Transmission dynamics and phylogeography of Mycobacterium tuberculosis in China based on whole-genome phylogenetic analysis.

OBJECTIVES: This study aimed to describe the lineage-specific transmissibility and epidemiological migration of Mycobacterium tuberculosis in China. METHODS: We curated a large set of whole-genome sequences from 3204 M. tuberculosis isolates, including thousands of newly sequenced genomes, and applied a series of metrics to compare the transmissibility of M. tuberculosis strains between lineages and sublineages. The countrywide transmission patterns of major lineages were explored. RESULTS: We found that lineage 2 (L2) was the most prevalent lineage in China (85.7%), with the major sublineage 2.2.1 (80.9%), followed by lineage 4 (L4) (13.8%), which comprises major sublineages 4.2 (1.5%), 4.4 (6.2%) and 4.5 (5.8%). We showed evidence for frequent cross-regional spread and large cluster formation of L2.2.1 strains, whereas L4 strains were relatively geographically restricted in China. Next, we applied a series of genomic indices to evaluate M. tuberculosis strain transmissibility and uncovered higher transmissibility of L2.2.1 compared with the L2.2.2 and L4 sublineages. Phylogeographic analysis showed that southern, eastern, and northern China were highly connected regions for countrywide L2.2.1 strain spread. CONCLUSIONS: The present study provides insights into the different transmission and migration patterns of the major M. tuberculosis lineages in China and highlights that transmissible L2.2.1 is a threat to tuberculosis control.

Mössbauer properties of the diferric cluster and the differential iron(II)-binding affinity of the iron sites in protein R2 of class Ia Escherichia coli ribonucleotide reductase: a DFT/electrostatics study.

The R2 subunit of class-Ia ribonucleotide reductase (RNR) from Escherichia coli (E. coli) contains a diiron active site. Starting from the apo-protein and Fe(II) in solution at low Fe(II)/apoR2 ratios, mononuclear Fe(II) binding is observed indicating possible different Fe(II) binding affinities for the two alternative sites. Further, based on their Mössbauer spectroscopy and two-iron-isotope reaction experiments, Bollinger et al. (J. Am. Chem. Soc., 1997, 119, 5976-5977) proposed that the site Fe1, which bonds to Asp84, should be associated with the higher observed (57)Fe Mössbauer quadrupole splitting (2.41 mm s(-1)) and lower isomer shift (0.45 mm s(-1)) in the Fe(III)Fe(III) state, site Fe2, which is further from Tyr122, should have a greater affinity for Fe(II) binding than site Fe1, and Fe(IV) in the intermediate X state should reside at site Fe2. In this paper, using 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) methodologies, we have demonstrated that the observed large quadrupole splitting for the diferric state R2 does come from site Fe1(III) and it is mainly caused by the binding position of the carboxylate group of the Asp84 sidechain. Further, a series of active site clusters with mononuclear Fe(II) binding at either site Fe1 or Fe2 have been studied, which show that with a single dielectric medium outside the active site quantum region, there is no energetic preference for Fe(II) binding at one site over another. However, when including the explicit extended protein environment in the PB-SCRF model, the reaction field favors the Fe(II) binding at site Fe2 rather than at site Fe1 by ~9 kcal mol(-1). Therefore our calculations support the proposal of the previous Mössbauer spectroscopy and two-iron-isotope reaction experiments by Bollinger et al.

Geometric and electrostatic study of the [4Fe-4S] cluster of adenosine-5'-phosphosulfate reductase from broken symmetry density functional calculations and extended X-ray absorption fine structure spectroscopy.

Adenosine-5'-phosphosulfate reductase (APSR) is an iron-sulfur protein that catalyzes the reduction of adenosine-5'-phosphosulfate (APS) to sulfite. APSR coordinates to a [4Fe-4S] cluster via a conserved CC-X(~80)-CXXC motif, and the cluster is essential for catalysis. Despite extensive functional, structural, and spectroscopic studies, the exact role of the iron-sulfur cluster in APS reduction remains unknown. To gain an understanding into the role of the cluster, density functional theory (DFT) analysis and extended X-ray fine structure spectroscopy (EXAFS) have been performed to reveal insights into the coordination, geometry, and electrostatics of the [4Fe-4S] cluster. X-ray absorption near-edge structure (XANES) data confirms that the cluster is in the [4Fe-4S](2+) state in both native and substrate-bound APSR while EXAFS data recorded at ~0.1 Å resolution indicates that there is no significant change in the structure of the [4Fe-4S] cluster between the native and substrate-bound forms of the protein. On the other hand, DFT calculations provide an insight into the subtle differences between the geometry of the cluster in the native and APS-bound forms of APSR. A comparison between models with and without the tandem cysteine pair coordination of the cluster suggests a role for the unique coordination in facilitating a compact geometric structure and "fine-tuning" the electronic structure to prevent reduction of the cluster. Further, calculations using models in which residue Lys144 is mutated to Ala confirm the finding that Lys144 serves as a crucial link in the interactions involving the [4Fe-4S] cluster and APS.

