Pulsed ELDOR spectroscopy measures the distance between the two tyrosyl dadicals in the R2 subunit of the E. coli ribonucleotide reductase

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). This RNR is composed of two homodimeric subunits: R1 and R2. R1 binds the NDPs in the active site, and R2 harbors the essential di-iron tyrosyl radical (Y*) cofactor. In this paper, we used PELDOR, a method that detects weak electron-electron dipolar coupling, to make the first direct measurement of the distance between the two Y*'s on each monomer of R2. In the crystal structure of R2, the Y*'s are reduced to tyrosines, and consequently R2 is inactive. In R2, where the Y*'s assume a well-defined geometry with respect to the protein backbone, the PELDOR method allows measurement of a distance of 33.1 +/- 0.2 A that compares favorably to the distance (32.4 A) between the center of mass of the spin density distribution of each Y* on each R2 monomer from the structure. The experiments provide the first direct experimental evidence for two Y*'s in a single R2 in solution.     

Site-specific replacement of Y356 with 3,4-dihydroxyphenylalanine in the beta2 subunit of E. coli ribonucleotide reductase

E. coli ribonucleotide reductase (RNR), composed of the homodimeric subunits alpha2 and beta2, catalyzes the conversion of nucleotides to deoxynucleotides via complex radical chemistry. The radical initiation process involves a putative proton-coupled electron transfer (PCET) pathway over 35 A between alpha2 and beta2. Y356 in beta2 has been proposed to lie on this pathway. To test this model, intein technology has been used to make beta2 semi-synthetically in which Y356 is replaced with a DOPA-amino acid. Analysis of this mutant with alpha2 and various combinations of substrate and effector by SF UV-vis spectroscopy and EPR methods demonstrates formation of a DOPA radical concomitant with disappearance of the tyrosyl radical, which initiates the reaction. The results reveal that Y356 lies on the PCET pathway and demonstrate the first kinetically competent conformational changes prior to ET. They further show that substrate binding brings about rapid conformational changes which place the complex into its active form(s) and suggest that the RNR complex is asymmetric.     

Rational polynomial representation of ribonucleotide reductase activity

Background  

Recent data suggest that ribonucleotide reductase (RNR) exists not only as a heterodimer R1₂R2₂ of R1₂ and R2₂ homodimers, but also as tetramers R1₄R2₄ and hexamers R1₆R2₆. Recent data also suggest that ATP binds the R1 subunit at a previously undescribed hexamerization site, in addition to its binding to previously described dimerization and tetramerization sites. Thus, the current view is that R1 has four NDP substrate binding possibilities, four dimerization site binding possibilities (dATP, ATP, dGTP, or dTTP), two tetramerization site binding possibilities (dATP or ATP), and one hexamerization site binding possibility (ATP), in addition to possibilities of unbound site states. This large number of internal R1 states implies an even larger number of quaternary states. A mathematical model of RNR activity which explicitly represents the states of R1 currently exists, but it is complicated in several ways: (1) it includes up to six-fold nested sums; (2) it uses different mathematical structures under different substrate-modulator conditions; and (3) it requires root solutions of high order polynomials to determine R1 proportions in mono-, di-, tetra- and hexamer states and thus RNR activity as a function of modulator and total R1 concentrations.     

Computational studies of reaction mechanisms of methane monooxygenase and ribonucleotide reductase

An overview of the computational efforts made by our group during the last few years in the field of nonheme diiron proteins is presented. Through application of ab initio methodology to a reasonable set of molecular models, significant progress is made in understanding how the soluble Methane Monooxygenase system achieves the hydroxylation of methane and how the catalytic cycle of Ribonucleotide Reductase is initiated. In particular, the current studies reveal in more detail (1) the nature of key intermediates in the reaction cycles of these two metalloenzymes, (2) details of how the iron centers regulate the systems, and (3) important aspects of how the carboxylate ligands in the active sites may tailor the enzymatic needs of the metalloprotein. This knowledge also leads to novel connections between the two enzymes. The coordinative unsaturation and carboxylate shifts investigated herein are two properties that are likely to be of more general impact in nonheme proteins. The control of the redox chemistry of the enzyme by the binuclear metal center, also analyzed here, should find common ground among other bimetallic systems as well.     

Site-specific insertion of 3-aminotyrosine into subunit alpha2 of E. coli ribonucleotide reductase: direct evidence for involvement of Y730 and Y731 in radical propagation

E. coli ribonucleotide reductase (RNR) catalyzes the production of deoxynucleotides using complex radical chemistry. Active RNR is composed of a 1:1 complex of two subunits: alpha2 and beta2. Alpha2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. Beta2 contains the stable diiron tyrosyl radical (Y122.) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 A between the Y122 in beta2 and C439 in the active site of alpha2. A docking model of alpha2beta2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y730 and Y731 in alpha2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-alpha2 and Y731NH2Y-alpha2 have been generated with high fidelity in yields of 4-6 mg/g of cell paste. These mutants have been examined by stopped flow UV-vis and EPR spectroscopies in the presence of beta2, CDP, and ATP. The results reveal formation of an NH2Y radical (NH2Y730. or NH2Y731.) in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-alpha2s make deoxynucleotides. These results show that the NH2Y. can oxidize C439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within alpha2. The observed NH2Y. may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover.     

