Resonance Raman spectra are reported for the 16O2 and 18O2 adducts of cobalt porphyrin complexes ... more Resonance Raman spectra are reported for the 16O2 and 18O2 adducts of cobalt porphyrin complexes with 3,5-dichloropyridine (DCP) for three different ‘picket-fence’ porphyrins. It is shown that the v(O—O) effectively couples with an internal mode of DCP giving rise to complicated spectral patterns. These spectral patterns were carefully analyzed to reveal the inherent wavenumbers of v(O—O) which are shown to vary as expected, given the known relative hydrogen bonding capabilities of the three different superstructured porphyrins.
Resonance Raman spectroscopy has been employed to investigate the structure of cyanide adducts of... more Resonance Raman spectroscopy has been employed to investigate the structure of cyanide adducts of the basic isoenzymes of horseradish peroxidase (HRP) in the pH range 5.5-12.5. Evidence for the binding of cyanide in two forms, characterized by the reversal of ordering of the Fe-CN stretching and Fe-C-N bending vibrations, is observed. Moreover, it is shown that both conformers exhibit an acid-alkaline transition in the pH range employed. In the first conformer, the Fe-C-N linkage is essentially linear, exhibiting axial Fe-CN stretching and Fe-C-N bending frequencies at 453 and 405 cm-1, respectively (at pH 5.5) (Lopez-Garriga et al., 1990). In the second conformer, the Fe-C-N fragment is bent, and the axial stretching and bending modes have been identified at 360 and 422 cm-1 at pH 5.5. At pH 12.5, the v[Fe-CN] stretching mode of the linear conformer shifts down by 9 cm-1 to 444 cm-1 while the bending frequency remains unchanged. For the bent conformer at this pH, the stretching mode shifts to 355 cm-1 (-5 cm-1), and the bending vibration shifts slightly to lower frequency by 2 cm-1 to 420 cm-1. The observed pH-dependent shift of the v[Fe-CN] stretching mode of the linear conformer is attributed to the direct effect of deprotonation of a distal-side amino acid residue while the shift of v[Fe-CN] of the bent conformer is most reasonably ascribable to indirect alteration of the iron-proximal histidine linkage induced by the distal-side deprotonation, a spectral response which reflects a protein-coupled "push-pull" mechanism for heterolytic O-O bond cleavage.
Journal of the American Chemical Society, Jan 14, 2015
The first step in the enzymatic cycle of mammalian peroxidases, including lactoperoxidase (LPO), ... more The first step in the enzymatic cycle of mammalian peroxidases, including lactoperoxidase (LPO), is binding of hydrogen peroxide to the ferric resting state to form a ferric-hydroperoxo intermediate designated as Compound 0, the residual proton temporarily associating with the distal pocket His109 residue. Upon delivery of this "stored" proton to the hydroperoxo fragment, it rapidly undergoes O-O bond cleavage, thereby thwarting efforts to trap it using rapid mixing methods. Fortunately, as shown herein, both the peroxo and the hydroperoxo (Compound 0) forms of LPO can be trapped by cryoradiolysis, with acquisition of their resonance Raman (rR) spectra now permitting structural characterization of their key Fe-O-O fragments. Studies were conducted under both acidic and alkaline conditions, revealing pH-dependent differences in relative populations of these intermediates. Furthermore, upon annealing, the low pH samples convert to two forms of a ferryl heme O-O bond-cleavage...
When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome ... more When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome P450 17A1 bound with either of the lyase substrates, 17-OH PREG or 17-OH PROG are shown here to generate the corresponding lyase products, dehydroepiandrosterone (DHEA) and androstenedione (AD) respectively. The current study uses gas chromatography-mass spectrometry (GC/MS) to document the presence of the initial substrates and products in extracts of the processed samples. A rapid and efficient method for the simultaneous determination of residual substrate and products by GC/MS is described without derivatization of the products. It is also shown that no lyase products were detected for similarly treated control samples containing no nanodisc associated CYP17 enzyme, demonstrating that the product is formed during the enzymatic reaction and not by GC/MS conditions nor the conditions produced by the cryoradiolysis process.
