Oleg Usov - Academia.edu (original) (raw)

Papers by Oleg Usov

Research paper thumbnail of ENDOR investigation of the liganding environment of mixed-spin ferric cytochrome c

Journal of the American Chemical Society, Jan 6, 2005

The electronic structure of the 5-coordinate quantum-mechanically mixed-spin (sextet-quartet) hem... more The electronic structure of the 5-coordinate quantum-mechanically mixed-spin (sextet-quartet) heme center in cytochrome c' was investigated by electron nuclear double resonance (ENDOR), a technique not previously applied to this mixed-spin system. Cytochrome c' was obtained from overexpressing variants of Rhodobacter sphaeroides 2.4.3. ENDOR for this study was done at the g(//) = 2.00 extremum where single-crystal-like, well-resolved spectra prevail. The heme meso protons of cytochrome c' showed a contact interaction that implied spin delocalization arising from the heme (d(z)(2)) orbital enhanced by iron out-of-planarity. An exchangeable proton ENDOR feature appeared from the proximal His123 Ndelta hydrogen. This Ndelta hydrogen, which crystallographically has no hydrogen-bonding partner and thus belongs to a neutral imidazole, showed a larger hyperfine coupling than the corresponding hydrogen-bonded Ndelta proton from metmyoglobin. The unique residue Phe14 occludes binding of a sixth ligand in cytochrome c', and ENDOR from a proton of the functionally important Phe14 ring, approximately 3.3 A away from the heme iron, was detected. ENDOR of the nitrogen ligand hyperfine structure is a direct probe into the sigma-antibonding (d(z)(2)) and (d(x)(2)-d(y)(2)) orbitals whose energies alter the relative stability and admixture of sextet and quartet states and whose electronic details were thus elucidated. ENDOR frequencies showed for cytochrome c' larger hyperfine couplings to the histidine nitrogen and smaller hyperfine couplings to the heme nitrogens than for high-spin ferric hemes. Both of these findings followed from the mixed-spin ground state, which has less (d(x)(2)-d(y)(2)) character than have fully high-spin ferric heme systems.

Research paper thumbnail of ENDOR of NO-ligated cytochrome c

Journal of the American Chemical Society, Jan 19, 2006

The five-coordinate NO-bound heme in cytochrome c&amp... more The five-coordinate NO-bound heme in cytochrome c' from an overexpressing variant of denitrifying R. sphaeroides 2.4.3 was investigated by proton, nitrogen, and deuterium Q-band ENDOR (electron nuclear double resonance). ENDOR was a direct probe of the unpaired electron density on the nitrogen of NO and, as measured across the EPR line shape, showed a hyperfine coupling range from 36 to 44 MHz for 14NO and 51 to 63 MHz for 15NO. The smallest NO coupling occurred at an electronic g-tensor axis perpendicular to the FeNO plane, and the largest hyperfine coupling occurred in the FeNO plane where the highest nitrogen valence spin density is located. The isotropic component of the NO hyperfine coupling indicated that the electron spin on the NO is not simply in a pi orbital having only 2p character but is in an orbital having 2s and 2p character in a 1:2 ratio. ENDOR frequencies from heme meso-protons, assigned with reference to porphyrin models, were determined to result from an anisotropic hyperfine tensor. This tensor indicated the orientation of the heme with respect to the FeNO plane and showed that the FeNO plane bisects the heme N-Fe-N 90 degrees angle. ENDOR provided additional structural information through dipolar couplings, as follows: (1) to the nearest proton of the Phe14 ring, approximately 3.1 A away from the heme iron, where Phe14 is positioned to occlude binding of NO as a 6th (distal) ligand; (2) to exchangeable deuterons assigned to Arg127 which may H-bond with the proximal NO ligand.

