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, M?ssbauer 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.     

Spectroscopic and computational studies of the de novo designed protein DF2t: correlation to the biferrous active site of ribonucleotide reductase and factors that affect O2 reactivity

DF2t, a de novo designed protein that mimics the active-site structure of many non-heme biferrous enzymes, has been studied using a combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature variable-field (VTVH) MCD. The active site of DF2t is found to have one five-coordinate iron and one four-coordinate iron, which are weakly antiferromagnetically coupled through a mu-1,3 carboxylate bridge. These results bear a strong resemblance to the spectra of Escherichia coli ribonucleotide reductase (R2), and density functional theory calculations were conducted on the W48F/D84E R2 mutant in order to determine the energetics of formation of a monodentate end-on-bound O2 to one iron in the binuclear site. The mu-1,3 carboxylate bridges found in O2-activating enzymes lack efficient superexchange pathways for the second electron transfer (i.e., the OH/oxo bridge in hemerythrin), and simulations of the binding of O2 in a monodentate end-on manner revealed that the bridging carboxylate ligands do not appear capable of transferring an electron to O2 from the remote Fe. Comparison of the results from previous studies of the mu-1,2 biferric-peroxo structure, which bridges both irons, finds that the end-on superoxide mixed-valent species is considerably higher in energy than the bridging peroxo-diferric species. Thus, one of the differences between O2-activating and O2-binding proteins appears to be the ability of O2 to bridge both Fe centers to generate a peroxo intermediate capable of further reactivity.     

Identification of iron(III) peroxo species in the active site of the superoxide reductase SOR from Desulfoarculus baarsii

The active site of superoxide reductase SOR consists of an Fe2+ center in an unusual [His4 Cys1] square-pyramidal geometry. It specifically reduces superoxide to produce H2O2. Here, we have reacted the SOR from Desulfoarculus baarsii directly with H2O2. We have found that its active site can transiently stabilize an Fe3+-peroxo species that we have spectroscopically characterized by resonance Raman. The mutation of the strictly conserved Glu47 into alanine results in a stabilization of this Fe3+-peroxo species, when compared to the wild-type form. These data support the hypothesis that the reaction of SOR proceeds through such a Fe3+-peroxo intermediate. This also suggests that Glu47 might serve to help H2O2 release during the reaction with superoxide.     

Spectroscopic and computational studies of α-keto acid binding to Dke1: understanding the role of the facial triad and the reactivity of β-diketones

The O(2) activating mononuclear nonheme iron enzymes generally have a common facial triad (two histidine and one carboxylate (Asp or Glu) residue) ligating Fe(II) at the active site. Exceptions to this motif have recently been identified in nonheme enzymes, including a 3His triad in the diketone cleaving dioxygenase Dke1. This enzyme is used to explore the role of the facial triad in directing reactivity. A combination of spectroscopic studies (UV-vis absorption, MCD, and resonance Raman) and DFT calculations is used to define the nature of the binding of the α-keto acid, 4-hydroxyphenlpyruvate (HPP), to the active site in Dke1 and the origin of the atypical cleavage (C2-C3 instead of C1-C2) pattern exhibited by this enzyme in the reaction of α-keto acids with dioxygen. The reduced charge of the 3His triad induces α-keto acid binding as the enolate dianion, rather than the keto monoanion, found for α-keto acid binding to the 2His/1 carboxylate facial triad enzymes. The mechanistic insight from the reactivity of Dke1 with the α-keto acid substrate is then extended to understand the reaction mechanism of this enzyme with its native substrate, acac. This study defines a key role for the 2His/1 carboxylate facial triad in α-keto acid-dependent mononuclear nonheme iron enzymes in stabilizing the bound α-keto acid as a monoanion for its decarboxylation to provide the two additional electrons required for O(2) activation.     