CYP17A1 is a key steroidogenic enzyme known to conduct several distinct chemical transformations ... more CYP17A1 is a key steroidogenic enzyme known to conduct several distinct chemical transformations on multiple substrates. In its hydroxylase activity, this enzyme adds a hydroxyl group at the 17α position of both pregnenolone and progesterone at approximately equal rates. However, the subsequent 17,20 carbon-carbon scission reaction displays variable substrate specificity in the numerous CYP17A1 isozymes operating in vertebrates, manifesting as different Kand kvalues when presented with 17α-hydroxypregnenlone (OHPREG) versus 17α-hydroxyprogesterone (OHPROG). Here we show that the identity of the residue at position 202 in human CYP17A1, thought to form a hydrogen bond with the A-ring alcohol substituent on the pregnene- nucleus, is a key driver of this enzyme's native preference for OHPREG. Replacement of asparagine 202 with serine completely reverses the preference of CYP17A1, more than doubling the rate of turnover of the OHPROG to androstenedione reaction and substantially dec...
Resonance Raman spectra with variable-wavelength excitation are reported for Ni{sup II} porphine ... more Resonance Raman spectra with variable-wavelength excitation are reported for Ni{sup II} porphine (NiP) and for the pyrrole-dâ, meso-dâ, and (pyrrole + meso)-dââ isotopomers, as well as for Ni{sup II} meso-tetraphenylporphine (NiTPP) and its pyrrole-¹âµNâ, pyrrole-dâ, ¹³Câ-meso, and phenyl-dââ isotopomers. All the Raman-active in-plane modes have been identified and are assigned to local coordinates which take into account the phasing of
Page 1. 3096 J. Phys. Chem. 1983, 87, 3096-3101 Infrared Spectra of Matrix-Isolated Metal Complex... more Page 1. 3096 J. Phys. Chem. 1983, 87, 3096-3101 Infrared Spectra of Matrix-Isolated Metal Complexes of Octaethylporphlne James R. Klncald," Chemlstry Department, Universlfy of Kentucky, Lexlngton, Kentucky 40506 Marek ...
DGCR8 is the RNA binding partner of the nuclease Drosha. Their complex (the "Microprocessor&... more DGCR8 is the RNA binding partner of the nuclease Drosha. Their complex (the "Microprocessor") is essential for processing of long, primary microRNAs (pri-miRNAs) in the nucleus. Heme binding to DGCR8 is essential for pri-miRNA processing. Based on the split Soret UV-visible (UV-vis) spectrum of ferric DGCR8, bis-thiolate sulfur (cysteinate, Cys-) heme iron coordination of DGCR8 heme iron was proposed. We have characterized DGCR8 heme ligation using the Δ276 DGCR8 variant and combined electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), electron nuclear double resonance, resonance Raman and electronic absorption spectroscopy. These studies indicate DGCR8 bis-Cys heme iron ligation, with conversion from bis-thiolate (Cys(-)/Cys(-)) axial coordination in ferric DGCR8 to bis-thiol (CysH/CysH) coordination in ferrous DGCR8. Pri-miRNA binding does not perturb ferric DGCR8's optical spectrum, consistent with the axial ligand environment being separated fr...