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, Mössbauer, 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.

Density functional theory analysis of structure, energetics, and spectroscopy for the Mn-Fe active site of Chlamydia trachomatis ribonucleotide reductase in four oxidation states.

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.

Quantum cluster size and solvent polarity effects on the geometries and Mössbauer properties of the active site model for ribonucleotide reductase intermediate X: a density functional theory study.

In studying the properties of metalloproteins using ab initio quantum mechanical methods, one has to focus on the calculations on the active site. The bulk protein and solvent environment is often neglected, or is treated as a continuum dielectric medium with a certain dielectric constant. The size of the quantum cluster of the active site chosen for calculations can vary by including only the first-shell ligands which are directly bound to the metal centers, or including also the second-shell residues which are adjacent to and normally have H-bonding interactions with the first-shell ligands, or by including also further hydrogen bonding residues. It is not well understood how the size of the quantum cluster and the value of the dielectric constant chosen for the calculations will influence the calculated properties. In this paper, we have studied three models (A, B, and C) of different sizes for the active site of the ribonucleotide reductase intermediate X, using density functional theory (DFT) OPBE functional with broken-symmetry methodology. Each model is studied in gas-phase and in the conductor-like screening (COSMO) solvation model with different dielectric constants ε = 4, 10, 20, and 80, respectively. All the calculated Fe-ligand geometries, Heisenberg J coupling constants, and the Mössbauer isomer shifts, quadrupole splittings, and the (57)Fe, (1)H, and (17)O hyperfine tensors are compared. We find that the calculated isomer shifts are very stable. They are virtually unchanged with respect to the size of the cluster and the dielectric constant of the environment. On the other hand, certain Fe-ligand distances are sensitive to both the size of the cluster and the value of ε. ε = 4, which is normally used for the protein environment, appears too small when studying the diiron active site geometry with only the first-shell ligands as seen by comparisons with larger models.

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

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.

Structural model studies for the peroxo intermediate P and the reaction pathway from P-->Q of methane monooxygenase using broken-symmetry density functional calculations.

Several structural models for the active site of the peroxo intermediate state "P" of the hydroxylase component of soluble methane monooxygenase (MMOH) have been studied, using two DFT functionals OPBE and PW91 with broken-symmetry methodology and the conductor-like screening (COSMO) solvation model. These active site models have different O2 binding modes to the diiron center, such as the mu-eta2,eta2, trans-mu-1,2 and cis-mu-1,2 conformations. The calculated properties, including optimized geometries, electronic energies, Fe net spin populations, and Mössbauer isomer shift and quadrupole splitting values, have been reported and compared with available experimental results. The high-spin antiferromagnetically (AF) coupled Fe3+ sites are correctly predicted by both OPBE and PW91 methods for all active site models. Our data analysis and comparisons favor a cis-mu-1,2 structure (model cis-mu-1,2a shown in Figure 9) likely to represent the active site of MMOH-P. Feasible structural changes from MMOH-P to another intermediate state MMOH-Q are also proposed, where the carboxylate group of Glu243 side chain has to open up from the mono-oxygen bridging position, and the dissociations of the terminal H2O ligand from Fe1 and of the oxygen atom in the carboxylate group of Glu144 from Fe2 are also necessary for the O2 binding mode changes from cis to trans. The O-O bond is proposed to break in the trans-conformation and forms two mu-oxo bridges in MMOH-Q. The terminal H2O molecule and the Glu144 side chain then rebind with Fe1 and Fe2, respectively, in Q.

Structural Model Studies for the High-Valent Intermediate Q of Methane Monooxygenase from Broken-Symmetry Density Functional Calculations.

