Alpha-hemoglobin stabilizing protein (AHSP) markedly decreases the redox potential and reactivity of alpha subunits of human HbA with hydrogen peroxide

Background: AHSP modifies redox properties of bound α subunits. Results: Isolated hemoglobin subunits exhibit significantly different redox properties compared to HbA. A significant decrease in the reduction potential of α subunits bound to AHSP results in preferential binding of ferric α. Conclusion: AHSP:α subunit complexes do not participate in ferric-ferryl heme redox cycling. Significance: AHSP binding to α subunits inhibits subunit pseudoperoxidase activity

Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in <i>Pectobacterium spp</i>

In order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; and &lt;i&gt;Pectobacterium atrosepticum&lt;/i&gt; with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that &lt;i&gt;Pectobacterium spp.&lt;/i&gt; carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; and &lt;i&gt;atrosepticum&lt;/i&gt; that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells

Thermodynamic Investigations of Metalloproteins: Metal as Probe and Protein as Probe

<p>In this dissertation several metalloproteins, both metal transport proteins and the classic metalloprotein hemoglobin, are investigated using a variety of biophysical and electrochemical techniques. In each case, thermodynamic measurements provide insight into the role and mode of action of the metalloprotein under investigation. In Chapters 2 and 3, we focus on the thermodynamic properties of the metal while bound by the protein. In Chapter 4, we focus on the thermodynamic properties of the protein with and without the metal. In Chapter 5, we utilize both the metal and the protein as our probe.</p> <p>In Chapter 2, we probe the thermodynamic properties of the heme-bound iron to elucidate the structure-function relationships underlying two important physiological responses of hemoglobin (Hb): the Root Effect of hemoglobin from certain fish and the different nitrite reactivities of hemoglobins from clams. Hemoglobins of some fish exhibit significantly lowered oxygen affinity at low pH, allowing for proton-mediated release of O₂. This phenomenon, known as the Root Effect, serves as a proton-driven pump delivering O₂ to the swim bladders and eyes of the fish. The clam, ,<italic>L. pectinata</italic>, expresses functionally distinct Hb I that transports H₂S and Hb II that transports O₂. These two hemoglobins differ widely in their reactivity with nitrite, a reactant of great importance to the study of vasodilation in humans. The structural basis of the extreme pH-sensitivity of the Root Effect Hbs and the extreme reactivities of the <italic>Lucina Hbs</italic> with nitrite are debated. Focusing on the metal as the probe, we investigate the reduction potentials of these Hbs using spectroelectrochemistry and compare our findings with oxygen binding studies performed by our collaborators. In both cases, our data strongly suggest that steric hindrance is the determining factor governing the respective physiological response of each hemoglobin. </p> <p>In Chapter 3, we again use the metal as the probe to determine the reduction potential of titanium bound by transferrin (Tf). Tf is the human iron transport protein that can also bind titanium. To address the possible mechanisms of titanium transport through the hypothesized redox-mediated Fe₂-Tf transport pathway, a modified spectroelectrochemistry (SEC) method was developed to measure the electrochemical properties of metalloproteins with very negative potentials. However, the reduction potential of Ti₂-Tf is far too negative to access with our system. As an alternative approach, the redox properties of several model titanium and iron compounds were characterized in order to develop a linear free energy relationship (LFER) allowing us to estimate the reduction potential of Ti₂-Tf to be ca. -900 mV vs. NHE. Our results indicate that the reduction potential of Ti₂-Tf is too low to be reduced by biological reducing agents and suggest that transferrin-mediated titanium transport follows a different mechanism than iron transport.</p> <p>In Chapter 4, our focus shifts to the thermodynamic properties of the protein. Some pathogenic Gram-negative bacteria such as <italic>N. gonorrhoeae</italic> steal iron from their human host by expressing a receptor (TbpA/TbpB), which binds the human iron transport protein transferrin (Tf). Once iron crosses the outer membrane, ferric binding protein (FbpA) transports it across the periplasm to the cytosol. Focusing on the protein, we investigated the protein-protein interactions involved in this transport process and the roles that TbpA and TbpB play with the use of an H/D exchange and mass spectrometry based method termed SUPREX. We report herein the first direct measurement of periplasmic FbpA binding to the outer membrane protein TbpA and we demonstrate that both TbpA and TbpB individually can deferrate Tf without energy supplied from TonB, resulting in sequestration by apo-FbpA.</p> <p>In Chapter 5, we extend our investigation of the <italic>N. gonorrhoeae</italic> iron uptake system by using the metal as the probe in one case and the protein as the probe in another case. TbpA, the &#946;-barrel receptor protein that is required for utilization of Fe₂-Tf as an iron source, has a plug domain which we hypothesize binds iron and interacts with FbpA on the periplasmic side of the outer membrane. Utilizing SUPREX to monitor the thermodynamic properties of protein folding, we investigate 1) the possible interactions between the TbpA-plug and FbpA and 2) the ability of the TbpA-plug to bind iron. </p> <p>Focusing on the metal as the probe, we designed an experimental apparatus to investigate the possible thermodynamic effects of the TbpA/TbpB receptor on the release of iron from Tf. We report the use of a competitive iron chelator and equilibrium dialysis allows for the spectroscopic monitoring of iron release from Tf in the absence of FbpA, but in the presence of opaque bacterial membrane preparations containing the receptor.</p>Dissertatio

