Radical-translocation intermediates and hurdling of pathway defects in "super-oxidized" (Mn(IV)/Fe(IV)) Chlamydia trachomatis ribonucleotide reductase.
ABSTRACT: A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y(•)) or a Mn(IV)/Fe(III) cluster in its ? subunit to oxidize a cysteine residue ?35 Å away in its ? subunit, generating a thiyl radical that abstracts hydrogen (H(•)) from the substrate. With either oxidant, the inter-subunit "hole-transfer" or "radical-translocation" (RT) process is thought to occur by a "hopping" mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wild-type (wt) RNR. The Mn(IV)/Fe(III) cofactor of Chlamydia trachomatis RNR assembles via a Mn(IV)/Fe(IV) intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when ? is present, accumulating radicals with EPR spectra characteristic of Y(•)'s. The dependence of Y(•) accumulation on the presence of substrate suggests that RT within this "super-oxidized" enzyme form is gated by the protein, and the failure of a ? variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y(•)(s) in the wt enzyme reside(s) within the RT pathway. Remarkably, two variant ? proteins having pathway substitutions rendering them inactive in their Mn(IV)/Fe(III) states can generate the pathway Y(•)'s in their Mn(IV)/Fe(IV) states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the "hurdling" of engineered defects in the RT pathway.
Project description:Ribonucleotide reductases (RNRs) catalyze conversion of ribonucleotides to deoxyribonucleotides in all organisms via a free-radical mechanism that is essentially conserved. In class I RNRs, the reaction is initiated and terminated by radical translocation (RT) between the ? and ? subunits. In the class Ic RNR from Chlamydia trachomatis (Ct RNR), the initiating event converts the active S = 1 Mn(IV)/Fe(III) cofactor to the S = 1/2 Mn(III)/Fe(III) "RT-product" form in the ? subunit and generates a cysteinyl radical in the ? active site. The radical can be trapped via the well-described decomposition reaction of the mechanism-based inactivator, 2'-azido-2'-deoxyuridine-5'-diphosphate, resulting in the generation of a long-lived, nitrogen-centered radical (N(•)) in ?. In this work, we have determined the distance between the Mn(III)/Fe(III) cofactor in ? and N(•) in ? to be 43 ± 1 Å by using double electron-electron resonance experiments. This study provides the first structural data on the Ct RNR holoenzyme complex and the first direct experimental measurement of the inter-subunit RT distance in any class I RNR.
Project description:The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) utilizes a Mn/Fe heterobinuclear cofactor, rather than the Fe/Fe cofactor found in the ? (R2) subunit of the class Ia enzymes, to react with O2. This reaction produces a stable Mn(IV)Fe(III) cofactor that initiates a radical, which transfers to the adjacent ? (R1) subunit and reacts with the substrate. We have studied the Mn(IV)Fe(III) cofactor using nuclear resonance vibrational spectroscopy (NRVS) and absorption (Abs)/circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD spectroscopies to obtain detailed insight into its geometric/electronic structure and to correlate structure with reactivity; NRVS focuses on the Fe(III), whereas MCD reflects the spin-allowed transitions mostly on the Mn(IV). We have evaluated 18 systematically varied structures. Comparison of the simulated NRVS spectra to the experimental data shows that the cofactor has one carboxylate bridge, with Mn(IV) at the site proximal to Phe127. Abs/CD/MCD/VTVH MCD data exhibit 12 transitions that are assigned as d-d and oxo and OH(-) to metal charge-transfer (CT) transitions. Assignments are based on MCD/Abs intensity ratios, transition energies, polarizations, and derivative-shaped pseudo-A term CT transitions. Correlating these results with TD-DFT calculations defines the Mn(IV)Fe(III) cofactor as having a ?-oxo, ?-hydroxo core and a terminal hydroxo ligand on the Mn(IV). From DFT calculations, the Mn(IV) at site 1 is necessary to tune the redox potential to a value similar to that of the tyrosine radical in class Ia RNR, and the OH(-) terminal ligand on this Mn(IV) provides a high proton affinity that could gate radical translocation to the ? (R1) subunit.
