Of central importance to the study of heme proteins are the effects imposed by axial ligand(s) on the heme structure and, therefore, on the overall activity of the protein. In this study, we confirm and extend the spectroscopic characterization of a mutated sperm whale myoglobin in which the proximal Histidine is replaced with a Tyrosine residue (MbH93Y). The MbH93Y, as well as wild-type sperm whale myoglobin and horse erythrocyte catalase (HEC), was purified and characterized by optical absorption and x-ray absorption (XAS) spectroscopies. Optical absorption spectra of HEC and the metmyoglobin, cyanometmyoglobin, reduced, oxy, and carbon-monoxy forms of both sperm whale myoglobin (SWMb) and MbH93Y were identical to previously reported values within the respective errors:. Extended x-ray absorption fine structure (EXAFS) studies revealed that the proximal bond length in MbH93Y was 2.13 ± 0.03 A, compared to 2.14 ± 0.02 A for sperm whale metmyoglobin and 1.90 ± 0.02 A for catalase. Additionally, the sixth coordination site normally occupied in wild type sperm whale metmyoglobin and in catalase at low temperatures was vacant in MbH93Y, a result corroborated by the optical absorption spectra and cyanogen bromide modification of the distal histidine. Measurements were also made on the cyanide complexes of the three proteins as well, among which, (i) the average iron-to-pyrrole nitrogen bond distance for MbH93Y-CN was 1.96 ± 0.015 A compared to 2.00 ± 0.015 A for WT SWMb-CN and HEC-CN and (ii) the proximal bond length in MbH93Y-CN was 2.07 ± 0.02 A, while that of WT SWMb-CN was 2.10 ± 0.02 A and that of HEC-CN was found to be 2.12 ± 0.02 A. Further, upon exposure to 2-molar equivalents of hydrogen peroxide, sperm whale myoglobin formed a Compound II -like spectrum, while the Soret absorbance of MbH93Y was rapidly, significantly, and irreversibly decreased. Furthermore, the dissociation constants for CN- binding to MbH93Y were found to be, on average, approximately three orders of magnitude higher than those of wild-type sperm whale myoglobin and are consistent with the many-fold higher cyanide binding kinetics for wild type, relative to the mutant protein. Finally, the PK. of the mutant was found to be more than three orders of magnitude higher than that of the native protein. Explanations focusing on probable electronic effects of the phenolate oxygen atom in the sperm whale myoglobin pocket are discussed.

The Gram-positive pathogens Corynebacterium diphtheriae and Streptococcus pyogenes both require iron for survival. These bacteria have developed sophisticated heme uptake and transport protein machinery responsible for the import of iron into the cell, in the form of heme from the human host. The heme utilization pathway (hmu) of C. diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. The axial ligation of the heme in HmuT was probed by examination of wild-type HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, R237A, Y272A, M292A, Y349A, and Y349F. Characterization by UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopies indicated that H136 and Y235 are the axial ligands in HmuT. Electrospray ionization mass spectrometry was also utilized to assess the roles of conserved residues in contribution to heme binding. The S. pyogenes streptococcal iron acquisition (sia)/heme transport system (hts) utilizes multiple proteins to bring host heme to the intracellular space. Both the substrate binding protein SiaA and the hemoprotein surface receptor Shr were investigated. The kinetic effects on SiaA heme release were probed through chemical unfolding of axial ligand mutants M79A and H229A, as well mutants thought to contribute to heme binding, K61A and C58A, and a control mutant, C47A. The unfolding pathways showed two processes for protein denaturation. This is consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket. The ease of protein unfolding is related to the strength of interaction of the residues with the heme. Shr contains two NEAT (near-iron transporter) domains (Shr-N1 and Shr-N2) which can both bind heme. Biophysical studies of both Shr-N1 and Shr-N2 indicated a new class of NEAT domains which utilize methionine as an axial ligand, rather than a tyrosine. Thermal and chemical unfolding showed ferrous Shr-N1 and Shr-N2 to be most resistant to denaturation. Shr-N2 was prone to autoreduction. Together, sequence alignment, homology modeling, and spectral signatures are all consistent with two methionines as the heme ligands of this novel type of NEAT heme-binding domain.

Some pathogenic bacteria derive significant amounts of iron heme from their hosts. In this study we investigated SiaA, a heme binding protein from Streptococcus pyogenes. The wildtype methionine79 putative axial ligand was mutated to alanine. SiaA M79A was expressed in E. coli in three production runs, lysed by sonication or French press, and purified by fast protein liquid chromatography (FPLC). Nickel affinity FPLC was found to give much purer SiaA when 30 mM imidazole was added to the binding buffer. The protocol using extensive sonication resulted in SiaA weighing 30464 Da. The protocol using French press resulted in SiaA weighting 33358 Da. Despite the difference in masses, the two forms of SiaA interacted with heme similarly.