Research paper thumbnail of EPR-ENDOR of the Cu(I)NO complex of nitrite reductase

Journal of the American Chemical Society, Jan 11, 2006

With limited reductant and nitrite under anaerobic conditions, copper-containing nitrite reductas... more With limited reductant and nitrite under anaerobic conditions, copper-containing nitrite reductase (NiR) of Rhodobacter sphaeroides yielded endogenous NO and the Cu(I)NO derivative of NiR. 14 N-and 15 N-nitrite substrates gave rise to characteristic 14 NO and 15 NO EPR hyperfine features indicating NO involvement, and enrichment of NiR with 63 Cu isotope caused an EPR line shape change showing copper involvement. A markedly similar Cu(I)NONiR complex was made by anaerobically adding a little endogenous NO gas to reduced protein and immediately freezing. The Cu(I)NONiR signal accounted for 60-90% of the integrated EPR intensity formerly associated with the Type 2 catalytic copper. Analysis of NO and Cu hyperfine couplings and comparison to couplings of inorganic Cu(I)NO model systems indicated ∼50% spin on the N of NO and ∼17% spin on Cu. ENDOR revealed weak nitrogen hyperfine coupling to one or more likely histidine ligands of copper. Although previous crystallography of the conservative I289V mutant had shown no structural change beyond the 289 position, this mutation, which eliminates the Cδ1 methyl of I289, caused the Cu(I)NONiR EPR spectrum to change and proton ENDOR features to be significantly altered. The proton hyperfine coupling that was significantly altered was consistent with a dipolar interaction between the Cδ1 protons of I289 and electron spin on the NO, where the NO would be located 3.0-3.7 Å from these protons. Such a distance positions the NO of Cu(

Research paper thumbnail of Hyperfine coupling to the bridging (17)O in the di-mu-oxo core of a Mn(III)-Mn(IV) model significant to the core electronic structure of the O(2)-evolving complex in photosystem II

Journal of the American Chemical Society, Jan 3, 2007

Research paper thumbnail of ChemInform Abstract: A New Family of Stable 2-Imidazoline Nitroxides

ChemInform, 1999

ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance t... more ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Research paper thumbnail of ChemInform Abstract: Heterocyclic Derivatives of Long-Chain Diacetylenic Acids

Research paper thumbnail of Cryogenic Photolysis of Activated Bleomycin to Ferric Bleomycin

The Journal of Physical Chemistry B, 2006

Activated bleomycin (ABLM) is a drug--Fe(III)-hydroperoxide complex kinetically competent in DNA ... more Activated bleomycin (ABLM) is a drug--Fe(III)-hydroperoxide complex kinetically competent in DNA attack (via H4' abstraction). This intermediate is relatively stable, but its spontaneous conversion to ferric bleomycin (Fe(III).BLM) is poorly characterized because no observable intermediate product accumulates. Light was shown to trigger ABLM attack on DNA in liquid at -30 degrees C, so ABLM was irradiated (at its 350 nm ligand-to-metal charge-transfer transition) at 77 K to stabilize possible intermediates. ABLM photolysis (quantum yield, Phi = 0.005) generates two kinds of product: Fe(III).BLM (with no detectable intermediate) and one or more minor (1-2%) radical O-Fe-BLM byproduct, photostable at 77 K. Adding DNA, even without its target H4', increases the quantum yield of ABLM conversion >10-fold while suppressing the observed radical yield. Since cryogenic solid-phase reactions can entail only constrained local rearrangement, the reaction(s) converting ABLM to Fe(III).BLM must be similarly constrained.

Research paper thumbnail of Highly Mobile Solvent Holes in Viscous Squalane Solutions As Detected by Quantum Beats and MARY Spectroscopy Techniques

The Journal of Physical Chemistry A, 1999

The quantum beats and magnetic field effect on the reaction yield spectroscopy techniques were em... more The quantum beats and magnetic field effect on the reaction yield spectroscopy techniques were employed to study the formation of diphenyl sulfide radical cations in squalane solutions under ionizing irradiation. It is demonstrated that the precursors of diphenyl sulfide radical cations are short-lived primary solvent radical cations (holes) with the electron spin resonance spectrum narrowed by the resonance charge transfer reaction. The rate constant of hole scavenging by diphenyl sulfide molecules was measured directly and amounts to 6.1 × 10 9 M -1 s -1 , exceeding the diffusion-controlled one several times. The obtained value is well in line with the data on pulse radiolysis of squalane solutions with optical monitoring of the highly mobile precursor, supporting the hypothesis about the hole nature of the latter.