Mössbauer isomer shift parameters have been obtained for both density functional theory (DFT) OPBE and OLYP functionals by linear regressions between the measured isomer shifts and calculated electron densities at Fe nuclei for a number of Fe(2+,2.5+) and Fe(2.5+,3+,3.5+,4+) complexes grouped separately. The calculated isomer shifts and quadrupole splittings on the sample Fe complexes from OPBE and OLYP functionals are similar to those of PW91 calculations (J. Comput. Chem. 27 (2006) 1292), however the fit parameters from the linear regressions differ between PW91 and OPBE, OLYP. Four models for the active site structure of the hydroxylase component of soluble methane monooxygenase (MMOH) have been studied, using three DFT functionals OPBE, OLYP, and PW91, incorporated with broken-symmetry methodology and the conductor-like screening (COSMO) solvation model. The calculated properties, including optimized geometries, electronic energies, pK(a)'s, Fe net spin populations, and Mössbauer isomer shifts and quadrupole splittings, have been reported and compared with available experimental values. The high-spin antiferromagnetically (AF) coupled Fe(4+) sites are correctly predicted by OPBE and OLYP methods for all active site models. PW91 potential overestimates the Fe-ligand covalencies for some of the models because of spin crossover. Our calculations and data analysis support the structure (our current model II shown in Figure 8) proposed by Friesner and Lippard's group (J. Am. Chem. Soc. 123 (2001) 3836-3837), which contains an Fe(4+)(µ-O)(2)Fe(4+) center, one axial water which also H-bonds to both side chains of Glu243 and Glu114, and one bidentate carboxylate group from the side chain of Glu144, which is likely to represent the active site of MMOH-Q. A new model structure (model IV shown in Figure 9), which has a terminal hydroxo and a protonated His147 which is dissociated from a nearby Fe, is more asymmetric in its Fe(µ-O)(2)Fe diamond core, and is another very good candidate for intermediate Q.

Experimental and DFT studies: novel structural modifications greatly enhance the solvent sensitivity of live cell imaging dyes.

Structural modifications of previously reported merocyanine dyes (Toutchkine, A.; Kraynov, V.; Hahn, K. J. Am. Chem. Soc. 2003, 125, 4132-4145) were found to greatly enhance the solvent dependence of their absorbance and fluorescence emission maxima. Density functional theory (DFT) calculations have been performed to understand the differences in optical properties between the new and previously synthesized dyes. Absorption and emission energies were calculated for several new dyes using DFT vertical self-consistent reaction field (VSCRF) methods. Geometries of ground and excited states were optimized with a conductor-like screening model (COSMO) and self-consistent field (SCF) methods. The new dyes have enhanced zwitterionic character in the ground state and much lower polarity in the excited state, as shown by the DFT-VSCRF calculations. Consistently, the position of the absorption bands are strongly blue-shifted in more polar solvent (methanol compared to benzene), as predicted by the DFT spectral calculations. Inclusion of explicit H-bonding solvent molecules within the quantum model further enhances the predicted shifts and is consistent with the observed spectral broadening. Smaller but significant spectral shifts in polar versus nonpolar solvent are predicted and observed for emission bands. The new dyes show large fluorescence quantum yields in polar hydrogen-bonding solvents; qualitatively, the longest bonds along the conjugated chain at the excited S1 state minimum are shorter in the more polar solvent, inhibiting photoisomerization. The loss of photostability of the dyes is a consequence of the reaction with and electron transfer to singlet oxygen, starting oxidative dye cleavage. The calculated vertical ionization potentials of three dyes I-SO, AI-SO(4), and AI-BA(4) in benzene and methanol are consistent with their relative photobleaching rates; the charge distributions along the conjugated chains for the three dyes are similarly predictive of higher reaction rates for AI-SO(4) and AI-BA(4) than for I-SO. Time-dependent DFT calculations were also performed on AI-BA(4); these were less accurate than the VSCRF method in predicting the absorption energy shift from benzene to methanol.

Density functional theory study of Fe(IV) d-d optical transitions in active-site models of class I ribonucleotide reductase intermediate X with vertical self-consistent reaction field methods.

