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ENDOR spectroscopy and DFT calculations: evidence for the hydrogen-bond network within ?2 in the PCET of E. coli ribonucleotide reductase.

ABSTRACT: Escherichia coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: ?2 and ?2. ?2 contains a stable di-iron tyrosyl radical (Y(122)(•)) cofactor required to generate a thiyl radical (C(439)(•)) in ?2 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: Y(122) ? W(48)[?] ? Y(356) in ?2, across the subunit interface to Y(731) ? Y(730) ? C(439) in ?2. Within ?2 a colinear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH(2)Y) replaced Y(730) in ?2, and this mutant was incubated with ?2, cytidine 5'-diphosphate, and adenosine 5'-triphosphate to generate a NH(2)Y(730)(•) in D(2)O. [(2)H]-Electron-nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained, and together with DFT models of ?2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C(439) and Y(731). 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 NH(2)Y(730)(•) are consistent with the combined effect of the hydrogen bonds to Cys(439) and Tyr(731), both nearly perpendicular to the ring plane of NH(2)Y(730.) The results provide the first experimental evidence for the hydrogen-bond network between the pathway residues in ?2 of the active RNR complex, for which no structural data are available.

SUBMITTER: Argirevic T

PROVIDER: S-EPMC4516058 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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ENDOR spectroscopy and DFT calculations: evidence for the hydrogen-bond network within α2 in the PCET of E. coli ribonucleotide reductase.

Argirević Tomislav T Riplinger Christoph C Stubbe JoAnne J Neese Frank F Bennati Marina M

Journal of the American Chemical Society 20121016 42

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 (Y(122)(•)) cofactor required to generate a thiyl radical (C(439)(•)) 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: Y(122) ⇆ W(4 ...[more]

PMID: 23072506

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Project description:Two models (I and II) for the active site structure of class-I ribonucleotide reductase (RNR) intermediate X in subunit R2 have been studied in this paper, using broken-symmetry density functional theory (DFT) incorporated with the conductor like screening (COSMO) solvation model and with the finite-difference Poisson-Boltzmann self-consistent reaction field (PB-SCRF) calculations. Only one of the bridging groups between the two iron centers is different between model-I and model-II. Model-I contains two mu-oxo bridges, while model-II has one bridging oxo and one bridging hydroxo. These are large active site models including up to the fourth coordination shell H-bonding residues. Mössbauer and ENDOR hyperfine property calculations show that model-I is more likely to represent the active site structure of RNR-X. However, energetically our pK(a) calculations at first highly favored the bridging oxo and hydroxo (in model-II) structure of the diiron center rather than having the di-oxo bridge (in model-I). Since the Arg236 and the nearby Lys42, which are very close to the diiron center, are on the protein surface of RNR-R2, it is highly feasible that one or two anion groups in solution would interact with the positively charged side chains of Arg236 and Lys42. The anion group(s) can be a reductant, phosphate, sulfate, nitrate, and other negatively charged groups existing in biological environments or in the buffer of the experiment. Since sulfate ions certainly exist in the buffer of the ENDOR experiment, we have examined the effect of the sulfate (SO(4)(2-), surrounded by explicit water molecules) H-bonding to the side chain of Arg236. We find that when sulfate interacts with Arg236, the carboxylate group of Asp237 tends to be protonated, and once Asp237 is protonated, the Fe(iii)Fe(iv) center in X favors the di-oxo bridge (model-I). This would explain that the ENDOR observed RNR-X active site structure is likely to be represented by model-I rather than model-II.

Dataset Information

ENDOR spectroscopy and DFT calculations: evidence for the hydrogen-bond network within ?2 in the PCET of E. coli ribonucleotide reductase.

Publications

ENDOR spectroscopy and DFT calculations: evidence for the hydrogen-bond network within α2 in the PCET of E. coli ribonucleotide reductase.

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