Research paper thumbnail of Highly Mobile Solvent Holes in Viscous Squalane Solutions As Detected by Quantum Beats and MARY Spectroscopy Techniques

The Journal of Physical Chemistry A, 1997

The quantum beats and magnetic field effect on the reaction yield spectroscopy techniques were em... more The quantum beats and magnetic field effect on the reaction yield spectroscopy techniques were employed to study the formation of diphenyl sulfide radical cations in squalane solutions under ionizing irradiation. It is demonstrated that the precursors of diphenyl sulfide radical cations are short-lived primary solvent radical cations (holes) with the electron spin resonance spectrum narrowed by the resonance charge transfer reaction. The rate constant of hole scavenging by diphenyl sulfide molecules was measured directly and amounts to 6.1 × 10 9 M -1 s -1 , exceeding the diffusion-controlled one several times. The obtained value is well in line with the data on pulse radiolysis of squalane solutions with optical monitoring of the highly mobile precursor, supporting the hypothesis about the hole nature of the latter.

Research paper thumbnail of A new family of stable 2-imidazoline nitroxides

Mendeleev Communications, 1998

Methods of synthesising stable 2-imidazoline nitroxides linked to a pyrazole moiety either direct... more Methods of synthesising stable 2-imidazoline nitroxides linked to a pyrazole moiety either directly or through a phenylethynylic bridge have been developed; an unusually strong temperature dependence of m eff for 2-(1-methylpyrazolyl-5)-4,4,5,5-tetramethyl-1-oxyl-2-imidazoline-3-oxide is observed.

Research paper thumbnail of Hyperfine Coupling to the Bridging 17 O in the Di-μ-oxo Core of a Mn III −Mn IV Model Significant to the Core Electronic Structure of the O 2 -Evolving Complex in Photosystem II

Journal of the American Chemical Society, 2007

Research paper thumbnail of Coating of Metal Powders with Polymers in Supercritical Carbon Dioxide

Industrial & Engineering Chemistry Research, 2001

Supercritical carbon dioxide was used as a solvent to produce polymeric films on fused silica pla... more Supercritical carbon dioxide was used as a solvent to produce polymeric films on fused silica plates and metal (Al, Mg) powders. Two polymers, poly(vinylidene fluoride) and poly(4vinylbiphenyl) (PVB), were used. Polymer-coated particles of metal powders exhibit enhanced resistance to the dissolution in aqueous basic and acidic solutions. The protective properties of the films were quantified based on the dissolution rate. The average thickness of the PVB films (that contain aromatic rings) was evaluated using UV absorption spectroscopy. A technique to measure the solubilities of poorly soluble polymers in supercritical carbon dioxide was developed. The effect of the coating conditions on the protective properties of the produced polymeric films was evaluated.

Research paper thumbnail of Resolution of the Spectroscopy versus Crystallography Issue for NO Intermediates of Nitrite Reductase from Rhodobacter sphaeroides

Journal of The American Chemical Society, 2007

Copper nitrite reductase (NiR) is a homotrimeric enzyme, containing a T1 copper site, which trans... more Copper nitrite reductase (NiR) is a homotrimeric enzyme, containing a T1 copper site, which transfers electrons to the T2 catalytic site, where nitrite is reduced by one electron to nitric oxide (NO 2

Research paper thumbnail of The Role of Arginine-127 at the Proximal NO-Binding Site in Determining the Electronic Structure and Function of 5-Coordinate NO-Heme in Cytochrome c ′ of Rhodobacter sphaeroides

