Wen-Ge Han

Density functional methods applied to metalloenzymes

Abstract Density functional calculations for structures, spin states, redox energetics and reaction pathways are presented for some selected metalloenzymes. The specific enzymes examined are: (1) Fe and Mn superoxide dismutase for redox energetics and the role of second shell residues; (2) galactose oxidase (Cu enzyme) and (3) glyoxalase I (Zn enzyme) for reaction pathways, mechanisms, intermediates, and transition states (reaction barriers); (4) iron-oxo dimer enzymes methane monooxygenase and ribonucleotide reductase for characterizing the oxidized and reduced forms in terms of structures and protonation states, and for a proposed structure for the high-valent intermediate Q in MMO. The interaction of the active site with the surrounding protein environment is also explored in a number of cases either by using expanded quantum mechanically treated clusters, or by using electrostatic/dielectric representations of the protein–solvent environment.

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, pKa, 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.

Density functional studies of the ground- and excited-state potential-energy curves of stilbene cis - trans isomerization

Using spin-unrestricted density functional theory (the VWN Becke-Perdew potential), including broken-symmetry and spin-projection methods, we have obtained the potential-energy curves as a function of the central torsional angle of stilbene in the ground (S0), the first excited triplet (T1), the first excited singlet (S1), and the doubly excited singlet (S2) states. The thermal trans-->cis isomerization of stilbene passes through a diradical broken-symmetry electronic structure around the twisted conformation (90 degrees central torsional angle) in the ground state. Our calculations support the proposed triplet mechanism for sensitized cis [symbol: see text] trans photoisomerization and the nonadiabatic singlet mechanism proposed by Orlandi and Siebrand. On the T1 potential-energy curve, the rotation of the C=C bond for both trans- and cis-stilbene will lead stilbene to the twisted conformation, from which the twisted stilbene will decay to the ground-state surface that is nearly isoenergetic with the T1 surface and has diradical electronic structure in the twisted region. On the S1 potential-energy curve, the energy increases in the direction from trans- to the twisted stilbene, and crosses with the neutral doubly excited S2 potential-energy curve, which has a minimum at the twisted structure and is lower in energy than the zwitterionic doubly excited state. The twisted stilbene around the energy minimum of the neutral doubly excited S2-state will decay onto the ground-state surface from where the rotation of the C=C bond leads the twisted stilbene to either the trans or cis configuration and the isomerization of stilbene is then completed. Similar studies have also been performed on a stilbene derivative with a substituent group, NHCOCH3.

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 (δexp) and DFT (PW91 potential with all‐electron triple‐ζ plus polarization basis sets) calculated electron densities at Fe nuclei [ρ(0)] for the Fe2+,2.5+ and Fe2.5+,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 δexp = α[ρ(0) − 11884.0] + C, the best fitting for 17 Fe2+,2.5+ complexes (totally 31 Fe sites) yields α = −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 Fe2.5+,3+,3.5+,4+ complexes (totally 30 Fe sites) yields α = −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 Fe2+,2.5+ fit from the Fe2.5+,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 (ΔEQ(cal)) and experimental (ΔEQ(exp)) quadrupole splittings based on |ΔEQ(exp)| = A |ΔEQ(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+(μ‐O)(μ‐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.

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.

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.

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.

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.

Multiple reactive immunization towards the hydrolysis of organophosphorus nerve agents: hapten design and synthesis.

We designed and synthesized a series of haptens to elicit catalytic antibodies with phosphatase activity against nerve agents. The design is based on the novel concept of multiple reactive immunization which aims to afford two or more catalytic residues within the antibodys binding cleft. The haptens showed the desired reactivity in vitro and were submitted for immunization.