Project description:Catalysis by a class I ribonucleotide reductase (RNR) begins when a cysteine (C) residue in the alpha(2) subunit is oxidized to a thiyl radical (C(*)) by a cofactor approximately 35 A away in the beta(2) subunit. In a class Ia or Ib RNR, a stable tyrosyl radical (Y(*)) is the C oxidant, whereas a Mn(IV)/Fe(III) cluster serves this function in the class Ic enzyme from Chlamydia trachomatis (Ct). It is thought that, in either case, a chain of Y residues spanning the two subunits mediates C oxidation by forming transient "pathway" Y(*)s in a multistep electron transfer (ET) process that is "gated" by the protein so that it occurs only in the ready holoenzyme complex. The drug hydroxyurea (HU) inactivates both Ia/b and Ic beta(2) subunits by reducing their C oxidants. Reduction of the stable cofactor Y(*) (Y122(*)) in Escherichia coli class Ia beta(2) is faster in the presence of alpha(2) and a substrate (CDP), leading to speculation that HU might intercept a transient ET pathway Y(*) under these turnover conditions. Here we show that this mechanism is one of two that are operant in HU inactivation of the Ct enzyme. HU reacts with the Mn(IV)/Fe(III) cofactor to give two distinct products: the previously described homogeneous Mn(III)/Fe(III)-beta(2) complex, which forms only under turnover conditions (in the presence of alpha(2) and the substrate), and a distinct, diamagnetic Mn/Fe cluster, which forms approximately 900-fold less rapidly as a second phase in the reaction under turnover conditions and as the sole outcome in the reaction of Mn(IV)/Fe(III)-beta(2) only. Formation of Mn(III)/Fe(III)-beta(2) also requires (i) either Y338, the subunit-interfacial ET pathway residue of beta(2), or Y222, the surface residue that relays the "extra electron" to the Mn(IV)/Fe(IV) intermediate during activation of beta(2) but is not part of the catalytic ET pathway, and (ii) W51, the cofactor-proximal residue required for efficient ET between either Y222 or Y338 and the cofactor. The combined requirements for the catalytic subunit, the substrate, and, most importantly, a functional surface-to-cofactor electron relay system imply that HU effects the Mn(IV)/Fe(III) --> Mn(III)/Fe(III) reduction by intercepting a Y(*) that forms when the ready holoenzyme complex is assembled, the ET gate is opened, and the Mn(IV) oxidizes either Y222 or Y338.
Project description:The beta(2) subunit of a class Ia or Ib ribonucleotide reductase (RNR) is activated when its carboxylate-bridged Fe(2)(II/II) cluster reacts with O(2) to oxidize a nearby tyrosine (Y) residue to a stable radical (Y(*)). During turnover, the Y(*) in beta(2) is thought to reversibly oxidize a cysteine (C) in the alpha(2) subunit to a thiyl radical (C(*)) by a long-distance ( approximately 35 A) proton-coupled electron-transfer (PCET) step. The C(*) in alpha(2) then initiates reduction of the 2' position of the ribonucleoside 5'-diphosphate substrate by abstracting the hydrogen atom from C3'. The class I RNR from Chlamydia trachomatis (Ct) is the prototype of a newly recognized subclass (Ic), which is characterized by the presence of a phenylalanine (F) residue at the site of beta(2) where the essential radical-harboring Y is normally found. We recently demonstrated that Ct RNR employs a heterobinuclear Mn(IV)/Fe(III) cluster for radical initiation. In essence, the Mn(IV) ion of the cluster functionally replaces the Y(*) of the conventional class I RNR. The Ct beta(2) protein also autoactivates by reaction of its reduced (Mn(II)/Fe(II)) metal cluster with O(2). In this reaction, an unprecedented Mn(IV)/Fe(IV) intermediate accumulates almost stoichiometrically and decays by one-electron reduction of the Fe(IV) site. This reduction is mediated by the near-surface residue, Y222, a residue with no functional counterpart in the well-studied conventional class I RNRs. In this review, we recount the discovery of the novel Mn/Fe redox cofactor in Ct RNR and summarize our current understanding of how it assembles and initiates nucleotide reduction.
