X-ray crystal structures have been obtained for all [NO2Y]-α2 mutants (2.1-3.1 Å resolution), which show minimal structural perturbation compared to wt-α2. Together with the pKa of the previously reported NO2Y356-β2 (7.5 in the α2/S/E complex; Yee, C. et al. Biochemistry 2003, 42, 14541-14552), these studies provide a picture of the protein environment of the ground state at each Y in the PCET pathway, and are the starting point for understanding differences in PCET mechanisms at each residue in the pathway. [ABSTRACT FROM AUTHOR]

How each metalloprotein assembles the correct metal at the proper binding site presents challenges to the cell. The di-iron enzyme ribonucleotide reductase (RNR) uses a diferric-tyrosyl radical (FeIII2-Y*) cofactor to initiate nucleotide reduction. Assembly of this cofactor requires O2, FeII, and a reducing equivalent. Recent studies show that RNR cofactor biosynthesis shares the same source of iron, in the form of [2Fe-2S]-GSH2 from the monothiol glutaredoxin Grx3/4, and the same electron source, in the form of the Dre2-Tah18 electron transfer chain, with the cytosolic iron-sulfur protein assembly (CIA) machinery required for maturation of [4Fe-4S] clusters in cytosolic and nuclear proteins. Here, we further investigated the interplay between the formation of the FeIII2-Y* cofactor in RNR and the cellular iron-sulfur (Fe-S) protein biogenesis pathways by examining both the iron loading into the RNR β subunit and the RNR catalytic activity in yeast mutants depleted of individual components of the mitochondrial iron-sulfur cluster assembly (ISC) and the CIA machineries. We found that both iron loading and cofactor assembly in RNR are dependent on the ISC machinery. Wealso found that Dre2 is required for RNR cofactor formation but appears to be dispensable for iron loading. None of the CIA components downstream of Dre2 was required for RNR cofactor formation. Thus, the pathways for RNR and Fe-S cluster biogenesis bifurcate after the Dre2-Tah18 step. We conclude that RNR cofactor biogenesis requires the ISC machinery to mature the Grx3/4 and Dre2 Fe-S proteins, which then function in iron and electron delivery to RNR, respectively. [ABSTRACT FROM AUTHOR]

Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (α and β), which form an α2β2 complex that catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleotides (dNDPs). β2 contains the essential tyrosyl radical (Y122•) that generates a thiyl radical (C439•) in α2 where dNDPs are made. This oxidation occurs over 35 Å through a pathway of amino acid radical intermediates (Y122 → [W48] → Y356 in β2 to Y731 → Y730 → C439 in α2). However, chemistry is preceded by a slow protein conformational change(s) that prevents observation of these intermediates. 2,3,5-Trifluorotyrosine site-specifically inserted at position 122 of β2 (F3Y•-β2) perturbs its conformation and the driving force for radical propagation, while maintaining catalytic activity (1.7 s-1). Rapid freeze-quench electron paramagnetic resonance spectroscopy and rapid chemical-quench analysis of the F3Y•-β2, α2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y356• and 0.5 equiv of dCDP, both at 30 s-1. In the absence of an external reducing system, Y356• reduction occurs concomitant with F3Y reoxidation (0.4 s-1) and subsequent to oxidation of all α2s. In the presence of a reducing system, a burst of dCDP (0.4 equiv at 22 s-1) is observed prior to steady-state turnover (1.7 s-1). The [Y356•] does not change, consistent with rate-limiting F3Y reoxidation. The data support a mechanism where Y122• is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active α2β2 complex to complete the catalytic cycle. [ABSTRACT FROM AUTHOR]

Tyrosyl radicals (Y·s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (FnYs, n = 1-4) are promising tools for studying the mechanism of Y· formation and reactivity, as their pKa values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F3Y) and demonstrate their ability to charge an orthogonal tRNA with a series of FnYs while maintaining high specificity over Y. An evolved aaRS is then used to incorporate FnYs site-specifically into the two subunits (α2 and β2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y·s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with FnY(s), and the resulting proteins are isolated in good yields. FnYs incorporated at position 122 of β2, the site of a stable Y· in wild-type RNR, generate long-lived FnY·s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y122(2,3,5)F3Y-β2 is energetically and/or conformationally modulated in such a way that the enzyme retains its activity but a new on-pathway Y· can accumulate. The distinct EPR properties of the 2,3,5-F3Y· facilitate spectral subtractions that make detection and identification of new Y·s straightforward [ABSTRACT FROM AUTHOR]

E. coil ribonucleotide reductase (ANA) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 Å from a tyrosyl radical (Y122•) within the β2 subunit to a cysteine residue (C439) within the α2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y122 → W48 → Y356 in β2, across the subunit interface to Y731 → Y730 → C439 in α2. Using the suppressor tRNAlaminoacyl-tRNA synthetase (AS) methodology, 3-aminotyrosine has been incorporated into position 730 in α2. Incubation of this mutant with β2, substrate, and allosteric effector resulted in loss of the Y122• and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH2Y•). In the current study [15N]- and [14N]-NH2Y730• have been generated in H2O and D2O and characterized by continuous wave 9 3Hz EPA and pulsed EPA spectroscopies at 9, 94, and 180 3Hz. The data give insight into the electronic and molecular structure of NH2Y730•. The g tensor (gx = 2.0052, gy = 2.0042, gz = 2.0022), the orientation of the β-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH2 moiety are consistent with an intramolecular hydrogen bond within NH2Y730•. This analysis is an essential first step in using the detailed structure of NH2Y730• to formulate a model for a PCET mechanism within α2 and for use of NH2Y in other systems where transient Y•s participate in catalysis. [ABSTRACT FROM AUTHOR]

Photochemical ribonucleotide reductases (photoRNRs) have been developed to study the proton-coupled electron transfer (PCET) mechanism of radical transport in Escherichia coli class I ribonucleotide reductase (RNR). The transport of the effective radical occurs along several conserved aromatic residues across two subunits: β2(.Y122 → W48 → Y356) → α2(Y731 → Y730 → C439). The current model for RNR activity suggests that radical transport is strongly controlled by conformational gating. The C-terminal tail peptide (Y-βC19) of β2 is the binding determinant of β2 to β2 and contains the redox active Y356 residue. A photoRNR has been generated synthetically by appending a Re(bpy)(CO)3CN ([Re]) photo-oxidant next to Y356 of the 20-mer peptide. Emission from the [Re] center dramatically increases upon peptide binding, serving as a probe for conformational dynamics and the protonation state of Y356. The diffusion coefficient of [Re]-Y-βC19 has been measured (kd1 = 6.1 x 10-7 cm-1 s-1), along with the dissociation rate constant for the [Re]-Y-βC19-α2 complex (7000 s-1 > koff > 400 s-1). Results from detailed time-resolved emission and absorption spectroscopy reveal biexponential kinetics, suggesting a large degree of conformational flexibility in the [Re]-Y-βC19-α2 complex that engenders partitioning of the N-terminus of the peptide into both bound and solvent-exposed fractions. [ABSTRACT FROM AUTHOR]

Focuses 2,3-difluorotyrosine at position 356 of ribonucleotide reductase R2. Probe of long-range proton-coupled electron transfer (PCET); Proposed PCET pathway and distanced based on crystal structures of R1 and R2.