Deciphering radical transport in the large subunit of class I ribonucleotide reductase

Incorporation of 2,3,6-trifluorotyrosine (F(3)Y) and a rhenium bipyridine ([Re]) photooxidant into a peptide corresponding to the C-terminus of the β protein (βC19) of Escherichia coli ribonucleotide reductase (RNR) allows for the temporal monitoring of radical transport into the α2 subunit of RNR. Injection of the photogenerated F(3)Y radical from the [Re]-F(3)Y-βC19 peptide into the surface accessible Y731 of the α2 subunit is only possible when the second Y730 is present. With the Y-Y established, radical transport occurs with a rate constant of 3 × 10(5) s(-1). Point mutations that disrupt the Y-Y dyad shut down radical transport. The ability to obviate radical transport by disrupting the hydrogen bonding network of the amino acids composing the colinear proton-coupled electron transfer pathway in α2 suggests a finely tuned evolutionary adaptation of RNR to control the transport of radicals in this enzyme.     

PELDOR study on the tyrosyl radicals in the R2 protein of mouse ribonucleotide reductase

Pulse electron-electron double resonance (PELDOR) has been employed to measure the distance between the putative tyrosyl radicals in the two halves of the R2 subunit from mouse ribonucleotide reductase. The results provide experimental evidence that the active, tyrosyl radical containing mouse R2 subunit forms a homodimeric form in solution. The distance between the two tyrosyl radicals present in the dimer was determined to be 3.25 +/- 0.05 nm.     

大肠杆菌异源表达pMMO活性中心pmoB及生物催化研究

颗粒甲烷单加氧酶（pMMO）是甲烷氧化菌中催化甲烷氧化生成甲醇的一种酶．Methylococcus capsulatus IMV 3021的pMMO活性位点是pmoB亚基,该亚基是一种可溶性蛋白．我们研究将pmoB亚基进行异源表达及生物催化活性的验证．当培养基中烟酰胺腺嘌呤二核苷酸（NADH）浓度为5 mmol／L时,可以观察到异源表达pmoB亚基具有催化甲烷氧化成甲醇活性,生成甲醇浓度为1．04 mmol／L．研究pMMO活性对于开发能直接将甲烷转化成甲醇的新型、环保催化剂有非常重要意义．     

EPR distance measurements support a model for long-range radical initiation in E. coli ribonucleotide reductase

The class I E. coli ribonucleotide reductase, composed of homodimers of R1 and R2, catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. The reduction process involves the tyrosyl radical on R2 that generates a transient thiyl radical on R1 over a proposed distance of 35 A. A mechanism-based inhibitor, 2'-azido-2'-deoxyuridine-5'-diphosphate, that reduces the tyrosyl radical on R2 and forms a nitrogen-centered radical on R1 has provided a method to measure the diagonal distance between the two subunits. PELDOR and DQC paramagnetic resonance methods give rise to a distance of 48 A, similar to that calculated from a docking model of the R1 and R2 structures.     

Variable coordination geometries at the diiron(II) active site of ribonucleotide reductase R2

The R2 subunit of Escherichia coli ribonucleotide reductase contains a dinuclear iron center that generates a catalytically essential stable tyrosyl radical by one electron oxidation of a nearby tyrosine residue. After acquisition of Fe(II) ions by the apo protein, the resulting diiron(II) center reacts with O(2) to initiate formation of the radical. Knowledge of the structure of the reactant diiron(II) form of R2 is a prerequisite for a detailed understanding of the O(2) activation mechanism. Whereas kinetic and spectroscopic studies of the reaction have generally been conducted at pH 7.6 with reactant produced by the addition of Fe(II) ions to the apo protein, the available crystal structures of diferrous R2 have been obtained by chemical or photoreduction of the oxidized diiron(III) protein at pH 5-6. To address this discrepancy, we have generated the diiron(II) states of wildtype R2 (R2-wt), R2-D84E, and R2-D84E/W48F by infusion of Fe(II) ions into crystals of the apo proteins at neutral pH. The structures of diferrous R2-wt and R2-D48E determined from these crystals reveal diiron(II) centers with active site geometries that differ significantly from those observed in either chemically or photoreduced crystals. Structures of R2-wt and R2-D48E/W48F determined at both neutral and low pH are very similar, suggesting that the differences are not due solely to pH effects. The structures of these "ferrous soaked" forms are more consistent with circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopic data and provide alternate starting points for consideration of possible O(2) activation mechanisms.     