X-ray crystal structures of reduced rubrerythrin and its azide adduct: a structure-based mechanism for a non-heme diiron peroxidase

Rubrerythrin (Rbr) is a 44-kDa homodimeric protein, found in many air-sensitive bacteria and archaea, which contains a unique combination of a rubredoxin-like [Fe(SCys)(4)] site and a non-sulfur, oxo/dicarboxylato-bridged diiron site. The diiron site structure resembles those found in O2-activating diiron enzymes. However, Rbr instead appears to function as a hydrogen peroxide reductase (peroxidase). The diferrous site in all-ferrous Rbr (Rbr(red)) shows a much greater reactivity with H2O2 than does the diferric site in all-ferric Rbr (Rbr(ox)), but only the latter structure has been reported. Here we report the X-ray crystal structures of the recombinant Rbr(red) from the sulfate reducing bacterium, Desulfovibrio vulgaris, as well as its azide adduct (Rbr(red)N3). We have also redetermined the structure of Rbr(ox) to a higher resolution than previously reported. The structural differences between Rbr(ox) and Rbr(red) are localized entirely at the diiron site. The most striking structural change upon reduction of the diferric to the diferrous site of Rbr is a 1.8-A movement of one iron away from a unique glutamate carboxylate ligand and toward a trans-disposed histidine side chain, which replaces the glutamate as a ligand. This movement increases the inter-iron distance from 3.3 to 4 A. Rbr(red)N(3) shows this same iron movement and His-->Glu ligand replacement relative to Rbr(ox), and, in addition, an azide coordinated to the diiron site in a cis mu-1,3 fashion, replacing two solvent ligands in Rbr(red). Relative to those in O2-activating enzymes, the bridging carboxylate ligation of the Rbr diiron site is less flexible upon diferric/diferrous interconversion. The diferrous site is also much more rigid, symmetrical, and solvent-exposed than those in O2-activating enzymes. On the basis of these unique structural features, a mechanism is proposed for facile reduction of hydrogen peroxide by Rbr involving a cis mu-eta(2) H2O2 diferrous intermediate.     

Factors governing the substitution of La3+ for Ca2+ and Mg2+ in metalloproteins: a DFT/CDM study

Trivalent lanthanide cations are extensively being used in biochemical experiments to probe various dication-binding sites in proteins; however, the factors governing the binding specificity of lanthanide cations for these binding sites remain unclear. Hence, we have performed systematic studies to evaluate the interactions between La3+ and model Ca2+ - and Mg2+ -binding sites using density functional theory combined with continuum dielectric methods. The calculations reveal the key factors and corresponding physical bases favoring the substitution of trivalent lanthanides for divalent Ca2+ and Mg2+ in holoproteins. Replacing Ca2+ or Mg2+ with La3+ is facilitated by (1) minimizing the solvent exposure and the flexibility of the metal-binding cavity, (2) freeing both carboxylate oxygen atoms of Asp/Glu side chains in the metal-binding site so that they could bind bidentately to La3+, (3) maximizing the number of metal-bound carboxylate groups in buried sites, but minimizing the number of metal-bound carboxylate groups in solvent-exposed sites, and (4) including an Asn/Gln side chain for sites lined with four Asp/Glu side chains. In proteins bound to both Mg2+ and Ca2+, La3+ would prefer to replace Ca2+, as compared to Mg2+. A second Mg2+-binding site with a net positive charge would hamper the Mg2+ --> La3+ exchange, as compared to the respective mononuclear site, although the La3+ substitution of the first native metal is more favorable than the second one. The findings of this work are in accord with available experimental data.     

Calibration of DFT Functionals for the Prediction of Fe Mössbauer Spectral Parameters in Iron-Nitrosyl and Iron-Sulfur Complexes: Accurate Geometries Prove Essential

