[Show abstract][Hide abstract] ABSTRACT: Fe(III)OOH and Fe(IV)O intermediates have now been documented in a number of nonheme iron active sites. In this Current Opinion we use spectroscopy combined with electronic structure calculations to define the frontier molecular orbitals (FMOs) of these species and their contributions to reactivity. For the low-spin Fe(III)OOH species in activated bleomycin we show that the reactivity of this nonheme iron intermediate is very different from that of the analogous Compound 0 of cytochrome P450. For Fe(IV)O S=1 model species we experimentally define the electronic structure and its contribution to reactivity, and computationally evaluate how this would change for the Fe(IV)O S=2 intermediates found in nonheme iron enzymes.
Current opinion in chemical biology 04/2009; 13(1):99-113. DOI:10.1016/j.cbpa.2009.02.011 · 6.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-valent FeIV=O species are key intermediates in the catalytic cycles of many mononuclear non-heme iron enzymes and have been structurally defined in model systems. Variable-temperature magnetic circular dichroism (VT-MCD) spectroscopy has been used to evaluate the electronic structures and in particular the Fe-O bonds of three FeIV=O (S = 1) model complexes, [FeIV(O)(TMC)(NCMe)]2+, [FeIV(O)(TMC)(OC(O)CF3)]+, and [FeIV(O)(N4Py)]2+. These complexes are characterized by their strong and covalent Fe-O pi-bonds. The MCD spectra show a vibronic progression in the nonbonding --> pi* excited state, providing the Fe-O stretching frequency and the Fe-O bond length in this excited state and quantifying the pi-contribution to the total Fe-O bond. Correlation of these experimental data to reactivity shows that the [FeIV(O)(N4Py)]2+ complex, with the highest reactivity toward hydrogen-atom abstraction among the three, has the strongest Fe-O pi-bond. Density functional calculations were correlated to the data and support the experimental analysis. The strength and covalency of the Fe-O pi-bond result in high oxygen character in the important frontier molecular orbitals (FMOs) for this reaction, the unoccupied beta-spin d(xz/yz) orbitals, that activates these for electrophilic attack. An extension to biologically relevant FeIV=O (S = 2) enzyme intermediates shows that these can perform electrophilic attack reactions along the same mechanistic pathway (pi-FMO pathway) with similar reactivity but also have an additional reaction channel involving the unoccupied alpha-spin d(z2) orbital (sigma-FMO pathway). These studies experimentally probe the FMOs involved in the reactivity of FeIV=O (S = 1) model complexes resulting in a detailed understanding of the Fe-O bond and its contributions to reactivity.
Journal of the American Chemical Society 12/2007; 129(51):15983-96. DOI:10.1021/ja074900s · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: (4-Hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) are two alpha-keto acid dependent mononuclear non-heme iron enzymes that use the same substrate, (4-hydroxyphenyl)pyruvate, but exhibit two different general reactivities. HmaS performs hydrogen-atom abstraction to yield benzylic hydroxylated product (S)-(4-hydroxy)mandelate, whereas HPPD utilizes an electrophilic attack mechanism that results in aromatic hydroxylated product homogentisate. These enzymes provide a unique opportunity to directly evaluate the similarities and differences in the reaction pathways used for these two reactivities. An Fe(II) methodology using CD, magnetic CD, and variable-temperature, variable-field magnetic CD spectroscopies was applied to HmaS and compared with that for HPPD to evaluate the factors that affect substrate interactions at the active site and to correlate these to the different reactivities exhibited by HmaS and HPPD to the same substrate. Combined with density functional theory calculations, we found that HmaS and HPPD have similar substrate-bound complexes and that the role of the protein pocket in determining the different reactivities exhibited by these enzymes (hydrogen-atom abstraction vs. aromatic electrophilic attack) is to properly orient the substrate, allowing for ligand field geometric changes along the reaction coordinate. Elongation of the Fe(IV) O bond in the transition state leads to dominant Fe(III) O(*-) character, which significantly contributes to the reactivity with either the aromatic pi-system or the C H sigma-bond.
Proceedings of the National Academy of Sciences 09/2006; 103(35):12966-73. DOI:10.1073/pnas.0605067103 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-valent iron-oxo intermediates are known or believed to be key oxidizing species in the catalytic mechanisms of many mononuclear and binuclear non-heme iron enzymes. So far only limited experimental data on their electronic structures are available. In this study we extend knowledge from the experimentally well characterized mononuclear Fe(IV)=O (S=1) biomimetic model system to computational insight into the spectroscopy and electronic structures of mono-and binuclear high-valent iron-oxo enzyme intermediates. In the mononuclear Fe(IV)=O complexes, we predict the spectroscopy and energies of the electronic transitions to be very different for the S=1 and S=2 spin states, but the iron-oxo bonding for both spin states to be very similar. A comparison of the S=2 mono- and binuclear high-valent iron-sites predicts similar electronic transitions. However, the bent iron-oxo bridge and interactions with the second iron-center in the dimer shift the transitions to higher energies and splits the d(xz/yz) orbital set. These electronic structure and TD-DFT results provide a basis for understanding the spectroscopy and electronic structures of high-valent intermediates in mono- and binuclear non-heme iron enzymes.
[Show abstract][Hide abstract] ABSTRACT: Bleomycin (BLM), a glycopeptide antibiotic chemotherapy agent, is capable of single- and double-strand DNA damage. Activated bleomycin (ABLM), a low-spin Fe(III)-OOH complex, is the last intermediate detected prior to DNA cleavage following hydrogen-atom abstraction from the C-4' of a deoxyribose sugar moiety. The mechanism of this C-H bond cleavage reaction and the nature of the active oxidizing species are still open issues. We have used kinetic measurements in combination with density functional calculations to study the reactivity of ABLM and the mechanism of the initial attack on DNA. Circular dichroism spectroscopy was used to directly monitor the kinetics of the ABLM reaction. These experiments yield a deuterium isotope effect, kH/kD approximately 3 for ABLM decay, indicating the involvement of a hydrogen atom in the rate-determining step. H-atom donors with relatively weak X-H bonds accelerate the reaction rate, establishing that ABLM is capable of hydrogen-atom abstraction. Density functional calculations were used to evaluate the two-dimensional potential energy surface for the direct hydrogen-atom abstraction reaction of the deoxyribose 4'-H by ABLM. The calculations confirm that ABLM is thermodynamically and kinetically competent for H-atom abstraction. The activation and reaction energies for this pathway are favored over both homolytic and heterolytic O-O bond cleavage. Direct H-atom abstraction by ABLM would generate a reactive Fe(IV)=O species, which would be capable of a second DNA strand cleavage, as observed in vivo. This study provides experimental and theoretical evidence for direct H-atom abstraction by ABLM and proposes an attractive mechanism for the role of ABLM in double-strand cleavage.
Journal of the American Chemical Society 05/2006; 128(14):4719-33. DOI:10.1021/ja057378n · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: (4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) is an alpha-keto-acid-dependent dioxygenase which catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate as part of tyrosine catabolism. While several di- and tri-ketone alkaloids are known as inhibitors of HPPD and used commercially as herbicides, one such inhibitor, [2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC), has also been used therapeutically to treat type I tyrosinemia and alkaptonuria in humans. To gain further insight into the mechanism of inhibition by NTBC, a combination of CD/MCD spectroscopy and DFT calculations of HPPD/Fe(II)/NTBC has been performed to evaluate the contribution of the Fe(II)-NTBC bonding interaction to the high affinity of this drug for the enzyme. The results indicate that the bonding of NTBC to Fe(II) is very similar to that for HPP, both involving similar pi-backbonding interactions between NTBC/HPP and Fe(II). Combined with the result that the calculated binding energy of NTBC is, in fact, approximately 3 kcal/mol less than that for HPP, the bidentate coordination of NTBC to Fe(II) is not solely responsible for its extremely high affinity for the enzyme. Thus, the pi-stacking interactions between the aromatic rings of NTBC and two phenyalanine residues, as observed in the crystallography of the HPPD/Fe(II)/NTBC complex, appear to be responsible for the observed high affinity of drug binding.
Biochemical and Biophysical Research Communications 01/2006; 338(1):206-14. DOI:10.1016/j.bbrc.2005.08.242 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enzymes containing heme, non-heme iron and copper active sites play important roles in the activation of dioxygen for substrate oxidation. One key reaction step is CH bond cleavage through H-atom abstraction. On the basis of the ligand environment and the redox properties of the metal, these enzymes employ different methods of dioxygen activation. Heme enzymes are able to stabilize the very reactive iron(IV)-oxo porphyrin-radical intermediate. This is generally not accessible for non-heme iron systems, which can instead use low-spin ferric-hydroperoxo and iron(IV)-oxo species as reactive oxidants. Copper enzymes employ still a different strategy and achieve H-atom abstraction potentially through a superoxo intermediate. This review compares and contrasts the electronic structures and reactivities of these various oxygen intermediates.
Current Opinion in Chemical Biology 05/2005; 9(2):152-63. DOI:10.1016/j.cbpa.2005.02.012 · 6.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: (Chemical Equation Presented) Extremely similar electronic structures and Fe-O bonding characterize FeIV=O S = 1 heme and non-heme species as a result of a decoupling of the oxoiron unit from the porphyrin (Por) π system in the heme complex (see scheme). However, along the reaction coordinate towards an FeIII-OH product, the Por π system interacts with the iron d orbitals, thus resulting in different reactivities for heme and non-heme complexes.
[Show abstract][Hide abstract] ABSTRACT: High valent FeIV=O species are key intermediates in the catalytic cycles of many mononuclear non-heme iron enzymes involving the binding and activation of dioxygen. Using variable temperature magnetic circular dichroism (VT MCD) spectroscopy and experimentally calibrated density functional calculations, we are able to present the first detailed description of the electronic structure of a non-heme FeIV=O S = 1 complex. These studies define the nature of the FeIV=O bond and present the basis for understanding high-valent oxygen intermediates in non-heme iron enzymes.
Journal of the American Chemical Society 06/2004; 126(17):5378-9. DOI:10.1021/ja0498033 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD, EC 126.96.36.199), has been studied using magnetic circular dichroism (MCD), variable-temperature variable-field (VTVH) MCD, X-ray absorption (XAS) pre-edge, and extended X-ray absorption fine structure (EXAFS) spectroscopies, which are analogous to methods used in earlier studies on the extradiol dioxygenase catechol 2,3-dioxygenase [Mabrouk et al. J. Am. Chem Soc. 1991, 113, 4053-4061]. For DHBD, the spectroscopic data can be correlated to the results of crystallography and with the results from density functional calculations to obtain detailed geometric and electronic structure descriptions of the resting and substrate (DHB) bound forms of the enzyme. The geometry of the active site of the resting enzyme, square pyramidal with a strong Fe-glutamate bond in the equatorial plane, localizes the redox active orbital in an orientation appropriate for O(2) binding. However, the O(2) reaction is not favorable, as it would produce a ferric superoxide intermediate with a weak Fe-O bond. Substrate binding leads to a new square pyramidal structure with the strong Fe-glutamate bond in the axial direction as indicated by a decrease in the (5)E(g) and increase in the (5)T(2g) splitting. Electronic structure calculations provide insight into the relative lack of dioxygen reactivity for the resting enzyme and its activation upon substrate binding.
Journal of the American Chemical Society 10/2003; 125(37):11214-27. DOI:10.1021/ja029746i · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bleomycin is an antibiotic used in cancer chemotherapy for its ability to achieve both single- and double-strand cleavage of DNA through abstraction of the deoxyribose C4'-H. Magnetic circular dichroism (MCD) and X-ray absorption (XAS) spectroscopies have been used to study the interaction of the biologically relevant FeIIBLM complex with DNA. Calf thymus DNA was used as the substrate as well as short oligonucleotides, including one with a preferred 5'-G-pyrimidine-3' cleavage site [d(GGAAGCTTCC)2] and one without [d(GGAAATTTCC)2]. DNA binding to FeIIBLM significantly perturbs the FeII active site, resulting in a change in intensity ratio of the d d transitions and a decrease in excited-state orbital splitting (5Eg). Although this effect is somewhat dependent on length and composition of the oligonucleotide, it is not correlated to the presence of a 5'-G-pyrimidine-3' cleavage site. No effect is observed on the charge-transfer transitions, indicating that the H-bonding recognition between the pyrimidine and guanine base does not perturb Fe-pyrimidine backbonding. Azide binding studies indicate that FeIIBLM bound to either oligomer has the same affinity for N3-. Parallel studies of BLM structural derivatives indicate that FeIIiso-PEPLM, in which the carbamoyl group is shifted on the mannose sugar, forms the same DNA-bound species as FeIIBLM. In contrast, FeIIDP-PEPLM, in which the -aminoalanine group is absent, forms a new species upon DNA binding. These data are consistent with a model in which the primary amine from the -aminoalanine is an FeII ligand and the mannose carbamoyl provides either a ligand to the FeII or significant second-sphere effects on the FeII site; intercalation of the bithiazole tail into the double helix likely brings the metal-bound complex close enough to the DNA to create steric interactions that remove the sugar groups from interaction with the FeII. The fact that the FeII active site is perturbed regardless of DNA sequence is consistent with the fact that cleavage is observed for both 5'-GC-3' and nonspecific oligomers and indicates that different reaction coordinates may be active, depending on orientation of the deoxyribose C4'-H.
Journal of the American Chemical Society 10/2003; 125(36):10810-21. DOI:10.1021/ja034579n · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-heme iron enzymes catalyze a wide range of O(2) reactions, paralleling those of heme systems. Non-heme iron active sites are, however, much more difficult to study because they do not exhibit the intense spectral features characteristic of the porphyrin ligand. A spectroscopic methodology was developed that provides significant mechanistic insight into the reactivity of non-heme ferrous active sites. These studies reveal a general mechanistic strategy used by these enzymes and differences in substrate and cofactor interactions dependent on their requirement for activation by iron. Contributions to O(2) activation have been elucidated for non-heme relative to heme ligand sets, and major differences in reactivity are defined with respect to the heterolytic and homolytic cleavage of O-O bonds.
Proceedings of the National Academy of Sciences 05/2003; 100(7):3589-94. DOI:10.1073/pnas.0336792100 · 9.67 Impact Factor