Low-frequency resonance Raman spectra of the cyanide and carbon monoxide adducts of lactoperoxida... more Low-frequency resonance Raman spectra of the cyanide and carbon monoxide adducts of lactoperoxidase are obtained with Soret excitation. The nu(Fe-CN) and delta(Fe-C-N) modes are detected at 360 and 453 cm-1, respectively. Upon the isotopic substitution of 13C14N, 12C15N, and 13C15N, the band at 453 cm-1 in the natural abundance adduct shifts to 448, 452, and 445 cm-1, while the 360-cm-1 peak shifts to 358, 357, and 356 cm-1, respectively. The 360-cm-1 band is shifted to 355 cm-1 when the pH is changed from 7.0 to 10.5. On the basis of a previous normal-mode analysis of the cyanoferric adduct of myeloperoxidase, a bent Fe-C-N linkage is suggested for the cyanide adduct of lactoperoxidase. The nu(Fe-CN) (374 cm-1) and delta(Fe-C-N) (480 cm-1) modes are observed for the cyanide adduct of reduced lactoperoxidase. For the carbon monoxide adduct, the nu(Fe-CO) (533 cm-1) and delta(Fe-C-O) (578 cm-1) modes at pH 7.0 are observed to shift to 498 and 570 cm-1 as the pH is raised from 7.0 to 10.0. The strong intensity of delta(Fe-C-O) at both acid and alkaline pHs, along with a suggested bent structure of the Fe-C-N moiety, implies a narrow heme pocket for lactoperoxidase.
Page 1. J. Am. Chem. SOC. 1991,113, 4815-4822 4815 self-diffusivities of 1.1 X 10-8 and 1.25 X IO... more Page 1. J. Am. Chem. SOC. 1991,113, 4815-4822 4815 self-diffusivities of 1.1 X 10-8 and 1.25 X IO4 m2 s-' were obtained. Hence it turns out that in the mixture the difference in the mobility of the two components is much less than for single-component adsorption. ...
Journal of the American Chemical Society, Aug 1, 1988
Page 1. 6006 J. Am. Chem. SOC. 1988, 110, 6006-6014 the present observations. The fact that no sy... more Page 1. 6006 J. Am. Chem. SOC. 1988, 110, 6006-6014 the present observations. The fact that no symmetrical three-electron-bond species are detected for the meso form can also not be explained by a possible wrong symmetry ...
Resonance Raman spectra are reported for the 16O2 and 18O2 adducts of cobalt porphyrin complexes ... more Resonance Raman spectra are reported for the 16O2 and 18O2 adducts of cobalt porphyrin complexes with 3,5-dichloropyridine (DCP) for three different ‘picket-fence’ porphyrins. It is shown that the v(O—O) effectively couples with an internal mode of DCP giving rise to complicated spectral patterns. These spectral patterns were carefully analyzed to reveal the inherent wavenumbers of v(O—O) which are shown to vary as expected, given the known relative hydrogen bonding capabilities of the three different superstructured porphyrins.
Resonance Raman spectroscopy has been employed to investigate the structure of cyanide adducts of... more Resonance Raman spectroscopy has been employed to investigate the structure of cyanide adducts of the basic isoenzymes of horseradish peroxidase (HRP) in the pH range 5.5-12.5. Evidence for the binding of cyanide in two forms, characterized by the reversal of ordering of the Fe-CN stretching and Fe-C-N bending vibrations, is observed. Moreover, it is shown that both conformers exhibit an acid-alkaline transition in the pH range employed. In the first conformer, the Fe-C-N linkage is essentially linear, exhibiting axial Fe-CN stretching and Fe-C-N bending frequencies at 453 and 405 cm-1, respectively (at pH 5.5) (Lopez-Garriga et al., 1990). In the second conformer, the Fe-C-N fragment is bent, and the axial stretching and bending modes have been identified at 360 and 422 cm-1 at pH 5.5. At pH 12.5, the v[Fe-CN] stretching mode of the linear conformer shifts down by 9 cm-1 to 444 cm-1 while the bending frequency remains unchanged. For the bent conformer at this pH, the stretching mode shifts to 355 cm-1 (-5 cm-1), and the bending vibration shifts slightly to lower frequency by 2 cm-1 to 420 cm-1. The observed pH-dependent shift of the v[Fe-CN] stretching mode of the linear conformer is attributed to the direct effect of deprotonation of a distal-side amino acid residue while the shift of v[Fe-CN] of the bent conformer is most reasonably ascribable to indirect alteration of the iron-proximal histidine linkage induced by the distal-side deprotonation, a spectral response which reflects a protein-coupled "push-pull" mechanism for heterolytic O-O bond cleavage.