Six popular density functionals in conjunction with the conductor-like screening (COSMO) solvation model have been used to obtain linear M?ssbauer isomer shift (IS) and quadrupole splitting (QS) parameters for a test set of 20 complexes (with 24 sites) comprised of nonheme nitrosyls (Fe-NO) and non-nitrosyl (Fe-S) complexes. For the first time in an IS analysis, the Fe electron density was calculated both directly at the nucleus, ρ(0)(N), which is the typical procedure, and on a small sphere surrounding the nucleus, ρ(0)(S), which is the new standard algorithm implemented in the ADF software package. We find that both methods yield (near) identical slopes from each linear regression analysis but are shifted with respect to ρ(0) along the x-axis. Therefore, the calculation of the Fe electron density with either method gives calibration fits with equal predictive value. Calibration parameters obtained from the complete test set for OLYP, OPBE, PW91, and BP86 yield correlation coefficients (r(2)) of approximately 0.90, indicating that the calibration fit is of good quality. However, fits obtained from B3LYP and B3LYP* with both Slater-type and Gaussian-type orbitals are generally found to be of poorer quality. For several of the complexes examined in this study, we find that B3LYP and B3LYP* give geometries that possess significantly larger deviations from the experimental structures than OLYP, OPBE, PW91 or BP86. This phenomenon is particularly true for the di- and tetranuclear Fe complexes examined in this study. Previous M?ssbauer calibration fit studies using these functionals have usually included mononuclear Fe complexes alone, where these discrepancies are less pronounced. An examination of spin expectation values reveals B3LYP and B3LYP* approach the weak-coupling limit more closely than the GGA exchange-correlation functionals. The high degree of variability in our calculated S(2) values for the Fe-NO complexes highlights their challenging electronic structure. Significant improvements to the isomer shift calibrations are obtained for B3LYP and B3LYP* when geometries obtained with the OLYP functional are used. In addition, greatly improved performance of these functionals is found if the complete test set is grouped separately into Fe-NO and Fe-S complexes. Calibration fits including only Fe-NO complexes are found to be excellent, while those containing the non-nitrosyl Fe-S complexes alone are found to demonstrate less accurate correlations. Similar trends are also found with OLYP, OPBE, PW91, and BP86. Correlations between experimental and calculated QSs were also investigated. Generally, universal and separate Fe-NO and Fe-S fit parameters obtained to determine QSs are found to be of good to excellent quality for every density functional examined, especially if [Fe(4)(NO)(4)(μ(3)-S)(4)](-) is removed from the test set.     

Structures of E. coli peptide deformylase bound to formate: insight into the preference for Fe2+ over Zn2+ as the active site metal

E. coli peptide deformylase (PDF) catalyzes the deformylation of nascent polypeptides generated during protein synthesis. While PDF was originally thought to be a zinc enzyme, subsequent studies revealed that the active site metal is iron. In an attempt to understand this unusual metal preference, high-resolution structures of Fe-, Co-, and Zn-PDF were determined in complex with its deformylation product, formate. In all three structures, the formate ion binds the metal and forms hydrogen-bonding interactions with the backbone nitrogen of Leu91, the amide side chain of Gln50, and the carboxylate side chain of Glu133. One key difference, however, is how the formate binds the metal. In Fe-PDF and Co-PDF, formate binds in a bidentate fashion, while in Zn-PDF, it binds in a monodentate fashion. Importantly, these structural results provide the first clues into the origins of PDF's metal-dependent activity differences. On the basis of these structures, we propose that the basis for the higher activity of Fe-PDF stems from the better ability of iron to bind and activate the tetrahedral transition state required for cleavage of the N-terminal formyl group.     

Ab initio molecular dynamics of heme in cytochrome c

Ab initio molecular dynamics (AIMD) calculations, based on the Car-Parrinello method, have been carried out for three models of heme c that is present in cytochrome c. Both the reduced (Fe(II)) and oxidized (Fe(III)) forms have been analyzed. The simplest models (1R and 1O, respectively) consist of a unsubstituted porphyrin (with no side chains) and two axially coordinated imidazole and ethylmethylthioether ligands. Density functional theory optimizations of these models confirm the basic electronic features and are the starting point for building more complex derivatives. AIMD simulations were performed after reaching the thermal stability at T = 300 K. The evolution of the Fe-L(ax) bond strengths is examined together with the relative rotations of the imidazole and methionine about the axial vector, which appear rather independent from each other. The next models (2R and 2O) contain side chains at the heme to better simulate the actual active site. It is observed that two adjacent propionate groups induce some important effects. The axial Fe-Sdelta bond is only weakened in 2R but is definitely cleaved in the oxidized species 2O. Also the mobility of the Im ligand seems to be reduced by the formation of a strong hydrogen bond that involves the Im Ndelta1-Hdelta1 bond and one carboxylate group. In 2O the interaction becomes so strong that a proton transfer occurs and the propionic acid is formed. Finally, the models 3 include a free N-methyl-acetamide molecule to mimic a portion of the protein backbone. This influences the orientation of carboxylate groups and limits the amount of their hydrogen bonding with the Im ligand. Residual electrostatic interactions are maintained, which are still able to modulate the dissociation of the methionine from the heme.     