Bordetella pertussis FbpA Binds Both Unchelated Iron and Iron Siderophore Complexes

Bordetella pertussis is the causative agent of whooping cough. This pathogenic bacterium can obtain the essential nutrient iron using its native alcaligin siderophore and by utilizing xeno-siderophores such as desferrioxamine B, ferrichrome, and enterobactin. Previous genome-wide expression profiling identified an iron repressible B. pertussis gene encoding a periplasmic protein (FbpA_Bp). A previously reported crystal structure shows significant similarity between FbpA_Bp and previously characterized bacterial iron binding proteins, and established its iron-binding ability. Bordetella growth studies determined that FbpA_Bp was required for utilization of not only unchelated iron, but also utilization of iron bound to both native and xeno-siderophores. In this <i>in vitro</i> solution study, we quantified the binding of unchelated ferric iron to FbpA_Bp in the presence of various anions and importantly, we demonstrated that FbpA_Bp binds all the ferric siderophores tested (native and xeno) with μM affinity. <i>In silico</i> modeling augmented solution data. FbpA_Bp was incapable of iron removal from ferric xeno-siderophores <i>in vitro</i>. However, when FbpA_Bp was reacted with native ferric-alcaligin, it elicited a pronounced change in the iron coordination environment, which may signify an early step in FbpA_Bp-mediated iron removal from the native siderophore. To our knowledge, this is the first time the periplasmic component of an iron uptake system has been shown to bind iron directly as Fe³⁺ and indirectly as a ferric siderophore complex

Borate as a Synergistic Anion for <i>Marinobacter algicola</i> Ferric Binding Protein, FbpA: A Role for Boron in Iron Transport in Marine Life

Boron in the ocean is generally considered a nonbiological element due to its relatively high concentration (0.4 mM) and depth independent concentration profile. Here we report an unexpected role for boron in the iron transport system of the marine bacterium <i>Marinobacter algicola</i>. Proteome analysis under varying boron concentrations revealed that the periplasmic ferric binding protein (Mb-FbpA) was among the proteins whose expression was most affected, strongly implicating the involvement of boron in iron utilization. Here we show that boron facilitates Fe³⁺ sequestration by Mb-FbpA at pH 8 (oceanic pH) by acting as a synergistic anion (B­(OH)₄^1–). Fe³⁺ sequestration does not occur at pH 6.5 where boric acid (B­(OH)₃; p<i>K</i>_a = 8.55) is the predominant species. Borate anion is also shown to bind to apo-Mb-FbpA with mM affinity at pH 8, consistent with the biological relevance implied from boron’s oceanic concentration (0.4 mM). Borate is among those synergistic anions tested which support the strongest Fe³⁺ binding to Mb-FbpA, where the range of anion dependent affinity constants is log <i>K′</i>_eff = 21–22. Since the p<i>K</i>_a of boric acid (8.55) lies near the pH of ocean water, changes in oceanic pH, as a consequence of fluctuations in atmospheric CO₂, may perturb iron uptake in many marine heterotrophic bacteria due to a decrease in oceanic borate anion concentration

Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens*

Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions