Triply Resonant Sum Frequency (TRSF) spectroscopy is a fully coherent mixed vibrational-electroni... more Triply Resonant Sum Frequency (TRSF) spectroscopy is a fully coherent mixed vibrational-electronic spectroscopic technique that is ideally suited for probing the vibrational-electronic couplings that become important in driving reactions. We have used cyanocobalamin (CNCbl) and deuterated aquacobalamin (D OCbl +) as model systems for demonstrating the feasibility of using the selectivity of coherent multidimensional spectroscopy to resolve electronic states within the broad absorption spectra of transition metal complexes and identify the nature of the vibrational and electronic state couplings. We resolve three short and long axis vibrational modes in the vibrationally congested 1400-1750 cm −1 region that are individually coupled to different electronic states in the 18,000-21,000 cm −1 region but have minimal coupling to each other. Double resonance with the individual vibrational fundamentals and their overtones selectively enhances the corresponding electronic resonances and resolves features within the broad absorption spectrum. This work demonstrates the feasibility of identifying coupling between different pairs of vibrational states with different electronic states that together form the reaction coordinate surface of transition metal enzymes.
Mononuclear nonheme iron complexes that serve as structural and functional mimics of the thiol di... more Mononuclear nonheme iron complexes that serve as structural and functional mimics of the thiol dioxygenases (TDOs), cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), have been prepared and characterized with crystallographic, spectroscopic, kinetic, and computational methods. The high-spin Fe(II) complexes feature the facially-coordinating tris(4,5-diphenyl-1methylimidazol-2-yl)phosphine (Ph2 TIP) ligand that replicates the three histidine (3His) triad of the TDO active sites. Further coordination with bidentate L-cysteine ethyl ester (CysOEt) or cysteamine (CysAm) anions yielded five-coordinate (5C) complexes that resemble the substratebound forms of CDO and ADO, respectively. Detailed electronic-structure descriptions of the [Fe(Ph2 TIP)(L S,N)]BPh 4 complexes, where L S,N = CysOEt (1) or CysAm (2), were generated through a combination of spectroscopic techniques [electronic absorption, magnetic circular dichroism (MCD)] and density functional theory (DFT). Complexes 1 and 2 decompose in the presence of O 2 to yield the corresponding sulfinic acid (RSO 2 H) products, thereby emulating the reactivity of the TDO enzymes and related complexes. Rate constants and activation parameters for the dioxygenation reactions were measured and interpreted with the aid of DFT calculations for O 2-bound intermediates. Treatment of the TDO models with nitric oxide (NO)-a wellestablished surrogate of O 2-led to a mixture of high-spin and low-spin {FeNO} 7 species at low temperature (−70 °C), as indicated by electron paramagnetic resonance (EPR) spectroscopy. At room temperature, these Fe/NO adducts convert to a common species with EPR and infrared (IR) features typical of cationic dinitrosyl iron complexes (DNICs). To complement these results, parallel spectroscopic, computational, and O 2 /NO reactivity studies were carried out using previously-reported TDO models that feature an anionic hydrotris(3-phenyl-5-methylpyrazolyl)borate (Ph,Me Tp −) ligand. Though the O 2 reactivities of the Ph2 TIP-and Ph,Me Tp-based complexes are quite similar, the supporting ligand perturbs the energies of Fe 3d-based molecular orbitals and modulates Fe-S bond covalency, suggesting possible rationales for the presence of neutral 3His coordination in CDO and ADO.
The cobalamin or B12 cofactor supports sulfur and one-carbon metabolism and the catabolism of odd... more The cobalamin or B12 cofactor supports sulfur and one-carbon metabolism and the catabolism of odd-chain fatty acids, branched-chain amino acids and cholesterol. CblC is a B12 processing enzyme involved in an early cytoplasmic step in the cofactor trafficking pathway. It catalyzes the glutathione (GSH)-dependent dealkylation of alkylcobalamins and the reductive decyanation of cyanocobalamin. CblC from Caenorhabdiitis elegans (ceCblC) also exhibits a robust thiol oxidase activity converting reduced GSH to oxidized GSSG with concomitant scrubbing of ambient dissolved O2 The mechanism of thiol oxidation catalyzed by ceCblC is not known. In this study, we demonstrate that novel coordination chemistry accessible to ceCblC-bound cobalamin, supports its thiol oxidase activity via a glutathionyl-cobalamin intermediate. Deglutathionylation of glutathionyl-cobalamin by a second molecule of GSH yields GSSG. The crystal structure of ceCblC provides insights into how architectural differences at ...