A molecular dynamics investigation of the resting, hydrogen peroxide-bound and compound II forms of cytochrome C peroxidase and Artromyces ramosus peroxidase

Molecular Dynamics (MD) simulations have been used to study structural and dynamic properties of the resting, hydrogen peroxide adduct and compound II forms of cytochrome C peroxidase (CCP) and Artromyces ramosus peroxidase (ARP). MD simulations of CCP show that: (i) hydrogen peroxide might form an outer sphere complex within the active site of the enzyme before the coordination to the iron centre takes place; (ii) Trp51 and His52 residues play a crucial role in the recognition and binding of hydrogen peroxide, while Arg48 is not directly involved; (iii) distal histidine (His52) allows an easy proton 1,2 shift within the H₂O₂ molecule, while Arg48 is not expected to play a role as crucial as His52 in promoting the heterolytic O–O bond breaking; (iv) the large mobility (about 2 Å) of the side chain of Arg48 in the compound II form allows the formation of a hydrogen bond (H-bond) with the ferryl oxygen, which contributes to the stabilisation of such an intermediate. The active site of the ARP enzyme is characterised by structural and dynamic features slightly different from the CCP active site. In particular, (i) the outer sphere complex with hydrogen peroxide occurring in CCP is not observed in ARP because of the substitution of Trp51 of CCP with the more hydrophobic residue Phe55 of ARP; (ii) His56 and the carbonyl group of Arg52 are determinant in controlling the hydrogen peroxide binding and its orientation in the active site. In ARP, both H₂O₂ and His56 have orientation different than in CCP, but still suited for an easy 1,2 proton shift. (iii) Arg52 in ARP is on average more distant from the heme-iron than in CCP, but its relative orientation is suited to promote an easy cleavage of H₂O₂. (iv) In compound II form of ARP, the Arg52 side chain is too far from the oxy-ferryl group to form a hydrogen bond and therefore ARP looses a stabilising factor, which is present in the corresponding form of CCP.     

Slow proton transfer from the hydrogen-labelled carboxylic acid side chain (Glu-165) of triosephosphate isomerase to imidazole buffer in D2O

The catalytic base at the active site of triosephosphate isomerase (TIM) was labelled with -H by abstraction of a proton from substrate d-glyceraldehyde 3-phosphate to form an enzyme-bound enediol(ate) in D2O solvent. The partitioning of this labelled enzyme between intramolecular transfer of -H to form dihydroxyacetone phosphate (DHAP), and irreversible exchange with -D from solvent was examined by determining the yields of H- and D-labelled products by 1H NMR spectroscopy. The yield of hydrogen-labelled product DHAP remains constant as the concentration of the basic form of imidazole buffer is increased from 0.014 to 0.56 M. This shows that the active site of free TIM, which has an open conformation needed to allow substrate binding, adopts a closed conformation at the enediolate-complex intermediate where the catalytic side chain is sequestered from interaction with imidazole dissolved in D2O.     

The true nature of the Di-iron(III) gamma-Keggin structure in water: catalytic aerobic oxidation and chemistry of an unsymmetrical trimer