Biochemistry, 2009

Cytochrome c′ is a heme protein from a denitrifying variant of R. sphaeroides which may serve to ... more Cytochrome c′ is a heme protein from a denitrifying variant of R. sphaeroides which may serve to store and transport metabolic NO while protecting against NO toxicity. Its heme site bears resemblance through its 5-coordinate NO-binding capability to the regulatory site in soluble guanylate cyclase. A conserved arginine (Arg-127) abuts the 5-coordinate NO-Heme binding site, and the alanine mutant R127A provided insight into the role of the Arg-127 in establishing the electronic structure of the heme-NO complex and in modifying the heme-centered redox potential and NO-binding affinity. By comparison to R127A, the wild-type Arg-127 was determined to increase the heme redox potential, diminish the NO binding affinity, perturb and diminish the 14 NO hyperfine coupling determined by ENDOR (electron nuclear double resonance), and increase the maximal electronic g-value. The larger isotropic NO hyperfine and the smaller maximal g-value of the R127A mutant together predicted that the Fe-N-O bond angle in the mutant is larger than that of the Arg-127-containing wild type protein. Deuterium ENDOR provided evidence for exchangeable H/D consistent with hydrogen bonding of Arg-127, but not Ala-127, to the O of the NO. Proton ENDOR features previously assigned to Phe-14 on the distal side of the heme were unperturbed by the proximal side R127A mutation, implying the localized nature of that mutational perturbation at the proximal, NO-binding side of the heme. From this Supporting Information Available: The information is provided: Cytochrome c′ Mutagenesis and Expression -Materials for Mutagenesis and Methods of Expression and Purification; . SDS PAGE gel electrophoresis of wild type and R127A mutants; . UV-Vis absorption spectra NO-bound Cyt c′ from wild type and R127A; . Comparison of first derivative Q-band EPR spectra of NO-ligated wild type and R127A; . the similarity of ferric heme EPR spectra from wild type and R127A; . the similarity of heme and histidine ENDOR features from the ferric heme center of wild type and R127A; . NO titration curves complementary to those in for R127A and wild type Cyt c′; . A plot of the ratio NO-Bound to NO-Unbound heme as a function of NO concentration for R127A and wild type Cyt c′. This material is available free of charge via the Internet at http://pubs.acs.org. 2 Rapid free-quench EPR work in progress shows that a 6-coordinate NO-Heme-His Complex ofCyt c′ arises within 10 ms of mixing reduced Cyt c′ and NO and continues to increase intensityfor at least 38 ms. The ratio of 5-coordinate to 6-coordinate NO-Cyt c′ increases during that time. 3 For a simple empirical comparison of NO affinity between wild type Cyt c′ and R127A, weconsidered NO binding as described by a simple mass action equilibrium, H•NO ↔ H + NO,where [H•NO] is the concentration of NO-bound heme, [H] is the concentration of unbound heme, and [NO] is the concentration of NO. Then K D = [H][NO]/[H•NO] is the dissociation constant. When [H•NO] = [H], (i.e., Y = ½), K D = [NO] ½ . where [NO] ½ . is the NO concentrationat half saturation. The free energy of NO binding is then RTln(K D ), where R is the gas constantand T is temperature, which was 293 K for our experiments NIH Public Access

Research paper thumbnail of ENDOR investigation of the liganding environment of mixed-spin ferric cytochrome c

Journal of the American Chemical Society, Jan 6, 2005

The electronic structure of the 5-coordinate quantum-mechanically mixed-spin (sextet-quartet) hem... more The electronic structure of the 5-coordinate quantum-mechanically mixed-spin (sextet-quartet) heme center in cytochrome c' was investigated by electron nuclear double resonance (ENDOR), a technique not previously applied to this mixed-spin system. Cytochrome c' was obtained from overexpressing variants of Rhodobacter sphaeroides 2.4.3. ENDOR for this study was done at the g(//) = 2.00 extremum where single-crystal-like, well-resolved spectra prevail. The heme meso protons of cytochrome c' showed a contact interaction that implied spin delocalization arising from the heme (d(z)(2)) orbital enhanced by iron out-of-planarity. An exchangeable proton ENDOR feature appeared from the proximal His123 Ndelta hydrogen. This Ndelta hydrogen, which crystallographically has no hydrogen-bonding partner and thus belongs to a neutral imidazole, showed a larger hyperfine coupling than the corresponding hydrogen-bonded Ndelta proton from metmyoglobin. The unique residue Phe14 occludes binding of a sixth ligand in cytochrome c', and ENDOR from a proton of the functionally important Phe14 ring, approximately 3.3 A away from the heme iron, was detected. ENDOR of the nitrogen ligand hyperfine structure is a direct probe into the sigma-antibonding (d(z)(2)) and (d(x)(2)-d(y)(2)) orbitals whose energies alter the relative stability and admixture of sextet and quartet states and whose electronic details were thus elucidated. ENDOR frequencies showed for cytochrome c' larger hyperfine couplings to the histidine nitrogen and smaller hyperfine couplings to the heme nitrogens than for high-spin ferric hemes. Both of these findings followed from the mixed-spin ground state, which has less (d(x)(2)-d(y)(2)) character than have fully high-spin ferric heme systems.

Research paper thumbnail of ENDOR of NO-ligated cytochrome c

Journal of the American Chemical Society, Jan 19, 2006

The five-coordinate NO-bound heme in cytochrome c&amp... more The five-coordinate NO-bound heme in cytochrome c' from an overexpressing variant of denitrifying R. sphaeroides 2.4.3 was investigated by proton, nitrogen, and deuterium Q-band ENDOR (electron nuclear double resonance). ENDOR was a direct probe of the unpaired electron density on the nitrogen of NO and, as measured across the EPR line shape, showed a hyperfine coupling range from 36 to 44 MHz for 14NO and 51 to 63 MHz for 15NO. The smallest NO coupling occurred at an electronic g-tensor axis perpendicular to the FeNO plane, and the largest hyperfine coupling occurred in the FeNO plane where the highest nitrogen valence spin density is located. The isotropic component of the NO hyperfine coupling indicated that the electron spin on the NO is not simply in a pi orbital having only 2p character but is in an orbital having 2s and 2p character in a 1:2 ratio. ENDOR frequencies from heme meso-protons, assigned with reference to porphyrin models, were determined to result from an anisotropic hyperfine tensor. This tensor indicated the orientation of the heme with respect to the FeNO plane and showed that the FeNO plane bisects the heme N-Fe-N 90 degrees angle. ENDOR provided additional structural information through dipolar couplings, as follows: (1) to the nearest proton of the Phe14 ring, approximately 3.1 A away from the heme iron, where Phe14 is positioned to occlude binding of NO as a 6th (distal) ligand; (2) to exchangeable deuterons assigned to Arg127 which may H-bond with the proximal NO ligand.

Research paper thumbnail of EPR-ENDOR of the Cu(I)NO complex of nitrite reductase

Journal of the American Chemical Society, Jan 11, 2006

With limited reductant and nitrite under anaerobic conditions, copper-containing nitrite reductas... more With limited reductant and nitrite under anaerobic conditions, copper-containing nitrite reductase (NiR) of Rhodobacter sphaeroides yielded endogenous NO and the Cu(I)NO derivative of NiR. 14 N-and 15 N-nitrite substrates gave rise to characteristic 14 NO and 15 NO EPR hyperfine features indicating NO involvement, and enrichment of NiR with 63 Cu isotope caused an EPR line shape change showing copper involvement. A markedly similar Cu(I)NONiR complex was made by anaerobically adding a little endogenous NO gas to reduced protein and immediately freezing. The Cu(I)NONiR signal accounted for 60-90% of the integrated EPR intensity formerly associated with the Type 2 catalytic copper. Analysis of NO and Cu hyperfine couplings and comparison to couplings of inorganic Cu(I)NO model systems indicated ∼50% spin on the N of NO and ∼17% spin on Cu. ENDOR revealed weak nitrogen hyperfine coupling to one or more likely histidine ligands of copper. Although previous crystallography of the conservative I289V mutant had shown no structural change beyond the 289 position, this mutation, which eliminates the Cδ1 methyl of I289, caused the Cu(I)NONiR EPR spectrum to change and proton ENDOR features to be significantly altered. The proton hyperfine coupling that was significantly altered was consistent with a dipolar interaction between the Cδ1 protons of I289 and electron spin on the NO, where the NO would be located 3.0-3.7 Å from these protons. Such a distance positions the NO of Cu(

Research paper thumbnail of Hyperfine coupling to the bridging (17)O in the di-mu-oxo core of a Mn(III)-Mn(IV) model significant to the core electronic structure of the O(2)-evolving complex in photosystem II

Journal of the American Chemical Society, Jan 3, 2007

Research paper thumbnail of ChemInform Abstract: A New Family of Stable 2-Imidazoline Nitroxides

ChemInform, 1999

ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance t... more ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Research paper thumbnail of ChemInform Abstract: Heterocyclic Derivatives of Long-Chain Diacetylenic Acids

Research paper thumbnail of Cryogenic Photolysis of Activated Bleomycin to Ferric Bleomycin