Journal of the American Chemical Society, Jan 14, 2015
The first step in the enzymatic cycle of mammalian peroxidases, including lactoperoxidase (LPO), ... more The first step in the enzymatic cycle of mammalian peroxidases, including lactoperoxidase (LPO), is binding of hydrogen peroxide to the ferric resting state to form a ferric-hydroperoxo intermediate designated as Compound 0, the residual proton temporarily associating with the distal pocket His109 residue. Upon delivery of this "stored" proton to the hydroperoxo fragment, it rapidly undergoes O-O bond cleavage, thereby thwarting efforts to trap it using rapid mixing methods. Fortunately, as shown herein, both the peroxo and the hydroperoxo (Compound 0) forms of LPO can be trapped by cryoradiolysis, with acquisition of their resonance Raman (rR) spectra now permitting structural characterization of their key Fe-O-O fragments. Studies were conducted under both acidic and alkaline conditions, revealing pH-dependent differences in relative populations of these intermediates. Furthermore, upon annealing, the low pH samples convert to two forms of a ferryl heme O-O bond-cleavage...
When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome ... more When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome P450 17A1 bound with either of the lyase substrates, 17-OH PREG or 17-OH PROG are shown here to generate the corresponding lyase products, dehydroepiandrosterone (DHEA) and androstenedione (AD) respectively. The current study uses gas chromatography-mass spectrometry (GC/MS) to document the presence of the initial substrates and products in extracts of the processed samples. A rapid and efficient method for the simultaneous determination of residual substrate and products by GC/MS is described without derivatization of the products. It is also shown that no lyase products were detected for similarly treated control samples containing no nanodisc associated CYP17 enzyme, demonstrating that the product is formed during the enzymatic reaction and not by GC/MS conditions nor the conditions produced by the cryoradiolysis process.
CYP17A1 is a key steroidogenic enzyme known to conduct several distinct chemical transformations ... more CYP17A1 is a key steroidogenic enzyme known to conduct several distinct chemical transformations on multiple substrates. In its hydroxylase activity, this enzyme adds a hydroxyl group at the 17α position of both pregnenolone and progesterone at approximately equal rates. However, the subsequent 17,20 carbon-carbon scission reaction displays variable substrate specificity in the numerous CYP17A1 isozymes operating in vertebrates, manifesting as different Kand kvalues when presented with 17α-hydroxypregnenlone (OHPREG) versus 17α-hydroxyprogesterone (OHPROG). Here we show that the identity of the residue at position 202 in human CYP17A1, thought to form a hydrogen bond with the A-ring alcohol substituent on the pregnene- nucleus, is a key driver of this enzyme's native preference for OHPREG. Replacement of asparagine 202 with serine completely reverses the preference of CYP17A1, more than doubling the rate of turnover of the OHPROG to androstenedione reaction and substantially dec...
Resonance Raman spectra with variable-wavelength excitation are reported for Ni{sup II} porphine ... more Resonance Raman spectra with variable-wavelength excitation are reported for Ni{sup II} porphine (NiP) and for the pyrrole-dâ, meso-dâ, and (pyrrole + meso)-dââ isotopomers, as well as for Ni{sup II} meso-tetraphenylporphine (NiTPP) and its pyrrole-¹âµNâ, pyrrole-dâ, ¹³Câ-meso, and phenyl-dââ isotopomers. All the Raman-active in-plane modes have been identified and are assigned to local coordinates which take into account the phasing of
Page 1. 3096 J. Phys. Chem. 1983, 87, 3096-3101 Infrared Spectra of Matrix-Isolated Metal Complex... more Page 1. 3096 J. Phys. Chem. 1983, 87, 3096-3101 Infrared Spectra of Matrix-Isolated Metal Complexes of Octaethylporphlne James R. Klncald," Chemlstry Department, Universlfy of Kentucky, Lexlngton, Kentucky 40506 Marek ...
DGCR8 is the RNA binding partner of the nuclease Drosha. Their complex (the "Microprocessor&... more DGCR8 is the RNA binding partner of the nuclease Drosha. Their complex (the "Microprocessor") is essential for processing of long, primary microRNAs (pri-miRNAs) in the nucleus. Heme binding to DGCR8 is essential for pri-miRNA processing. Based on the split Soret UV-visible (UV-vis) spectrum of ferric DGCR8, bis-thiolate sulfur (cysteinate, Cys-) heme iron coordination of DGCR8 heme iron was proposed. We have characterized DGCR8 heme ligation using the Δ276 DGCR8 variant and combined electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), electron nuclear double resonance, resonance Raman and electronic absorption spectroscopy. These studies indicate DGCR8 bis-Cys heme iron ligation, with conversion from bis-thiolate (Cys(-)/Cys(-)) axial coordination in ferric DGCR8 to bis-thiol (CysH/CysH) coordination in ferrous DGCR8. Pri-miRNA binding does not perturb ferric DGCR8's optical spectrum, consistent with the axial ligand environment being separated fr...
Low-frequency resonance Raman spectra of the cyanide and carbon monoxide adducts of lactoperoxida... more Low-frequency resonance Raman spectra of the cyanide and carbon monoxide adducts of lactoperoxidase are obtained with Soret excitation. The nu(Fe-CN) and delta(Fe-C-N) modes are detected at 360 and 453 cm-1, respectively. Upon the isotopic substitution of 13C14N, 12C15N, and 13C15N, the band at 453 cm-1 in the natural abundance adduct shifts to 448, 452, and 445 cm-1, while the 360-cm-1 peak shifts to 358, 357, and 356 cm-1, respectively. The 360-cm-1 band is shifted to 355 cm-1 when the pH is changed from 7.0 to 10.5. On the basis of a previous normal-mode analysis of the cyanoferric adduct of myeloperoxidase, a bent Fe-C-N linkage is suggested for the cyanide adduct of lactoperoxidase. The nu(Fe-CN) (374 cm-1) and delta(Fe-C-N) (480 cm-1) modes are observed for the cyanide adduct of reduced lactoperoxidase. For the carbon monoxide adduct, the nu(Fe-CO) (533 cm-1) and delta(Fe-C-O) (578 cm-1) modes at pH 7.0 are observed to shift to 498 and 570 cm-1 as the pH is raised from 7.0 to 10.0. The strong intensity of delta(Fe-C-O) at both acid and alkaline pHs, along with a suggested bent structure of the Fe-C-N moiety, implies a narrow heme pocket for lactoperoxidase.
Page 1. J. Am. Chem. SOC. 1991,113, 4815-4822 4815 self-diffusivities of 1.1 X 10-8 and 1.25 X IO... more Page 1. J. Am. Chem. SOC. 1991,113, 4815-4822 4815 self-diffusivities of 1.1 X 10-8 and 1.25 X IO4 m2 s-' were obtained. Hence it turns out that in the mixture the difference in the mobility of the two components is much less than for single-component adsorption. ...
Journal of the American Chemical Society, Aug 1, 1988
Page 1. 6006 J. Am. Chem. SOC. 1988, 110, 6006-6014 the present observations. The fact that no sy... more Page 1. 6006 J. Am. Chem. SOC. 1988, 110, 6006-6014 the present observations. The fact that no symmetrical three-electron-bond species are detected for the meso form can also not be explained by a possible wrong symmetry ...
Uploads
Papers by James Kincaid