The complex [gamma(1,2)-SiW(10){Fe(OH(2))}(2)O(38)](6)(-) (1) has been reported to catalyze the much sought reductant-free selective O(2)-based epoxidation of alkenes (Nishiyama, Y.; Nakagawa, Y.; Mizuno, N. Angew. Chem. Int. Ed. 2001, 40, 3639-3641) in chlorocarbon-acetonitrile solution. The challenge of reproducing catalysis by 1 led us to examine this chemistry in detail. In H(2)O, a desirable solvent for catalysis, 1, does not exist in the proposed organic-medium form in which the two iron atoms are in the binding pocket defined by the equatorial oxygens and, importantly, by two oxygens bound to the central Si heteroatom. Instead, 1 in H(2)O initially forms an unusual trimer [{Fe(2)(OH)(3)(H(2)O)(2)}(3)(gamma-SiW(10)O(36))(3)](15)(-) (2). The X-ray structure of 2 shows that the Fe-O(Si) bonds are cleaved and new bonds (mu-hydroxo bridges) form between these Fe centers and those of the neighboring [gamma(1,2)-SiW(10)Fe(2)] units. Structural, physical, and computational evidence indicate that if the bonds between the d-electron center, M (Fe in the case of 1 and 2), and the terminal ligands on M are stronger than the M-O(x)() bonds, then the out-of-pocket form is more stable and is the one observed. Significantly, 2 in H(2)O forms an intermediate that catalyzes the effective aerobic oxidation of sulfur compounds (mercaptoethanol is oxidized to the corresponding disulfide by O(2) at ambient pressure and temperature). All experimental findings are consistent with dissociation of a gamma-SiW(10) Keggin unit from the trimer, 2, to form the catalytically active species.     

Spectroscopic definition of the ferroxidase site in M ferritin: comparison of binuclear substrate vs cofactor active sites

Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a nonheme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly antiferromagnetically coupled, consistent with a mu-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure--including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin--coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites.     

Rational reprogramming of the R2 subunit of Escherichia coli ribonucleotide reductase into a self-hydroxylating monooxygenase.

The outcome of O2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122*) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine)2-(carboxylate)4 ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a mu-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H(peroxo)) in O2 activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the "extra" electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the mu-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122* and which converts very slowly (t1/2 approximately 7 h) upon incubation at 0 degrees C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone epsilon-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with 16O2 or 18O2 show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O2. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and M?ssbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(III)-phenolate species is ascribed to a ligand rearrangement in which mu-O2- is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon-hydroxyphenylalanine is formed and pathways leading to Y122* formation predominate in both R2-D84E and R2-W48F.     

Density functional theory study of trans-dioxo complexes of iron, ruthenium, and osmium with saturated amine ligands, trans-[M(O)2(NH3)2(NMeH2)2]2+ (M=Fe, Ru, Os), and detection of [Fe(qpy)(O)2]n+ (n=1, 2) by high-resolution ESI mass spectrometry

Density functional theory (DFT) calculations on trans-dioxo metal complexes containing saturated amine ligands, trans-[M(O)2(NH3)2(NMeH2)2]2+ (M=Fe, Ru, Os), were performed with different types of density functionals (DFs): 1) pure generalized gradient approximations (pure GGAs): PW91, BP86, and OLYP; 2) meta-GGAs: VSXC and HCTH407; and 3) hybrid DFs: B3LYP and PBE1PBE. With pure GGAs and meta-GGAs, a singlet d2 ground state for trans-[Fe(O)2(NH3)2(NMeH2)2]2+ was obtained, but a quintet ground state was predicted by the hybrid DFs B3LYP and PBE1PBE. The lowest transition energies in water were calculated to be at lambda approximately 509 and 515 nm in the respective ground-state geometries from PW91 and B3LYP calculations. The nature of this transition is dependent on the DFs used: a ligand-to-metal charge-transfer (LMCT) transition with PW91, but a pi(Fe-O)-->pi*(Fe-O) transition with B3LYP, in which pi and pi* are the bonding and antibonding combinations between the dpi(Fe) and ppi(O(2-)) orbitals. The FeVI/V reduction potential of trans-[Fe(O)2(NH3)2NMeH2)2]2+ was estimated to be +1.30 V versus NHE based on PW91 results. The [Fe(qpy)(O)2](n+) (qpy=2,2':6',2':6',2':6',2'-quinquepyridine; n=1 and 2) ions, tentatively assigned to dioxo iron(V) and dioxo iron(VI), respectively, were detected in the gas phase by high-resolution ESI-MS spectroscopy.     

How can a single second sphere amino acid substitution cause reduction midpoint potential changes of hundreds of millivolts?