JBIC Journal of Biological Inorganic Chemistry, 2016
The human-type ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri (LrPduO) catalyz... more The human-type ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri (LrPduO) catalyzes the adenosylation of Co(II)rrinoids to generate adenosylcobalamin (AdoCbl) or adenosylcobinamide (AdoCbi +). This process requires the formation of "supernucleophilic" Co(I)rrinoid intermediates in the enzyme active site that are properly positioned to abstract the adeonsyl moiety from co-substrate ATP. Previous magnetic circular dichroism (MCD) spectroscopic and X-ray crystallographic analyses revealed that LrPduO achieves the thermodynamically challenging reduction of Co(II)rrinoids by displacing the axial ligand with a non-coordinating phenylalanine residue to produce a four-coordinate species. However, relatively little is currently known about the interaction between the tetradentate equatorial ligand of Co(II)rrinoids (the corrin ring) and the enzyme active site. To address this issue, we have collected resonance Raman (rR) data of Co(II)rrinoids free in solution and bound to the LrPduO active site. The relevant resonance-enhanced vibrational features of the free Co(II)rrinoids are assigned on the basis of rR intensity calculations using density functional theory to establish a suitable framework for interpreting rR spectral changes that occur upon Co(II)rrinoid binding to the LrPduO/ATP complex in terms of structural perturbations of the corrin ring. To complement our rR data, we have also obtained MCD spectra of Co(II)rrinoids bound to LrPduO complexed with the ATP analogue UTP. Collectively, our results provide compelling evidence that in the LrPduO active site, the corrin ring of Co(II)rrinoids is firmly locked in place by several amino acid side chains so as to facilitate the dissociation of the axial ligand.
Journal of the American Chemical Society, Jul 30, 2016
Enzymes in the sulfur network generate the signaling molecule, hydrogen sulfide (H2S), from the a... more Enzymes in the sulfur network generate the signaling molecule, hydrogen sulfide (H2S), from the amino acids cysteine and homocysteine. Since it is toxic at elevated concentrations, cells are equipped to clear H2S. A canonical sulfide oxidation pathway operates in mitochondria, converting H2S to thiosulfate and sulfate. We have recently discovered the ability of ferric hemoglobin to oxidize sulfide to thiosulfate and iron-bound hydropolysulfides. In this study, we report that myoglobin exhibits a similar capacity for sulfide oxidation. We have trapped and characterized iron-bound sulfur intermediates using cryo-mass spectrometry and X-ray absorption spectroscopy. Further support for the postulated intermediates in the chemically challenging conversion of H2S to thiosulfate and iron-bound catenated sulfur products is provided by EPR and resonance Raman spectroscopy in addition to density functional theory computational results. We speculate that the unusual sensitivity of skeletal mus...
Journal of the American Chemical Society, Jan 23, 2016
EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosylt... more EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosyltransferases (ACATs), implicated in the biosynthesis of adenosylcobalamin (AdoCbl). In the presence of cosubstrate ATP, ACATs raise the Co(II)/Co(I) reduction potential of their cob(II)alamin [Co(II)Cbl] substrate by >250 mV via the formation of a unique four-coordinate (4c) Co(II)Cbl species, thereby facilitating the formation of a "supernucleophilic" cob(I)alamin intermediate required for the formation of the AdoCbl product. Previous kinetic studies of EutT revealed the importance of a HX11CCX2C(83) motif for catalytic activity and have led to the proposal that residues in this motif serve as the binding site for a divalent transition metal cofactor [e.g., Fe(II) or Zn(II)]. This motif is absent in other ACAT families, suggesting that EutT employs a distinct mechanism for AdoCbl formation. To assess how metal ion binding to the HX11CCX2C(83) motif affects the relative yield...
Vitamin B12 and its biologically active derivatives, 5′-deoxyadenosylcobalamin (AdoCbl) and methy... more Vitamin B12 and its biologically active derivatives, 5′-deoxyadenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl), have long fascinated chemists with their elaborate structures and unusual reactivities in enzymatic systems. Due to their large size and complex electronic structures, these cofactors have posed a major challenge for computational chemists. Yet, recent insights gained from kinetic, spectroscopic, and X-ray crystallographic studies, have established an excellent
Because of their complex geometric and electronic structures, the active sites and cofactors of b... more Because of their complex geometric and electronic structures, the active sites and cofactors of bioorganometallic enzymes, which are characterized by their metal-carbon bonds, pose a major challenge for computational chemists. However, recent progress in computer technology and theoretical chemistry, along with insights gained from mechanistic, spectroscopic, and X-ray crystallographic studies, have established an excellent foundation for the successful completion of computational studies aimed at elucidating the electronic structures and catalytic cycles of these species. This chapter briefly reviews the most popular computational approaches employed in theoretical studies of bioorganometallic species and summarizes important information obtained from computational studies of (i) the enzymatic formation and cleavage of the Co-C bond of coenzyme B(12); (ii) the catalytic cycle of methyl-coenzyme M reductase and its nickel-containing cofactor F(430); (iii) the polynuclear active-site...
Proceedings of the National Academy of Sciences, 2007
M olecular oxygen (O 2) is the principal oxidant used by aerobic organisms to carry out a wide ra... more M olecular oxygen (O 2) is the principal oxidant used by aerobic organisms to carry out a wide range of metabolic reactions and transformations. The enzymes involved in these processes use a rich variety of different active sites to activate O 2 , such as heme cofactors, mono-and binuclear nonheme iron centers, mono-and binuclear copper complexes, and heteronuclear heme
We previously reported a preliminary mechanistic study of aerobic Cu(OAc) 2-catalyzed methoxylati... more We previously reported a preliminary mechanistic study of aerobic Cu(OAc) 2-catalyzed methoxylation of 4-tolylboronic ester (King, et al. J. Am. Chem. Soc., 2009, 131, 5044-5045), which revealed that aryl transmetalation from the boronic ester to Cu II is the turnover-limiting step. In the present study, more-thorough kinetic and spectroscopic studies provide additional insights into transmetalation pathway and the identity of the Cu II catalyst resting state(s). EPR spectroscopic studies show that at least two copper(II) species are present under catalytic conditions and their relative populations vary as a function of reaction time and acidity of the arylboronic ester, and are influenced by addition of acetic acid or acetate to the reaction mixture. Analysis of kinetic data and 11 B NMR and EPR spectra under diverse reaction conditions suggests that aryl transmetalation occurs from a tetracoordinate, anionic boronate to a cationic Cu II species, mediated by a methoxide-bridge.
The cobalamin-dependent methionine synthase (MetH) from Escherichia coli is a modular enzyme that... more The cobalamin-dependent methionine synthase (MetH) from Escherichia coli is a modular enzyme that catalyzes a methyl-group transfer from methyltetrahydrofolate to homocysteine via a methylcob(III)alamin (MeCbl) intermediate, generating tetrahydrofolate and methionine (Met). Once every ~2000 turnovers, the cobalamin cofactor is converted to the inactive cob(II)alamin (Co 2+ Cbl) form, from which MeCbl has to be recovered in order for MetH to re-enter the catalytic cycle. A particularly puzzling aspect of this reactivation process is that it requires the reduction of the Co 2+ Cbl species to cob(I)alamin (Co 1+ Cbl) by flavodoxin, a reaction that would appear to be endergonic based upon the corresponding reduction potentials. To explore how MetH may overcome this apparent thermodynamic challenge, we have prepared the I690C/G743C variant of a C-terminal fragment of MetH (MetH CT) so as to lock the enzyme into the activation conformation without perturbing any of the residues in the vicinity of the active site. A detailed spectroscopic characterization of this species and the I690C/G743C/Y1139F MetH CT triple mutant reveals that the strategy employed by MetH to activate Co 2+ Cbl for Co 2+ → Co 1+ reduction likely involves (i) an axial ligand switch to generate a five-coordinate species with an axially coordinated water molecule and (ii) a significant lengthening, or perhaps complete rupture, of the Co-OH 2 bond of the cofactor, thereby causing a large stabilization of the Co 3d z 2-based "redox-active" molecular orbital. The lengthening of the Co-OH 2 bond is mediated by the Y1139 active-site residue and becomes much more dramatic when the S-adenosylmethionine substrate is present in the enzyme active site. This substrate requirement provides MetH a means to suppress deleterious side reactions involving the transiently formed Co 1+ Cbl "supernucleophile".
CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo bios... more CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo biosynthesis of adenosylcobalamin (AdoCbl), catalyzing the formation of the essential Co-C bond by transferring the adenosyl group from a molecule of ATP to a transient Co 1+ corrinoid species generated in the enzyme active site. A particularly fascinating aspect of this reaction is that the flavodoxin in vivo reducing agent that serves as the electron donor to CobA possesses a reduction potential that is considerably more positive than that of the Co 2+/1+ couple of the corrinoid substrate. To explore how CobA may overcome this challenge, we have employed electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to probe the interaction between Co 3+-and Co 2+ corrinoids and the enzyme active site. Our data reveal that while Co 3+ corrinoids interact only weakly with CobA, Co 2+ corrinoids undergo partial conversion to a new paramagnetic species that can be obtained in nearly quantitative yield when CobA is preincubated with the co-substrate ATP. This "activated" species is characterized by a distinct set of ligand field transitions in the near-IR spectral region and EPR parameters that are unprecedented for Co 2+ corrinoids. Analysis of these data on the basis of qualitative spectral correlations and density functional theory computations reveals that this unique Co 2+ corrinoid species possesses an essentially square-planar Co 2+ center that lacks any significant axial bonding interactions. Possible implications of these findings for the mechanism of Co 2+ f Co 1+ reduction employed by CobA and Co-C bond-forming enzymes in general are explored.
Abstract The (3d) 2 ions Cr 4+ , Mn 5+ and Fe 6+ show sharp-line luminescence in the NIR around 1... more Abstract The (3d) 2 ions Cr 4+ , Mn 5+ and Fe 6+ show sharp-line luminescence in the NIR around 1.2, 1.2 and 1.6 μm, respectively. An attempt is made to find trends and similarities for these three ions and to provide an appropriate model.
We have prepared and thoroughly characterized, using X-ray crystallographic, spectroscopic, and c... more We have prepared and thoroughly characterized, using X-ray crystallographic, spectroscopic, and computational methods, the diazide adduct of [Fe III (dapsox)(H 2 O) 2 ] 1+ [dapsox=2,6diacetylpyridinebis(semioxamazide)] (1), alow-molecular weight, functional analogue of iron superoxide dismutase (FeSOD). The X-ray crystal structure of the dimeric form of 1, (Na[Fe III (dapsox)(N 3) 2 ] DMF) 2 (2) shows two axially coordinated, symmetry inequivalent azides with differing Fe-N 3 bond lengths and Fe-N-N 2 bond angles. This inequivalence of the azide ligands likely reflects the presence of an inter-dimer H-bonding interaction between a dapsox NH group and the coordinated nitrogen of one of the two azide ligands. Resonance Raman (rR) data obtained for frozen aqueous solution and solid-state samples of 2 indicate that the azides remain inequivalent in solution, suggesting that one of the azide ligands of 1 engages in an intermolecular hydrogen bonding interaction with a water molecule. Density functional theory (DFT) and timedependent DFT calculations have been used to study two different computational models of 1, one using coordinates taken from the X-ray crystal structure of 2, and the other generated via DFT geometry optimization. An evaluation of these models on the basis of electronic absorption, magnetic circular dichroism, and rR data indicates that the crystal structure based model provides a more accurate electronic structure description of 1, providing further support for the proposed intermolecular hydrogen bonding of 1 in the solid state and in solution. An analysis of the experimentally validated DFT results for this model reveals that the azides have both σand πbonding interactions with the Fe III center and that more negative charge is located on the Febound, rather than on the terminal, nitrogen atom of each azide. These observations are reminiscent of the results previously reported for the azide adduct of FeSOD and provide clues regarding the origin the high catalytic activity of Fe-dapsox for superoxide disproportionation.
Triply Resonant Sum Frequency (TRSF) spectroscopy is a fully coherent mixed vibrational-electroni... more Triply Resonant Sum Frequency (TRSF) spectroscopy is a fully coherent mixed vibrational-electronic spectroscopic technique that is ideally suited for probing the vibrational-electronic couplings that become important in driving reactions. We have used cyanocobalamin (CNCbl) and deuterated aquacobalamin (D OCbl +) as model systems for demonstrating the feasibility of using the selectivity of coherent multidimensional spectroscopy to resolve electronic states within the broad absorption spectra of transition metal complexes and identify the nature of the vibrational and electronic state couplings. We resolve three short and long axis vibrational modes in the vibrationally congested 1400-1750 cm −1 region that are individually coupled to different electronic states in the 18,000-21,000 cm −1 region but have minimal coupling to each other. Double resonance with the individual vibrational fundamentals and their overtones selectively enhances the corresponding electronic resonances and resolves features within the broad absorption spectrum. This work demonstrates the feasibility of identifying coupling between different pairs of vibrational states with different electronic states that together form the reaction coordinate surface of transition metal enzymes.
Mononuclear nonheme iron complexes that serve as structural and functional mimics of the thiol di... more Mononuclear nonheme iron complexes that serve as structural and functional mimics of the thiol dioxygenases (TDOs), cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), have been prepared and characterized with crystallographic, spectroscopic, kinetic, and computational methods. The high-spin Fe(II) complexes feature the facially-coordinating tris(4,5-diphenyl-1methylimidazol-2-yl)phosphine (Ph2 TIP) ligand that replicates the three histidine (3His) triad of the TDO active sites. Further coordination with bidentate L-cysteine ethyl ester (CysOEt) or cysteamine (CysAm) anions yielded five-coordinate (5C) complexes that resemble the substratebound forms of CDO and ADO, respectively. Detailed electronic-structure descriptions of the [Fe(Ph2 TIP)(L S,N)]BPh 4 complexes, where L S,N = CysOEt (1) or CysAm (2), were generated through a combination of spectroscopic techniques [electronic absorption, magnetic circular dichroism (MCD)] and density functional theory (DFT). Complexes 1 and 2 decompose in the presence of O 2 to yield the corresponding sulfinic acid (RSO 2 H) products, thereby emulating the reactivity of the TDO enzymes and related complexes. Rate constants and activation parameters for the dioxygenation reactions were measured and interpreted with the aid of DFT calculations for O 2-bound intermediates. Treatment of the TDO models with nitric oxide (NO)-a wellestablished surrogate of O 2-led to a mixture of high-spin and low-spin {FeNO} 7 species at low temperature (−70 °C), as indicated by electron paramagnetic resonance (EPR) spectroscopy. At room temperature, these Fe/NO adducts convert to a common species with EPR and infrared (IR) features typical of cationic dinitrosyl iron complexes (DNICs). To complement these results, parallel spectroscopic, computational, and O 2 /NO reactivity studies were carried out using previously-reported TDO models that feature an anionic hydrotris(3-phenyl-5-methylpyrazolyl)borate (Ph,Me Tp −) ligand. Though the O 2 reactivities of the Ph2 TIP-and Ph,Me Tp-based complexes are quite similar, the supporting ligand perturbs the energies of Fe 3d-based molecular orbitals and modulates Fe-S bond covalency, suggesting possible rationales for the presence of neutral 3His coordination in CDO and ADO.
The cobalamin or B12 cofactor supports sulfur and one-carbon metabolism and the catabolism of odd... more The cobalamin or B12 cofactor supports sulfur and one-carbon metabolism and the catabolism of odd-chain fatty acids, branched-chain amino acids and cholesterol. CblC is a B12 processing enzyme involved in an early cytoplasmic step in the cofactor trafficking pathway. It catalyzes the glutathione (GSH)-dependent dealkylation of alkylcobalamins and the reductive decyanation of cyanocobalamin. CblC from Caenorhabdiitis elegans (ceCblC) also exhibits a robust thiol oxidase activity converting reduced GSH to oxidized GSSG with concomitant scrubbing of ambient dissolved O2 The mechanism of thiol oxidation catalyzed by ceCblC is not known. In this study, we demonstrate that novel coordination chemistry accessible to ceCblC-bound cobalamin, supports its thiol oxidase activity via a glutathionyl-cobalamin intermediate. Deglutathionylation of glutathionyl-cobalamin by a second molecule of GSH yields GSSG. The crystal structure of ceCblC provides insights into how architectural differences at ...
JBIC Journal of Biological Inorganic Chemistry, 2016
The human-type ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri (LrPduO) catalyz... more The human-type ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri (LrPduO) catalyzes the adenosylation of Co(II)rrinoids to generate adenosylcobalamin (AdoCbl) or adenosylcobinamide (AdoCbi +). This process requires the formation of "supernucleophilic" Co(I)rrinoid intermediates in the enzyme active site that are properly positioned to abstract the adeonsyl moiety from co-substrate ATP. Previous magnetic circular dichroism (MCD) spectroscopic and X-ray crystallographic analyses revealed that LrPduO achieves the thermodynamically challenging reduction of Co(II)rrinoids by displacing the axial ligand with a non-coordinating phenylalanine residue to produce a four-coordinate species. However, relatively little is currently known about the interaction between the tetradentate equatorial ligand of Co(II)rrinoids (the corrin ring) and the enzyme active site. To address this issue, we have collected resonance Raman (rR) data of Co(II)rrinoids free in solution and bound to the LrPduO active site. The relevant resonance-enhanced vibrational features of the free Co(II)rrinoids are assigned on the basis of rR intensity calculations using density functional theory to establish a suitable framework for interpreting rR spectral changes that occur upon Co(II)rrinoid binding to the LrPduO/ATP complex in terms of structural perturbations of the corrin ring. To complement our rR data, we have also obtained MCD spectra of Co(II)rrinoids bound to LrPduO complexed with the ATP analogue UTP. Collectively, our results provide compelling evidence that in the LrPduO active site, the corrin ring of Co(II)rrinoids is firmly locked in place by several amino acid side chains so as to facilitate the dissociation of the axial ligand.
Journal of the American Chemical Society, Jul 30, 2016
Enzymes in the sulfur network generate the signaling molecule, hydrogen sulfide (H2S), from the a... more Enzymes in the sulfur network generate the signaling molecule, hydrogen sulfide (H2S), from the amino acids cysteine and homocysteine. Since it is toxic at elevated concentrations, cells are equipped to clear H2S. A canonical sulfide oxidation pathway operates in mitochondria, converting H2S to thiosulfate and sulfate. We have recently discovered the ability of ferric hemoglobin to oxidize sulfide to thiosulfate and iron-bound hydropolysulfides. In this study, we report that myoglobin exhibits a similar capacity for sulfide oxidation. We have trapped and characterized iron-bound sulfur intermediates using cryo-mass spectrometry and X-ray absorption spectroscopy. Further support for the postulated intermediates in the chemically challenging conversion of H2S to thiosulfate and iron-bound catenated sulfur products is provided by EPR and resonance Raman spectroscopy in addition to density functional theory computational results. We speculate that the unusual sensitivity of skeletal mus...
Journal of the American Chemical Society, Jan 23, 2016
EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosylt... more EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosyltransferases (ACATs), implicated in the biosynthesis of adenosylcobalamin (AdoCbl). In the presence of cosubstrate ATP, ACATs raise the Co(II)/Co(I) reduction potential of their cob(II)alamin [Co(II)Cbl] substrate by >250 mV via the formation of a unique four-coordinate (4c) Co(II)Cbl species, thereby facilitating the formation of a "supernucleophilic" cob(I)alamin intermediate required for the formation of the AdoCbl product. Previous kinetic studies of EutT revealed the importance of a HX11CCX2C(83) motif for catalytic activity and have led to the proposal that residues in this motif serve as the binding site for a divalent transition metal cofactor [e.g., Fe(II) or Zn(II)]. This motif is absent in other ACAT families, suggesting that EutT employs a distinct mechanism for AdoCbl formation. To assess how metal ion binding to the HX11CCX2C(83) motif affects the relative yield...
Vitamin B12 and its biologically active derivatives, 5′-deoxyadenosylcobalamin (AdoCbl) and methy... more Vitamin B12 and its biologically active derivatives, 5′-deoxyadenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl), have long fascinated chemists with their elaborate structures and unusual reactivities in enzymatic systems. Due to their large size and complex electronic structures, these cofactors have posed a major challenge for computational chemists. Yet, recent insights gained from kinetic, spectroscopic, and X-ray crystallographic studies, have established an excellent
Because of their complex geometric and electronic structures, the active sites and cofactors of b... more Because of their complex geometric and electronic structures, the active sites and cofactors of bioorganometallic enzymes, which are characterized by their metal-carbon bonds, pose a major challenge for computational chemists. However, recent progress in computer technology and theoretical chemistry, along with insights gained from mechanistic, spectroscopic, and X-ray crystallographic studies, have established an excellent foundation for the successful completion of computational studies aimed at elucidating the electronic structures and catalytic cycles of these species. This chapter briefly reviews the most popular computational approaches employed in theoretical studies of bioorganometallic species and summarizes important information obtained from computational studies of (i) the enzymatic formation and cleavage of the Co-C bond of coenzyme B(12); (ii) the catalytic cycle of methyl-coenzyme M reductase and its nickel-containing cofactor F(430); (iii) the polynuclear active-site...
Proceedings of the National Academy of Sciences, 2007
M olecular oxygen (O 2) is the principal oxidant used by aerobic organisms to carry out a wide ra... more M olecular oxygen (O 2) is the principal oxidant used by aerobic organisms to carry out a wide range of metabolic reactions and transformations. The enzymes involved in these processes use a rich variety of different active sites to activate O 2 , such as heme cofactors, mono-and binuclear nonheme iron centers, mono-and binuclear copper complexes, and heteronuclear heme
We previously reported a preliminary mechanistic study of aerobic Cu(OAc) 2-catalyzed methoxylati... more We previously reported a preliminary mechanistic study of aerobic Cu(OAc) 2-catalyzed methoxylation of 4-tolylboronic ester (King, et al. J. Am. Chem. Soc., 2009, 131, 5044-5045), which revealed that aryl transmetalation from the boronic ester to Cu II is the turnover-limiting step. In the present study, more-thorough kinetic and spectroscopic studies provide additional insights into transmetalation pathway and the identity of the Cu II catalyst resting state(s). EPR spectroscopic studies show that at least two copper(II) species are present under catalytic conditions and their relative populations vary as a function of reaction time and acidity of the arylboronic ester, and are influenced by addition of acetic acid or acetate to the reaction mixture. Analysis of kinetic data and 11 B NMR and EPR spectra under diverse reaction conditions suggests that aryl transmetalation occurs from a tetracoordinate, anionic boronate to a cationic Cu II species, mediated by a methoxide-bridge.
The cobalamin-dependent methionine synthase (MetH) from Escherichia coli is a modular enzyme that... more The cobalamin-dependent methionine synthase (MetH) from Escherichia coli is a modular enzyme that catalyzes a methyl-group transfer from methyltetrahydrofolate to homocysteine via a methylcob(III)alamin (MeCbl) intermediate, generating tetrahydrofolate and methionine (Met). Once every ~2000 turnovers, the cobalamin cofactor is converted to the inactive cob(II)alamin (Co 2+ Cbl) form, from which MeCbl has to be recovered in order for MetH to re-enter the catalytic cycle. A particularly puzzling aspect of this reactivation process is that it requires the reduction of the Co 2+ Cbl species to cob(I)alamin (Co 1+ Cbl) by flavodoxin, a reaction that would appear to be endergonic based upon the corresponding reduction potentials. To explore how MetH may overcome this apparent thermodynamic challenge, we have prepared the I690C/G743C variant of a C-terminal fragment of MetH (MetH CT) so as to lock the enzyme into the activation conformation without perturbing any of the residues in the vicinity of the active site. A detailed spectroscopic characterization of this species and the I690C/G743C/Y1139F MetH CT triple mutant reveals that the strategy employed by MetH to activate Co 2+ Cbl for Co 2+ → Co 1+ reduction likely involves (i) an axial ligand switch to generate a five-coordinate species with an axially coordinated water molecule and (ii) a significant lengthening, or perhaps complete rupture, of the Co-OH 2 bond of the cofactor, thereby causing a large stabilization of the Co 3d z 2-based "redox-active" molecular orbital. The lengthening of the Co-OH 2 bond is mediated by the Y1139 active-site residue and becomes much more dramatic when the S-adenosylmethionine substrate is present in the enzyme active site. This substrate requirement provides MetH a means to suppress deleterious side reactions involving the transiently formed Co 1+ Cbl "supernucleophile".
CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo bios... more CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo biosynthesis of adenosylcobalamin (AdoCbl), catalyzing the formation of the essential Co-C bond by transferring the adenosyl group from a molecule of ATP to a transient Co 1+ corrinoid species generated in the enzyme active site. A particularly fascinating aspect of this reaction is that the flavodoxin in vivo reducing agent that serves as the electron donor to CobA possesses a reduction potential that is considerably more positive than that of the Co 2+/1+ couple of the corrinoid substrate. To explore how CobA may overcome this challenge, we have employed electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to probe the interaction between Co 3+-and Co 2+ corrinoids and the enzyme active site. Our data reveal that while Co 3+ corrinoids interact only weakly with CobA, Co 2+ corrinoids undergo partial conversion to a new paramagnetic species that can be obtained in nearly quantitative yield when CobA is preincubated with the co-substrate ATP. This "activated" species is characterized by a distinct set of ligand field transitions in the near-IR spectral region and EPR parameters that are unprecedented for Co 2+ corrinoids. Analysis of these data on the basis of qualitative spectral correlations and density functional theory computations reveals that this unique Co 2+ corrinoid species possesses an essentially square-planar Co 2+ center that lacks any significant axial bonding interactions. Possible implications of these findings for the mechanism of Co 2+ f Co 1+ reduction employed by CobA and Co-C bond-forming enzymes in general are explored.
Abstract The (3d) 2 ions Cr 4+ , Mn 5+ and Fe 6+ show sharp-line luminescence in the NIR around 1... more Abstract The (3d) 2 ions Cr 4+ , Mn 5+ and Fe 6+ show sharp-line luminescence in the NIR around 1.2, 1.2 and 1.6 μm, respectively. An attempt is made to find trends and similarities for these three ions and to provide an appropriate model.
We have prepared and thoroughly characterized, using X-ray crystallographic, spectroscopic, and c... more We have prepared and thoroughly characterized, using X-ray crystallographic, spectroscopic, and computational methods, the diazide adduct of [Fe III (dapsox)(H 2 O) 2 ] 1+ [dapsox=2,6diacetylpyridinebis(semioxamazide)] (1), alow-molecular weight, functional analogue of iron superoxide dismutase (FeSOD). The X-ray crystal structure of the dimeric form of 1, (Na[Fe III (dapsox)(N 3) 2 ] DMF) 2 (2) shows two axially coordinated, symmetry inequivalent azides with differing Fe-N 3 bond lengths and Fe-N-N 2 bond angles. This inequivalence of the azide ligands likely reflects the presence of an inter-dimer H-bonding interaction between a dapsox NH group and the coordinated nitrogen of one of the two azide ligands. Resonance Raman (rR) data obtained for frozen aqueous solution and solid-state samples of 2 indicate that the azides remain inequivalent in solution, suggesting that one of the azide ligands of 1 engages in an intermolecular hydrogen bonding interaction with a water molecule. Density functional theory (DFT) and timedependent DFT calculations have been used to study two different computational models of 1, one using coordinates taken from the X-ray crystal structure of 2, and the other generated via DFT geometry optimization. An evaluation of these models on the basis of electronic absorption, magnetic circular dichroism, and rR data indicates that the crystal structure based model provides a more accurate electronic structure description of 1, providing further support for the proposed intermolecular hydrogen bonding of 1 in the solid state and in solution. An analysis of the experimentally validated DFT results for this model reveals that the azides have both σand πbonding interactions with the Fe III center and that more negative charge is located on the Febound, rather than on the terminal, nitrogen atom of each azide. These observations are reminiscent of the results previously reported for the azide adduct of FeSOD and provide clues regarding the origin the high catalytic activity of Fe-dapsox for superoxide disproportionation.
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Papers by Thomas Brunold