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DFT calculations of comparative energetics and ENDOR/Mossbauer properties for two protonation states of the iron dimer cluster of ribonucleotide reductase intermediate X.

ABSTRACT: 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.

SUBMITTER: Han WG

PROVIDER: S-EPMC2746754 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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DFT calculations of comparative energetics and ENDOR/Mössbauer properties for two protonation states of the iron dimer cluster of ribonucleotide reductase intermediate X.

Han Wen-Ge WG Noodleman Louis L

Dalton transactions (Cambridge, England : 2003) 20090623 30

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 con ...[more]

PMID: 19623405

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Project description:The Fea3(3+)···CuB(2+) dinuclear center (DNC) structure of the as-isolated oxidized ba3 cytochrome c oxidase (CcO) from Thermus thermophilus (Tt) is still not fully understood. When the proteins are initially crystallized in the oxidized state, they typically become radiolyticly reduced through X-ray irradiation. Several X-ray crystal structures of reduced ba3 CcO from Tt are available. However, depending on whether the crystals were prepared in a lipidic cubic phase environment or in detergent micelles, and whether the CcO's were chemically or radiolyticly reduced, the X-ray diffraction analysis of the crystals showed different Fea3(2+)···CuB(+) DNC structures. On the other hand, Mössbauer spectroscopic experiments on reduced and oxidized ba3 CcOs from Tt (Zimmermann et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5779-5783) revealed multiple (57)Fea3(2+) and (57)Fea3(3+) components. Moreover, one of the (57)Fea3(3+) components observed at 4.2 K transformed from a proposed "low-spin" state to a different high-spin species when the temperature was increased above 190 K, whereas the other high-spin (57)Fea3(3+) component remained unchanged. In the current Article, in order to understand the heterogeneities of the DNC in both Mössbauer spectra and X-ray crystal structures, the spin crossover of one of the (57)Fea3(3+) components, and how the coordination and spin states of the Fea3(3+/2+) and Cu(2+/1+) sites relate to the heterogeneity of the DNC structures, we have applied density functional OLYP calculations to the DNC clusters established based on the different X-ray crystal structures of ba3 CcO from Tt. As a result, specific oxidized and reduced DNC structures related to the observed Mössbauer spectra and to spectral changes with temperature have been proposed. Our calculations also show that, in certain intermediate states, the His233 and His283 ligand side chains may dissociate from the CuB(+) site, and they may become potential proton loading sites during the catalytic cycle.

Project description:We previously used a combination of continuous-wave (CW) and pulsed electron-nuclear double resonance (ENDOR) protocols to identify the types of protonated oxygen (OH(x)) species and their disposition within the Fe(III)/Fe(IV) cluster of intermediate X, the direct precursor of the essential diferric-tyrosyl radical cofactor of the beta2 subunit of Escherichia coli ribonucleotide reductase (RNR). We concluded that X contains the [(H(x)O)Fe(III)OFe(IV)] fragment (T model), and does not contain a mu-hydroxo bridge. When combined with a subsequent (17)O ENDOR study of X prepared with H(2)(17)O and with (17)O(2), the results led us to suggest that this fragment is the entire inorganic core of X. This has been questioned by recent reports, but these reports do not themselves agree on the core of X. One concluded that X possesses a di-mu-oxo Fe(III)/Fe(IV) core plus a terminal (H(2)O) bound to Fe(III) [e.g., Han, W.-G.; Liu, T.; Lovell, T.; Noodleman, L. J. Am. Chem. Soc. 2005, 127, 15778-15790]. The other [Mitic, N.; Clay, M. D.; Saleh, L.; Bollinger, J. M.; Solomon, E. I. J. Am. Chem. Soc. 2007, 129, 9049-9065] concluded that X contains only a single oxo bridge and postulated the presence of an additional hydroxo bridge plus a terminal hydroxyl bound to Fe(III). In this report we take advantage of improvements in 35 GHz pulsed ENDOR performance to reexamine the protonation state of oxygenic ligands of the inorganic core of X by directly probing the exchangeable proton(s) with (2)H pulsed ENDOR spectroscopy. These (2)H ENDOR measurements confirm that X contains an Fe(III)-bound terminal aqua ligand (H(x)O), but the spectra contain none of the features that would be required for the proton of a bridging hydroxyl. Thus, we confirm that X contains a terminal aqua (most likely hydroxo) ligand to Fe(III) in addition to one or two mu-oxo bridges but does not contain a mu-hydroxo bridge. The (2)H ENDOR measurements further demonstrate that this conclusion is applicable to both wild type and Y122F-beta2 mutant, and in fact we detect no difference between the properties of protons on the terminal oxygens in the two variants; likewise, (14)N ENDOR measurements of histidyl ligands bound to Fe show no difference between the two variants.

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DFT calculations of comparative energetics and ENDOR/Mossbauer properties for two protonation states of the iron dimer cluster of ribonucleotide reductase intermediate X.

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DFT calculations of comparative energetics and ENDOR/Mössbauer properties for two protonation states of the iron dimer cluster of ribonucleotide reductase intermediate X.

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