The active site metal ion of superoxide dismutase (SOD) is reduced and reoxidized as it disproportionates superoxide to dioxygen and hydrogen peroxide. Thus, the reduction midpoint potential (Em) is a critical determinant of catalytic activity. In E. coli Fe-containing SOD (FeSOD), reduction of Fe3+ is accompanied by protonation of a coordinated OH-, to produce Fe2+ coordinated by H2O. The coordinated solvent's only contact with the protein beyond the active site is a conserved Gln residue. Mutation of this Gln to His or Glu resulted in elevation of the Em by 220 mV and more than 660 mV, respectively [Yikilmaz et al., Biochemistry 2006, 45, 1151-1161], despite the fact that overall protein structure was preserved, His is a chemically conservative replacement for Gln, and neutral Glu is isostructural and isoelectronic with Gln. Therefore, we have investigated several possible bases for the elevated Em's, including altered Fe electronic structure, altered active site electrostatics, altered H-bonding and altered redox-coupled proton transfer. Using EPR, MCD, and NMR spectroscopies, we find that the active site electronic structures of the two mutants resemble that of the WT enzyme, for both oxidation states, and Q69E-FeSOD's apparent deviation from WT-like Fe3+ coordination in the oxidized state can be explained by increased affinity for a small anion. Spontaneous coordination of an exogenous anion can only stabilize oxidized Q69E-Fe3+SOD and, therefore, cannot account for the increased Em of Q69E FeSOD. WT-like anion binding affinities and active site pK's indicate that His69 of Q69H-FeSOD is neutral in both oxidation states, like Gln69 of WT-FeSOD, whereas Glu69 appears to be neutral in the oxidized state but ionized in the reduced state of Q69E-FeSOD. A 1.1 A resolution crystal structure of Q69E-Fe2+SOD indicates that Glu69 accepts a strong H-bond from coordinated solvent in the reduced state, in contrast to the case in WT-FeSOD where Gln69 donates an H-bond. These data and DFT calculations lead to the proposal that the elevated Em of Q69E-FeSOD can be substantially explained by (1) relief from enforced H-bond donation in the reduced state, (2) Glu69's capacity to provide a proton for proton-coupled Fe3+ reduction, and (3) strong hydrogen bond acceptance in the reduced state, which stabilizes coordinated H2O. Our results thus support the hypothesis that the protein matrix can apply significant redox tuning via its influence over redox-coupled proton transfer and the energy associated with it.     

DFT investigation on the mechanism of the deacetylation reaction catalyzed by LpxC

LpxC is a key enzyme in the biochemical synthesis of Lipid A, an important outer cell-membrane component found in a number of pathogenic bacteria. Using DFT, we have investigated the binding of the substrate within its active site as well as the deacetylation mechanism it catalyzes. The substrate is found to preferentially coordinate to the active site Zn2+ via its carbonyl oxygen between a Zn2+-bound H2O and an adjacent threonine (Thr191). Furthermore, upon substrate binding a nearby Glu78 residue is found to readily deprotonate the remaining Zn2+-bound H2O. Unlike several related metallopeptidases, the mechanism of LpxC is found to proceed via four steps; (i) initial hydroxylation of the substrates' carbonyl carbon to give a gem-diolate intermediate, (ii) protonation of the amide nitrogen by the histidine His265-H+, (iii) a barrier-less change in the active site-intermediate hydrogen-bond network and finally, (iv) C-N bond cleavage. Notably, the rate-determining step of the mechanism of LpxC is found to be the initial hydroxylation while the final C-N bond cleavage occurs with an overall barrier of 23.6 kJ mol-1. Furthermore, LpxC uses a general acid/base pair mechanism as indicated by the fact that both His265-H+ and Glu78 are accordingly involved.     

Preparation of high purity 1,2-diols by catalytic oxidation of linear terminal alkenes with H202 in the presence of carboxylic acids under solvent-free conditions

The oxidation of terminal alkenes was smoothly catalyzed by a recyclable and environmentally friendly catalytic system: [(C18H37)2N(CH3)2]3[PW4O16]/H2O2/formic acid. This new catalytic system is not only capable of catalyzing oxidation of terminal alkenes with a phase-transfer character, but also under solvent-free conditions, avoiding the use of chlorinated solvents. Many different kinds of terminal alkenes could be converted to the corresponding 1,2-diols of high purity in high yields. The catalyst could be easily separated and reused after reaction. Both fresh and used [(C18H37)2N(CH3)2]3[PW4O16] catalyst was characterized by Raman and FTIR.