The active form of Chlamydia trachomatis ribonucleotide reductase R2 protein contains a heterodinuclear Mn(IV)/Fe(III) cluster with S = 1 ground state.
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ABSTRACT:
SUBMITTER: Jiang W
PROVIDER: S-EPMC3870001 | biostudies-literature | 2007 Jun
REPOSITORIES: biostudies-literature
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The active form of Chlamydia trachomatis ribonucleotide reductase R2 protein contains a heterodinuclear Mn(IV)/Fe(III) cluster with S = 1 ground state.
Jiang Wei W Bollinger J Martin JM Krebs Carsten C
Journal of the American Chemical Society 20070527 24
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Project description: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.
| S-EPMC3931446 | biostudies-literature
Project description:The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) employs a Mn(IV)/Fe(III) cofactor in each monomer of its β2 subunit to initiate nucleotide reduction. The cofactor forms by reaction of Mn(II)/Fe(II)-β2 with O2. Previously, in vitro cofactor assembly from apo β2 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 β2. 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)-β2 complex reveals additional structural determinants for activation of the cofactor, including a proposed site for side-on (η(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.
| S-EPMC3821933 | biostudies-literature
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) β 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 β 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-μ-oxo diamond core structure with a terminal hydroxide ligand to the Mn(IV).
| S-EPMC5518598 | biostudies-literature
Project description:Models for the Mn-Fe active site structure of ribonucleotide reductase (RNR) from pathogenic bacteria Chlamydia trachomatis (Ct) in different oxidation states have been studied in this paper, using broken-symmetry density functional theory (DFT) incorporated with the conductor like screening (COSMO) solvation model and also with finite-difference Poisson-Boltzmann self-consistent reaction field (PB-SCRF) calculations. The detailed structures for the reduced Mn(II)-Fe(II), the met Mn(III)-Fe(III), the oxidized Mn(IV)-Fe(III) and the superoxidized Mn(IV)-Fe(IV) states are predicted. The calculated properties, including geometries, (57)Fe Mossbauer isomer shifts and quadrupole splittings, and (57)Fe and (55)Mn electron nuclear double resonance (ENDOR) hyperfine coupling constants, are compared with the available experimental data. The Mössbauer and energetic calculations show that the (mu-oxo, mu-hydroxo) models better represent the structure of the Mn(IV)-Fe(III) state than the di-mu-oxo models. The predicted Mn(IV)-Fe(III) distances (2.95 and 2.98 A) in the (mu-oxo, mu-hydroxo) models are in agreement with the extended X-ray absorption fine structure (EXAFS) experimental value of 2.92 A (Younker et al. J. Am. Chem. Soc. 2008, 130, 15022-15027). The effect of the protein and solvent environment on the assignment of the Mn metal position is examined by comparing the relative energies of alternative mono-Mn(II) active site structures. It is proposed that if the Mn(II)-Fe(II) protein is prepared with prior addition of Mn(II) or with Mn(II) richer than Fe(II), Mn is likely positioned at metal site 2, which is further from Phe127.
| S-EPMC2919573 | biostudies-literature
Project description:Ribonucleotide reductases (RNRs) are the sole de novo source of deoxyribonucleotides for DNA synthesis and repair across all organisms and carry out their reaction via a radical mechanism. RNR from Chlamydia trachomatis generates its turnover-initiating cysteinyl radical by long-range reduction of a Mn(IV)/Fe(III) cofactor, producing a Mn(III)/Fe(III) intermediate. Herein, we characterize the protonation states of the inorganic ligands in this reduced state using advanced pulse electron paramagnetic resonance (EPR) spectroscopy and 2H-isotope labeling. A strongly coupled deuteron is observed by hyperfine sublevel correlation (HYSCORE) spectroscopy experiments and indicates the presence of a bridging hydroxo ligand. Isotope-dependent EPR line broadening analysis and the magnitude of the estimated Mn-Fe exchange coupling constant together suggest a μ-oxo/μ-hydroxo core. Two distinct signals detected in electron-nuclear double resonance (ENDOR) spectra are attributable to less strongly coupled hydrons of a terminal water ligand to Mn(III). Together, these experiments imply that the reduced cofactor has a mixed μ-oxo/μ-hydroxo core with a terminal water ligand on Mn(III). This structural assignment sheds light generally on the reactivity of Mn/Fe heterobimetallic sites and, more specifically, on the proton-coupling in the electron transfer that initiates ribonucleotide reduction in this subclass of RNRs.
| S-EPMC11883743 | biostudies-literature
Project description:The class Ic ribonucleotide reductase from Chlamydia trachomatis ( Ct) uses a stable Mn(IV)/Fe(III) cofactor to initiate nucleotide reduction by a free-radical mechanism. Extended X-ray absorption fine structure (EXAFS) spectroscopy and density functional theory (DFT) calculations are used to postulate a structure for this cofactor. Fe and Mn K-edge EXAFS data yield an intermetallic distance of approximately 2.92 A. The Mn data also suggest the presence of a short 1.74 A Mn-O bond. These metrics are compared to the results of DFT calculations on 12 cofactor models derived from the crystal structure of the inactive Fe 2(III/III) form of the protein. Models are differentiated by the protonation states of their bridging and terminal OH X ligands as well as the location of the Mn(IV) ion (site 1 or 2). The models that agree best with experimental observation feature a mu-1,3-carboxylate bridge (E120), terminal solvent (H 2O/OH) to site 1, one mu-O bridge, and one mu-OH bridge. The site-placement of the metal ions cannot be discerned from the available data.
| S-EPMC3890746 | biostudies-literature
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.
| S-EPMC2693138 | biostudies-literature
Project description:Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O-O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.
| S-EPMC7615503 | biostudies-literature
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.
| S-EPMC5840866 | biostudies-literature
Project description:The ferritin superfamily contains several protein groups that share a common fold and metal coordinating ligands. The different groups utilize different dinuclear cofactors to perform a diverse set of reactions. Several groups use an oxygen-activating di-iron cluster, while others use di-manganese or heterodinuclear Mn/Fe cofactors. Given the similar primary ligand preferences of Mn and Fe as well as the similarities between the binding sites, the basis for metal specificity in these systems remains enigmatic. Recent data for the heterodinuclear cluster show that the protein scaffold per se is capable of discriminating between Mn and Fe and can assemble the Mn/Fe center in the absence of any potential assembly machineries or metal chaperones. Here we review the current understanding of the assembly of the heterodinuclear cofactor in the two different protein groups in which it has been identified, ribonucleotide reductase R2c proteins and R2-like ligand-binding oxidases. Interestingly, although the two groups form the same metal cluster they appear to employ partly different mechanisms to assemble it. In addition, it seems that both the thermodynamics of metal binding and the kinetics of oxygen activation play a role in achieving metal specificity.
| S-EPMC4118035 | biostudies-literature