DFT calculations for intermediate and active states of the diiron center with a tryptophan or tyrosine radical in Escherichia coli ribonucleotide reductase

Class Ia ribonucleotide reductase subunit R2 contains a diiron active site. In this paper, active-site models for the intermediate X-Trp48(?+) and X-Tyr122(?), the active Fe(III)Fe(III)-Tyr122(?), and the met Fe(III)Fe(III) states of Escherichia coli R2 are studied, using broken-symmetry density functional theory incorporated with the conductor-like screening solvation model. Different structural isomers and different protonation states have been explored. Calculated geometric, energetic, Mo?ssbauer, hyperfine, and redox properties are compared with available experimental data. Feasible detailed structures of these intermediate and active states are proposed. Asp84 and Trp48 are most likely the main contributing residues to the result that the transient Fe(IV)Fe(IV) state is not observed in wild-type class Ia E. coli R2. Asp84 is proposed to serve as a proton-transfer conduit between the diiron cluster and Tyr122 in both the tyrosine radical activation pathway and the first steps of the catalytic proton-coupled electron-transfer pathway. Proton-coupled and simple redox potential calculations show that the kinetic control of proton transfer to Tyr122(?) plays a critical role in preventing reduction from the active Fe(III)Fe(III)-Tyr122(?) state to the met state, which is potentially the reason why Tyr122(?) in the active state can be stable over a very long period.     

Local and global effects of metal binding within the small subunit of ribonucleotide reductase

Each beta-protomer of the small betabeta subunit of Escherichia coli ribonucleotide reductase (R2) contains a binuclear iron cluster with inequivalent binding sites: Fe(A) and Fe(B). In anaerobic Fe(II) titrations of apoprotein under standard buffer conditions, we show that the majority of the protein binds only one Fe(II) atom per betabeta subunit. Additional iron occupation can be achieved upon exposure to O2 or in high glycerol buffers. The differential binding affinity of the A- and B-sites allows us to produce heterobinuclear Mn(II)Fe(II) and novel Mn(III)Fe(III) clusters within a single beta-protomer of R2. The oxidized species are produced with H2O2 addition. We demonstrate that no significant exchange of metal occurs between the A- and B-sites, and thus the binding of the first metal is under kinetic control, as has been suggested previously. The binding of first Fe(II) atom to the active site in a beta-protomer (betaI) induces a global protein conformational change that inhibits access of metal to the active site in the other beta-protomer (betaII). The binding of the same Fe(II) atom also induces a local effect at the active site in betaI-protomer, which lowers the affinity for metal in the A-site. The mixed metal FeMn species are quantitatively characterized with electron paramagnetic resonance spectroscopy. The previously reported catalase activity of Mn2(II)R2 is shown not to be associated with Mn.     

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.     

Effects of the protein environment on the structure and energetics of active sites of metalloenzymes. ONIOM study of methane monooxygenase and ribonucleotide reductase.

As the first application of our recently developed ONIOM2(QM:MM) and ONIOM3(QM:QM:MM) codes to the metalloenzymes with a large number of protein residues, two members of the non-heme protein family, methane monooxygenause and ribonucleotide reductase, have been chosen. The "active-site + four alpha-helical fragments" model was adopted which includes about 1000 atoms from 62 residues around the Fe-centered spheres. Comparison of the active-site geometries of MMOH and R2 units optimized with this model with those obtained with the "active site only" (with only 39-46 atoms) model and the X-ray results clearly demonstrates the crucial role of the active site-protein interaction in the enzymatic activities.     

Structure of the nitrogen-centered radical formed during inactivation of E. coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine-5'-diphosphate: trapping of the 3'-ketonucleotide

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 (N(3)UDP), have provided much insight into the reduction mechanism. N(3)UDP 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. N(2) 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'-[(17)O]-N(3)UDP 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 [(17)O] hyperfine tensor. DFT calculations were employed to determine which structures are in best agreement with the simulated hyperfine 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.     

PELDOR spectroscopy with DOPA-beta2 and NH2Y-alpha2s: distance measurements between residues involved in the radical propagation pathway of E. coli ribonucleotide reductase

Escherichia coli ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'-deoxynucleotides. The active enzyme is a 1:1 complex of two homodimeric subunits, alpha2 and beta2. The alpha2 is the site of nucleotide reduction, and beta2 harbors a diferric tyrosyl radical (Y122*) cofactor. Turnover requires formation of a cysteinyl radical (C439*) in the active site of alpha2 at the expense of the Y122* in beta2. A docking model for the alpha2beta2 interaction and a pathway for radical transfer from beta2 to alpha2 have been proposed. This pathway contains three Ys: Y356 in beta2 and Y731/Y730 in alpha2. We have previously incorporated 3-hydroxytyrosine and 3-aminotyrosine into these residues and showed that they act as radical traps. In this study, we use these alpha2/beta2 variants and PELDOR spectroscopy to measure the distance between the Y122* in one alphabeta pair and the newly formed radical in the second alphabeta pair. The results yield distances that are similar to those predicted by the docking model for radical transfer. Further, they support a long-range radical initiation process for C439* generation and provide a structural constraint for residue Y356, which is thermally labile in all beta2 structures solved to date.