Project description:The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the ? subunit oxidizes a cysteine residue ~35 Å away in the ? subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn(IV) ion of a Mn(IV)/Fe(III) cluster, which assembles in a reaction between O(2) and the Mn(II)/Fe(II) complex of ?. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn(IV) ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn(IV) ion most likely resides in this site (i.e., (1)Mn(IV)/(2)Fe(III)), but a subsequent computational study favored its occupation of site 2 ((1)Fe(III)/(2)Mn(IV)). In this work, we have sought to resolve the location of the Mn(IV) ion in Ct RNR-? by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn(IV)/Fe(III) clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both (1)Mn(II)/(2)Fe(II) and (1)Fe(II)/(2)Mn(II) complexes are competent to react with O(2) to produce the corresponding oxidized states. However, with diminished Mn(II) loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these "low-Mn" samples on a per-Mn basis implies that the (1)Mn(IV)/(2)Fe(III)-? is at least the more active of the two oxidized forms and may be the only active form.
Project description:Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y(•)) generated by oxidation of a reduced dinuclear metal cluster. The Fe(III)2-Y(•) cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe(II)2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn(III)2-Y(•) cofactor in their NrdF subunit. Mn(II)2-NrdF does not react with O2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdIhq, which, in the presence of O2, reacts to form the Mn(III)2-Y(•) cofactor. Here we investigate the mechanism of assembly of the Mn(III)2-Y(•) cofactor in Bacillus subtilis NrdF. Cluster assembly from Mn(II)2-NrdF, NrdI(hq), and O2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdI(hq) reduces O2 to O2(•-) (40-48 s(-1), 0.6 mM O2), the O2(•-) channels to and reacts with Mn(II)2-NrdF to form a Mn(III)Mn(IV) intermediate (2.2 ± 0.4 s(-1)), and the Mn(III)Mn(IV) species oxidizes tyrosine to Y(•) (0.08-0.15 s(-1)). Controlled production of O2(•-) by NrdIhq during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn(II)2 cluster with O2 and satisfies the requirement for an "extra" reducing equivalent in Y(•) generation.
Project description:Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides into deoxyribonucleotides necessary for DNA biosynthesis. Unlike the conventional class Ia RNRs which use a diiron cofactor in their subunit R2, the active site of the RNR-R2 from Chlamydia trachomatis (Ct) contains a Mn/Fe cofactor. The detailed structure of the Mn/Fe core has yet to be established. In this paper we evaluate six different structural models of the Ct RNR active site in the Mn(iv)/Fe(iii) state by using Mössbauer parameter calculations and simulations of Mn/Fe extended X-ray absorption fine structure (EXAFS) spectroscopy, and we identify a structure similar to a previously proposed DFT-optimized model that shows quantitative agreement with both EXAFS and Mössbauer spectroscopic data.