The Journal of Physical Chemistry B, 2006

Activated bleomycin (ABLM) is a drug--Fe(III)-hydroperoxide complex kinetically competent in DNA ... more Activated bleomycin (ABLM) is a drug--Fe(III)-hydroperoxide complex kinetically competent in DNA attack (via H4' abstraction). This intermediate is relatively stable, but its spontaneous conversion to ferric bleomycin (Fe(III).BLM) is poorly characterized because no observable intermediate product accumulates. Light was shown to trigger ABLM attack on DNA in liquid at -30 degrees C, so ABLM was irradiated (at its 350 nm ligand-to-metal charge-transfer transition) at 77 K to stabilize possible intermediates. ABLM photolysis (quantum yield, Phi = 0.005) generates two kinds of product: Fe(III).BLM (with no detectable intermediate) and one or more minor (1-2%) radical O-Fe-BLM byproduct, photostable at 77 K. Adding DNA, even without its target H4', increases the quantum yield of ABLM conversion >10-fold while suppressing the observed radical yield. Since cryogenic solid-phase reactions can entail only constrained local rearrangement, the reaction(s) converting ABLM to Fe(III).BLM must be similarly constrained.

Research paper thumbnail of Highly Mobile Solvent Holes in Viscous Squalane Solutions As Detected by Quantum Beats and MARY Spectroscopy Techniques

The Journal of Physical Chemistry A, 1999

The quantum beats and magnetic field effect on the reaction yield spectroscopy techniques were em... more The quantum beats and magnetic field effect on the reaction yield spectroscopy techniques were employed to study the formation of diphenyl sulfide radical cations in squalane solutions under ionizing irradiation. It is demonstrated that the precursors of diphenyl sulfide radical cations are short-lived primary solvent radical cations (holes) with the electron spin resonance spectrum narrowed by the resonance charge transfer reaction. The rate constant of hole scavenging by diphenyl sulfide molecules was measured directly and amounts to 6.1 × 10 9 M -1 s -1 , exceeding the diffusion-controlled one several times. The obtained value is well in line with the data on pulse radiolysis of squalane solutions with optical monitoring of the highly mobile precursor, supporting the hypothesis about the hole nature of the latter.

Research paper thumbnail of Highly Mobile Solvent Holes in Viscous Squalane Solutions As Detected by Quantum Beats and MARY Spectroscopy Techniques

The Journal of Physical Chemistry A, 1997

The quantum beats and magnetic field effect on the reaction yield spectroscopy techniques were em... more The quantum beats and magnetic field effect on the reaction yield spectroscopy techniques were employed to study the formation of diphenyl sulfide radical cations in squalane solutions under ionizing irradiation. It is demonstrated that the precursors of diphenyl sulfide radical cations are short-lived primary solvent radical cations (holes) with the electron spin resonance spectrum narrowed by the resonance charge transfer reaction. The rate constant of hole scavenging by diphenyl sulfide molecules was measured directly and amounts to 6.1 × 10 9 M -1 s -1 , exceeding the diffusion-controlled one several times. The obtained value is well in line with the data on pulse radiolysis of squalane solutions with optical monitoring of the highly mobile precursor, supporting the hypothesis about the hole nature of the latter.

Research paper thumbnail of A new family of stable 2-imidazoline nitroxides

Mendeleev Communications, 1998

Methods of synthesising stable 2-imidazoline nitroxides linked to a pyrazole moiety either direct... more Methods of synthesising stable 2-imidazoline nitroxides linked to a pyrazole moiety either directly or through a phenylethynylic bridge have been developed; an unusually strong temperature dependence of m eff for 2-(1-methylpyrazolyl-5)-4,4,5,5-tetramethyl-1-oxyl-2-imidazoline-3-oxide is observed.

Research paper thumbnail of Hyperfine Coupling to the Bridging 17 O in the Di-μ-oxo Core of a Mn III −Mn IV Model Significant to the Core Electronic Structure of the O 2 -Evolving Complex in Photosystem II

Journal of the American Chemical Society, 2007

Research paper thumbnail of Coating of Metal Powders with Polymers in Supercritical Carbon Dioxide

Industrial & Engineering Chemistry Research, 2001

Supercritical carbon dioxide was used as a solvent to produce polymeric films on fused silica pla... more Supercritical carbon dioxide was used as a solvent to produce polymeric films on fused silica plates and metal (Al, Mg) powders. Two polymers, poly(vinylidene fluoride) and poly(4vinylbiphenyl) (PVB), were used. Polymer-coated particles of metal powders exhibit enhanced resistance to the dissolution in aqueous basic and acidic solutions. The protective properties of the films were quantified based on the dissolution rate. The average thickness of the PVB films (that contain aromatic rings) was evaluated using UV absorption spectroscopy. A technique to measure the solubilities of poorly soluble polymers in supercritical carbon dioxide was developed. The effect of the coating conditions on the protective properties of the produced polymeric films was evaluated.