Some pathogenic bacteria derive significant amounts of iron heme from their hosts. In this study we investigated SiaA, a heme binding protein from Streptococcus pyogenes. The wildtype methionine79 putative axial ligand was mutated to alanine. SiaA M79A was expressed in E. coli in three production runs, lysed by sonication or French press, and purified by fast protein liquid chromatography (FPLC). Nickel affinity FPLC was found to give much purer SiaA when 30 mM imidazole was added to the binding buffer. The protocol using extensive sonication resulted in SiaA weighing 30464 Da. The protocol using French press resulted in SiaA weighting 33358 Da. Despite the difference in masses, the two forms of SiaA interacted with heme similarly.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18291号 / 工博第3883号 / 新制||工||1596(附属図書館) / 31149 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 伊藤 紳三郎, 教授 木村 俊作, 教授 辻井 敬亘 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

The correlation between NMR chemical shifts of interstitial atoms and electronic structures of boron- and carbon-centered hexazirconium halide clusters was investigated by density functional theory (DFT) calculation. The influences of bridging halide and terminal ligand variations on electronic structure were examined respectively. Inverse proportionality was found between the chemical shifts and the calculated energy gaps between two Kohn-Sham orbitals of t1u symmetry, which arose from the bonding and antibonding interaction between the zirconium cage bonding orbitals and the interstitial 2p orbitals. Chemical shielding properties of the interstitial atoms were calculated with Gauge Including Atomic Orbital (GIAO) method. Stepwise ligand substitution of terminal chlorides on [(Zr6CCl12)Cl6]4-cluster by tri(n-butyl)-phosphine oxide (Bu3PO) was conducted with the aid of TlPF6. Composition of the reaction mixtures was analyzed by use of both 13C and 31P NMR. A preliminary scheme for synthesis and separation of [(Zr6CCl12)Cl6-x(Bu3PO)x]x-4 (x = 3 ?? 5) mixture based on solubility difference was reevaluated. Three 1,10-phenanthroline based bidentate ligands, namely, 2,9-Bis(diphenyl-phosphinyl)-1,10-phenanthroline, 2,9-Bis(diethoxyphosphoryl)-1,10-phenanthroline, and 2,9-Bis(di-n-butoxyphosphoryl)-1,10-phenantholine, were synthesized for bridge-chelating the hexazirconium clusters. Coordination chemistry of these ligands with the [Zr6BCl12] and [Zr6CCl12] clusters was subject to preliminary investigation.

Many pathogenic bacteria use their iron acquisition mechanisms to live inside hosts. Streptococcus pyogenes is a pathogenic bacterium that uses streptococcal iron acquisition ABC transporter to obtain heme. SiaA (HtsA, spy1795), a lipoprotein located on the cell surface, serves as a heme binding protein. To understand the iron-uptake mechanism, histidine 229, one of the two proposed axial ligands in SiaA, was mutated to alanine. SiaA H229A was expressed in E. coli, lysed by French Press, and purified by fast protein liquid chromatography (FPLC). SDS-PAGE indicated that pure protein was isolated. Nickel affinity FPLC gave purer H229A when 0.5 M imidazole was added to the binding buffer. Overall, histidine 229 is likely to be an axial ligand in wild type SiaA, as shown by the fact the mutant readily lost heme as evidenced by UV-vis spectra.

The correlation between NMR chemical shifts of interstitial atoms and electronic structures of boron- and carbon-centered hexazirconium halide clusters was investigated by density functional theory (DFT) calculation. The influences of bridging halide and terminal ligand variations on electronic structure were examined respectively. Inverse proportionality was found between the chemical shifts and the calculated energy gaps between two Kohn-Sham orbitals of t1u symmetry, which arose from the bonding and antibonding interaction between the zirconium cage bonding orbitals and the interstitial 2p orbitals. Chemical shielding properties of the interstitial atoms were calculated with Gauge Including Atomic Orbital (GIAO) method. Stepwise ligand substitution of terminal chlorides on [(Zr6CCl12)Cl6]4-cluster by tri(n-butyl)-phosphine oxide (Bu3PO) was conducted with the aid of TlPF6. Composition of the reaction mixtures was analyzed by use of both 13C and 31P NMR. A preliminary scheme for synthesis and separation of [(Zr6CCl12)Cl6-x(Bu3PO)x]x-4 (x = 3 ?? 5) mixture based on solubility difference was reevaluated. Three 1,10-phenanthroline based bidentate ligands, namely, 2,9-Bis(diphenyl-phosphinyl)-1,10-phenanthroline, 2,9-Bis(diethoxyphosphoryl)-1,10-phenanthroline, and 2,9-Bis(di-n-butoxyphosphoryl)-1,10-phenantholine, were synthesized for bridge-chelating the hexazirconium clusters. Coordination chemistry of these ligands with the [Zr6BCl12] and [Zr6CCl12] clusters was subject to preliminary investigation.

Sulfonated phthalocyanines were characterized using capillary electrophoresis and mass spectrometry. Derivatives investigated included the copper, cobalt, zinc and metal-free sulfonated phthalocyanines. The electropherograms of commercially available copper phthalocyanine-3,4',4'',4'''-tetrasulfonic acid and 4,4',4'',4'''-tetrasulfonic acid were very different, consistent with the latter compound having a structure that is not fully sulfonated. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) were used to characterize the sulfonated phthalocyanines. Mass spectral evidence was obtained for a pentasulfonated species of both the metal-free phthalocyanine and zinc phthalocyanine when these species were made by sulfonation of the metal-free phthalocyanine (followed by zinc insertion in the latter case). Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The heme binding protein (HBP) of this transporter is SiaA (HtsA). Several biophysical techniques were used to determine the coordination state, and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggested that the heme is six-coordinate and low spin in both oxidation states of the protein, with methionine and histidine as axial ligands. The pKa of SiaA was determined, as were the reductive and oxidative midpoint potentials. Guanidinium titration studies of wild-type SiaA showed that the ferric state is less stable than the ferrous state. Free energy of unfolding values [ÄG(H2O)] for the oxidized and reduced proteins were 7.3 ± 0.8 and 16.0 ± 3.6 kcal mol−1, respectively. Denaturation of the histidine mutant H229A was not able to be followed via absorbance spectrometry, possibly due to the large amount of apoprotein present or to non-specific binding of the heme in the binding pocket. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.