Project description:High-valent iron and manganese complexes effect some of the most challenging biochemical reactions known, including hydrocarbon and water oxidations associated with the global carbon cycle and oxygenic photosynthesis, respectively. Their extreme reactivity presents an impediment to structural characterization, but their biological importance and potential chemical utility have, nevertheless, motivated extensive efforts toward that end. Several such intermediates accumulate during activation of class I ribonucleotide reductase (RNR) ? subunits, which self-assemble dimetal cofactors with stable one-electron oxidants that serve to initiate the enzyme's free-radical mechanism. In the class I-c ? subunit from Chlamydia trachomatis, a heterodinuclear Mn(II)/Fe(II) complex reacts with dioxygen to form a Mn(IV)/Fe(IV) intermediate, which undergoes reduction of the iron site to produce the active Mn(IV)/Fe(III) cofactor. Herein, we assess the structure of the Mn(IV)/Fe(IV) activation intermediate using Fe- and Mn-edge extended X-ray absorption fine structure (EXAFS) analysis and multifrequency pulse electron paramagnetic resonance (EPR) spectroscopy. The EXAFS results reveal a metal-metal vector of 2.74-2.75 Å and an intense light-atom (C/N/O) scattering interaction 1.8 Å from the Fe. Pulse EPR data reveal an exchangeable deuterium hyperfine coupling of strength |T| = 0.7 MHz, but no stronger couplings. The results suggest that the intermediate possesses a di-?-oxo diamond core structure with a terminal hydroxide ligand to the Mn(IV).
Project description:The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) employs a Mn(IV)/Fe(III) cofactor in each monomer of its ?2 subunit to initiate nucleotide reduction. The cofactor forms by reaction of Mn(II)/Fe(II)-?2 with O2. Previously, in vitro cofactor assembly from apo ?2 and divalent metal ions produced a mixture of two forms, with Mn at site 1 (Mn(IV)/Fe(III)) or site 2 (Fe(III)/Mn(IV)), of which the more active Mn(IV)/Fe(III) product predominates. Here we have addressed the basis for metal site selectivity by determining X-ray crystal structures of apo, Mn(II), and Mn(II)/Fe(II) complexes of Ct ?2. A structure obtained anaerobically with equimolar Mn(II), Fe(II), and apoprotein reveals exclusive incorporation of Mn(II) at site 1 and Fe(II) at site 2, in contrast to the more modest site selectivity achieved previously. Site specificity is controlled thermodynamically by the apoprotein structure, as only minor adjustments of ligands occur upon metal binding. Additional structures imply that, by itself, Mn(II) binds in either site. Together, the structures are consistent with a model for in vitro cofactor assembly in which Fe(II) specificity for site 2 drives assembly of the appropriately configured heterobimetallic center, provided that Fe(II) is substoichiometric. This model suggests that use of a Mn(IV)/Fe(III) cofactor in vivo could be an adaptation to Fe(II) limitation. A 1.8 Å resolution model of the Mn(II)/Fe(II)-?2 complex reveals additional structural determinants for activation of the cofactor, including a proposed site for side-on (?(2)) addition of O2 to Fe(II) and a short (3.2 Å) Mn(II)-Fe(II) interionic distance, promoting formation of the Mn(IV)/Fe(IV) activation intermediate.
Project description:Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates in iron-limited and oxidative stress conditions. We have recently demonstrated in vitro that this RNR is active with both diferric-tyrosyl radical (Fe(III)(2)-Y(•)) and dimanganese(III)-Y(•) (Mn(III)(2)-Y(•)) cofactors in the ?2 subunit, NrdF [Cotruvo, J. A., Jr., and Stubbe, J. (2010) Biochemistry 49, 1297-1309]. Here we demonstrate, by purification of this protein from its endogenous levels in an E. coli strain deficient in its five known iron uptake pathways and grown under iron-limited conditions, that the Mn(III)(2)-Y(•) cofactor is assembled in vivo. This is the first definitive determination of the active cofactor of a class Ib RNR purified from its native organism without overexpression. From 88 g of cell paste, 150 ?g of NrdF was isolated with ?95% purity, with 0.2 Y(•)/?2, 0.9 Mn/?2, and a specific activity of 720 nmol min(-1) mg(-1). Under these conditions, the class Ib RNR is the primary active RNR in the cell. Our results strongly suggest that E. coli NrdF is an obligate manganese protein in vivo and that the Mn(III)(2)-Y(•) cofactor assembly pathway we have identified in vitro involving the flavodoxin-like protein NrdI, present inside the cell at catalytic levels, is operative in vivo.