Research paper thumbnail of Resolution of the Spectroscopy versus Crystallography Issue for NO Intermediates of Nitrite Reductase from Rhodobacter sphaeroides

Journal of The American Chemical Society, 2007

Copper nitrite reductase (NiR) is a homotrimeric enzyme, containing a T1 copper site, which trans... more Copper nitrite reductase (NiR) is a homotrimeric enzyme, containing a T1 copper site, which transfers electrons to the T2 catalytic site, where nitrite is reduced by one electron to nitric oxide (NO 2

Research paper thumbnail of The Role of Arginine-127 at the Proximal NO-Binding Site in Determining the Electronic Structure and Function of 5-Coordinate NO-Heme in Cytochrome c ′ of Rhodobacter sphaeroides

Biochemistry, 2009

Cytochrome c′ is a heme protein from a denitrifying variant of R. sphaeroides which may serve to ... more Cytochrome c′ is a heme protein from a denitrifying variant of R. sphaeroides which may serve to store and transport metabolic NO while protecting against NO toxicity. Its heme site bears resemblance through its 5-coordinate NO-binding capability to the regulatory site in soluble guanylate cyclase. A conserved arginine (Arg-127) abuts the 5-coordinate NO-Heme binding site, and the alanine mutant R127A provided insight into the role of the Arg-127 in establishing the electronic structure of the heme-NO complex and in modifying the heme-centered redox potential and NO-binding affinity. By comparison to R127A, the wild-type Arg-127 was determined to increase the heme redox potential, diminish the NO binding affinity, perturb and diminish the 14 NO hyperfine coupling determined by ENDOR (electron nuclear double resonance), and increase the maximal electronic g-value. The larger isotropic NO hyperfine and the smaller maximal g-value of the R127A mutant together predicted that the Fe-N-O bond angle in the mutant is larger than that of the Arg-127-containing wild type protein. Deuterium ENDOR provided evidence for exchangeable H/D consistent with hydrogen bonding of Arg-127, but not Ala-127, to the O of the NO. Proton ENDOR features previously assigned to Phe-14 on the distal side of the heme were unperturbed by the proximal side R127A mutation, implying the localized nature of that mutational perturbation at the proximal, NO-binding side of the heme. From this Supporting Information Available: The information is provided: Cytochrome c′ Mutagenesis and Expression -Materials for Mutagenesis and Methods of Expression and Purification; . SDS PAGE gel electrophoresis of wild type and R127A mutants; . UV-Vis absorption spectra NO-bound Cyt c′ from wild type and R127A; . Comparison of first derivative Q-band EPR spectra of NO-ligated wild type and R127A; . the similarity of ferric heme EPR spectra from wild type and R127A; . the similarity of heme and histidine ENDOR features from the ferric heme center of wild type and R127A; . NO titration curves complementary to those in for R127A and wild type Cyt c′; . A plot of the ratio NO-Bound to NO-Unbound heme as a function of NO concentration for R127A and wild type Cyt c′. This material is available free of charge via the Internet at http://pubs.acs.org. 2 Rapid free-quench EPR work in progress shows that a 6-coordinate NO-Heme-His Complex ofCyt c′ arises within 10 ms of mixing reduced Cyt c′ and NO and continues to increase intensityfor at least 38 ms. The ratio of 5-coordinate to 6-coordinate NO-Cyt c′ increases during that time. 3 For a simple empirical comparison of NO affinity between wild type Cyt c′ and R127A, weconsidered NO binding as described by a simple mass action equilibrium, H•NO ↔ H + NO,where [H•NO] is the concentration of NO-bound heme, [H] is the concentration of unbound heme, and [NO] is the concentration of NO. Then K D = [H][NO]/[H•NO] is the dissociation constant. When [H•NO] = [H], (i.e., Y = ½), K D = [NO] ½ . where [NO] ½ . is the NO concentrationat half saturation. The free energy of NO binding is then RTln(K D ), where R is the gas constantand T is temperature, which was 293 K for our experiments NIH Public Access