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.

Haemophilus ducreyi is a gram-negative and heme-dependent bactreia. H. ducreyi is the responsible of causing chancroid, a sexually transmitted infection forming genital ulcers. Infection with H. ducreyi is associated with an increased risk of acquiring HIV-1 as well as increasing the risk of the HIV-1 transmission. Heme acquisition in H. ducreyi occur through a receptor mediated process in which it start with binding of hemoglobin and heme to their cognate outer membrane receptors, HgbA and TdhA, respectively. The receptors are energized by the TonB complex. Following that the deposition of heme into the periplasmic area is unclear. Profiling of the periplasmic proteome of the H. ducreyi resulted in the identification of a periplasmic- binding protein that highly expressed in heme limitation conditions, and it has been called hHbp. This protein is encoded by a gene resides in a locus of four genes displaying genetic features of an ABC transporter. The gene cluster is organized as an operon comprising an internal membrane protein (IntPro), a sulphate reductase gamma subunit (dsvC), a heme dependant periplasmic bind-ing protein (hHBP), and an ATPase. The purified periplasmic binding protein, hHbp, bind heme in a dose-dependent and saturable manner. Moreover, the binding between heme and hHbp was specifically competitively inhibited by heme. The proposal planned to cre-ate an isogenic hhbp mutant by insertional inactivation using a kanamycin cassette, to genotypically and phenotypically characterize the mutant and thereby to confirm the cru-cial role of the hhbp gene in heme transport in H. ducreyi. Several attempts to ligate a kanamycin resistance cassette into hhbp to construct such a mutant were unsuccessful de-spite the systematic alteration of the ligation conditions and the use of kanamycin re-sistant genes derived from a variety of different plasmids. The explanations for this fail-ure are uncertain. In future work, two other approaches to construct an hhbp mutant in-clude the FRT-FLP recombinase technology and the use of overlapping extension PCR with a chloramphenicol cassette.

No description available.

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.