The Fe(IV) d-d transition energies for four active-site structural models of class I ribonucleotide reductase (RNR) intermediate X have been calculated using broken-symmetry density functional theory incorporated with the Slater transition state vertical self-consistent reaction field methodology. Our model I (Figure 1), which contains two mu-oxo bridges, one terminal water, and one bidentate carboxylate group, yields the best Fe(IV) d-d transition energies compared with experiment. Our previous study (J. Am. Chem. Soc. 2005, 127, 15778-15790) also shows that most of the other calculated properties of model I in both native and mutant Y122F forms, including geometries, spin states, pKa's, 57Fe, 1H, and 17O hyperfine tensors, and 57Fe Mössbauer isomer shifts and quadrupole splittings, are also the best in agreement with the available experimental data. This model is likely to represent the active-site structure of the intermediate state X of RNR.

Structure, redox, pKa, spin. A golden tetrad for understanding metalloenzyme energetics and reaction pathways.

After a review of the current status of density functional theory (DFT) for spin-polarized and spin-coupled systems, we focus on the resting states and intermediates of redox-active metalloenzymes and electron transfer proteins, showing how comparisons of DFT-calculated spectroscopic parameters with experiment and evaluation of related energies and geometries provide important information. The topics we examine include (1) models for the active-site structure of methane monooxygenase intermediate Q and ribonucleotide reductase intermediate X; (2) the coupling of electron transfer to proton transfer in manganese superoxide dismutase, with implications for reaction kinetics; (3) redox, pK(a), and electronic structure issues in the Rieske iron-sulfur protein, including their connection to coupled electron/proton transfer, and an analysis of how partial electron delocalization strongly alters the electron paramagnetic resonance spectrum; (4) the connection between protein-induced structural distortion and the electronic structure of oxidized high-potential 4Fe4S proteins with implications for cluster reactivity; (5) an analysis of cluster assembly and central-atom insertion into the FeMo cofactor center of nitrogenase based on DFT structural and redox potential calculations.

DFT calculations of 57Fe Mössbauer isomer shifts and quadrupole splittings for iron complexes in polar dielectric media: applications to methane monooxygenase and ribonucleotide reductase.

To predict the isomer shifts of Fe complexes in different oxidation and spin states more accurately, we have performed linear regression between the measured isomer shifts (delta(exp)) and DFT (PW91 potential with all-electron triple-zeta plus polarization basis sets) calculated electron densities at Fe nuclei [rho(0)] for the Fe(2+,2.5+) and Fe(2.5+,3+,3.5+,4+) complexes separately. The geometries and electronic structures of all complexes in the training sets are optimized within the conductor like screening (COSMO) solvation model. Based on the linear correlation equation delta(exp) = alpha[rho(0) - 11884.0] + C, the best fitting for 17 Fe(2+,2.5+) complexes (totally 31 Fe sites) yields alpha = -0.405 +/- 0.042 and C = 0.735 +/- 0.047 mm s(-1). The correlation coefficient is r = -0.876 with a standard deviation of SD = 0.075 mm s(-1). In contrast, the linear fitting for 19 Fe(2.5+,3+,3.5+,4+) complexes (totally 30 Fe sites) yields alpha = -0.393 +/- 0.030 and C = 0.435 +/- 0.014 mm s(-1), with the correlation coefficient r = -0.929 and a standard deviation SD = 0.077 mm s(-1). We provide a physical rationale for separating the Fe(2+,2.5+) fit from the Fe(2.5+,3+,3.5+,4+) fit, which also is clearly justified on a statistical empirical basis. Quadrupole splittings have also been calculated for these systems. The correlation between the calculated (DeltaE(Q(cal))) and experimental (DeltaE(Q(exp))) quadrupole splittings based on |DeltaE(Q(exp))| = A |DeltaE(Q(cal))| + B yields slope A, which is almost the ideal value 1.0 (A = 1.002 +/- 0.030) and intercept B almost zero (B = 0.033 +/- 0.068 mm s(-1)). Further calculations on the reduced diferrous and oxidized diferric active sites of class-I ribonucleotide reductase (RNR) and the hydroxylase component of methane monooxygenase (MMOH), and on a mixed-valent [(tpb)Fe3+(mu-O)(mu-CH3CO2)Fe4+(Me3[9]aneN3)]2+ (S = 3/2) complex and its corresponding diferric state have been performed. Calculated results are in very good agreement with the experimental data.

Seven clues to the origin and structure of class-I ribonucleotide reductase intermediate X.

Class-I ribonucleotide reductases (RNRs) are aerobic enzymes that catalyze the reduction of ribonucleotides to deoxyribonucleotides providing the required building blocks for DNA replication and repair. These ribonucleotide-to-deoxyribonucleotide reactions occur by a long range radical (or proton-coupled-electron-transfer) propagation mechanism initiated by a fairly stable tyrosine radical ("the pilot light"). When this pilot light goes out, the tyrosine radical is regenerated by a high-oxidation-state enzyme intermediate, called X. The active site of class-I RNR-X has been recognized as a spin coupled Fe(III)Fe(IV) center with S(total)=1/2 ground state. Although several clues have been obtained from Mössbauer, (57)Fe, (1)H, (17)O(2), and H(2)(17)O ENDOR (electron-nuclear double resonance), EXAFS (extended X-ray absorption fine structure), and MCD (magnetic circular dichroism) experiments, the detailed structure of the intermediate X is still unknown. In the past three years, we have been studying the properties of a set of model clusters for RNR-X using broken-symmetry density functional theory (DFT), and have compared them with the available experimental results. Based on the detailed analysis and comparisons, we have proposed a definite form for the active site structure of class-I RNR intermediate X. The puzzle is now set: can you find any flaws in the argument or evidence? Can you add anything further to the current experimental picture? The argument is formulated from seven experimental clues with associated calculations and models.

Active site structure of class I ribonucleotide reductase intermediate X: a density functional theory analysis of structure, energetics, and spectroscopy.

Several models for the active site structure of class I ribonucleotide reductase (RNR) intermediate X have been studied in the work described in this paper, using broken-symmetry density functional theory (DFT) incorporated with the conductor-like screening (COSMO) solvation model. The calculated properties, including geometries, spin states, 57Fe, 1H, and 17O hyperfine tensors, Mössbauer isomer shifts, and quadrupole splittings, and the estimation of the Fe(IV) d-d transition energies have been compared with the available experimental values. On the basis of the detailed analysis and comparisons, we propose a definite form for the active site structure of class I RNR intermediate X, which contains an Fe1(III)Fe2(IV) center (where Fe1 is the iron site closer to Tyr122, and the two iron sites are high-spin antiferromagnetically coupled to give a total 1/2 net spin), two mu-oxo bridges, one terminal water which binds to Fe1(III) and also H-bonds to both side chains of Asp84 and Glu238, and one bidentate carboxylate group from the side chain of Glu115.

Density functional study of a micro-1,1-carboxylate bridged Fe(III)-O-Fe(IV) model complex. 2. Comparison with ribonucleotide reductase intermediate X.

Using broken-symmetry density functional theory, we have studied an experimentally proposed model for ribonucleotide reductase (RNR) intermediate X, which contains a single oxo bridge, one terminal H(2)O or OH(-) ligand, a bidentate carboxylate from Glu115, and a mono-oxygen bridge provided by Glu238. For the models proposed here, the terminal H(2)O/OH(-) ligand binds to site Fe1 which is closer to Tyr122. The diiron centers are assigned as high-spin Fe(III)Fe(IV) and antiferromagnetically coupled to give the S(total) = (1)/(2) ground state. Calculations show that the model with a terminal hydroxide in the antiferromagnetic [S(Fe1) = 2, S(Fe2) = (5)/(2)] state (Fe1 = Fe(IV), Fe2 = Fe(III)) is the lowest energy state, and the calculated isomer shift and quadrupole splitting values for this cluster are also the best among the four clusters studied here when compared with the experimental values. However, the DFT-calculated (1)H proton and (17)O hyperfine tensors for this state do not show good agreement with the experiments. The calculated Fe1-Fe2 distances for this and the other three clusters at >2.9 A are much longer than the 2.5 A which was predicted by the EXAFS measurements. The mono-oxygen bridge provided by Glu238 tends to be closer to one of the Fe sites in all clusters studied here, and it does not function as a bridge in helping to produce a short Fe-Fe distance. Overall, the models tested here are not likely to represent the core structure of RNR intermediate X. The model with the terminal OH(-) binding to the Fe1(III) center shows the best calculated (1)H proton and (17)O hyperfine tensors compared with the experimental values. This supports the earlier proposal based on analysis of ENDOR spectra (Willems et al.(16)) that the terminal oxygen group binds to the Fe(III) site in RNR-X.

A theoretical study of the UV/visible absorption and emission solvatochromic properties of solvent-sensitive dyes.

Using the density-functional vertical self-consistent reaction field (VSCRF) solvation model, incorporated with the conductor-like screening model (COSMO) and the self-consistent reaction field (SCRF) methods, we have studied the solvatochromic shifts of both the absorption and emission bands of four solvent-sensitive dyes in different solutions. The dye molecules studied here are: S-TBA merocyanine, Abdel-Halim's merocyanine, the rigidified amino-coumarin C153, and Nile red. These dyes were selected because they exemplify different structural features likely to impact the solvent-sensitive fluorescence of "push-pull", or merocyanine, fluorophores. All trends of the blue or red shifts were correctly predicted, comparing with the experimental observations. Explict H-bonding interactions were also considered in several protic solutions like H2O, methanol and ethanol, showing that including explicit H-bonding solvent molecule(s) in the calculations is important to obtain the correct order of the excitation and emission energies. The geometries, electronic structures, dipole moments, and intra- and intermolecular charge transfers of the dyes in different solvents are also discussed.

Temperature-dependent behavior of protein-chromophore interactions: a theoretical study of a blue fluorescent antibody.

The unusual temperature-dependent excited-state dynamics of a stilbene-antibody complex reported by Simeonov et al. are explored using theoretical methods. The anomalous temperature-dependent fluorescence emission and lifetime are shown to be the result of interplay among temperature-modulated protein flexibility, the excited-state potential surface for the stilbene central twist, and changes in the stilbene charge distribution upon excitation. Stilbene is found to possess a similar geometry and orientation within the antibody binding pocket at all temperatures in the ground state and at low temperatures (approximately 240 K) in the excited state. At higher temperatures (approximately 260 K), the excited-state conformation twists around the central double bond and adopts an alternate orientation within the binding pocket. These changes result from protein side chain and loop motions that are frozen out at lower temperatures and account for the observed red shift of the fluorescence emission spectrum (a calculated shift of 3.8 kcal mol-1 compares favorably with the approximately 5 kcal.mol-1 observed experimentally). Approximately 3.0 kcal mol-1 of this stabilization is global in nature and is not attributable to specific local interactions. Local interactions between stilbene and Tyr-B39 contribute approximately 0.8 kcal mol-1 to the fluorescence shift. The primary structural change in simulations of the high temperature excited state involves a decrease in the stilbene-tyrosine distance and a relative change in orientation of the aromatic rings. We identify several nearby charged residues that contribute to the fluorescence emission shift and provide targets for mutagenesis to probe the temperature-dependent dynamics of protein-chromophore interactions.

DFT calculations of isomer shifts and quadrupole splitting parameters in synthetic iron-oxo complexes: applications to methane monooxygenase and ribonucleotide reductase.

To predict isomer shifts and quadrupole splitting parameters of Fe atoms in the protein active sites of methane monooxygenase and ribonucleotide reductase, a correlation between experimental isomer shifts ranging 0.1-1.5 mm s(-)(1) for Fe atoms in a training set with the corresponding density functional theory (DFT) calculated electron densities at the Fe nuclei in those complexes is established. The geometries of the species in the training set, consisting of synthetic polar monomeric and dimeric iron complexes, are taken from the Cambridge structural database. A comparison of calculated Mössbauer parameters for Fe atoms from complexes in the training set with their corresponding experimental values shows very good agreement (standard deviation of 0.11 mm/s, correlation coefficient of -0.94). However, for the Fe atoms in the active sites of the structurally characterized proteins of methane monooxygenase and ribonucleotide reductase, the calculated Mössbauer parameters deviate more from their experimentally measured values. The high correlation that exists between calculated and observed quadrupole splitting and isomer shift parameters for the synthetic complexes leads us to conclude that the main source of the error arising for the protein active sites is due to the differing degrees of atomic-level resolution for the protein structural data, compared to the synthetic complexes in the training set. Much lower X-ray resolutions associated with the former introduce uncertainty in the accuracy of several bond lengths. This is ultimately reflected in the calculated isomer shifts and quadrupole splitting parameters of the Fe sites in the proteins. For the proteins, the closest correspondence between predicted and observed Mössbauer isomer shifts follows the order MMOH(red), RNR(red), MMOH(ox), and RNR(ox), with average deviations from experiment of 0.17, 0.17, 0.17-0.20, and 0.32 mm/s, but this requires DFT geometry optimization of the iron-oxo dimer complexes.