Heme Exposure Research Articles

Discovery of Intracellular Heme-binding Protein HrtR, Which Controls Heme Efflux by the Conserved HrtB-HrtA Transporter in Lactococcus lactis

Most commensal and food bacteria lack heme biosynthesis genes. For several of these, the capture of environmental heme is a means of activating aerobic respiration metabolism. Our previous studies in the Gram-positive bacterium Lactococcus lactis showed that heme exposure strongly induced expression of a single operon, called here hrtRBA, encoding an ortholog of the conserved membrane hrt (heme-regulated transporter) and a unique transcriptional regulator that we named HrtR. We show that HrtR expressed as a fusion protein is a heme-binding protein. Heme iron interaction with HrtR is non-covalent, hexacoordinated, and involves two histidines, His-72 and His-149. HrtR specifically binds a 15-nt palindromic sequence in the hrtRBA promoter region, which is needed for hrtRBA repression. HrtR-DNA binding is abolished by heme addition, which activates expression of the HrtB-HrtA (HrtBA) transporter in vitro and in vivo. The use of HrtR as an intracellular heme sensor appears to be conserved among numerous commensal bacteria, in contrast with numerous Gram-positive pathogens that use an extracellular heme-sensing system, HssRS, to regulate hrt. Finally, we show for the first time that HrtBA permease controls heme toxicity by its direct and specific efflux. The use of an intracellular heme sensor to control heme efflux constitutes a novel paradigm for bacterial heme homeostasis.

Interaction between heme oxygenase-1 genotypes and exposure to pesticides in Parkinson's disease

Interaction between heme oxygenase-1 genotypes and exposure to pesticides in Parkinson's disease

The non-native conformations of cytochrome c in sodium dodecyl sulfate and their modulation by ATP

To understand the interaction of cytochrome c (cyt c) with membranes, a systematic investigation of sodium dodecyl sulfate (SDS)-induced conformational alterations in native horse heart ferricytochrome c (pH 7.0) was carried out using heme absorbance, tryptophan fluorescence and circular dichroism (CD) spectroscopy. ATP interaction with membrane-bound cyt c is known to regulate the process of apoptosis. To understand the effect of nucleotide phosphates on membrane-bound cyt c, we also carried out studies of the interaction of ATP with cyt c in the presence of SDS. Fluorescence and UV-Vis data suggest that SDS induces two different transitions (F to C1, C1 to C2) in cyt c, one in the pre-micellar region and the other in the post-micellar region. The fluorescence data further indicated the increase in distance between Trp 59 and heme in the intermediates in both the regions, suggesting loosening up of cyt c on titration with SDS. The far-UV and near-UV CD data suggest partial loss of secondary and tertiary structure in C1, but complete loss of tertiary structure and no further loss of secondary structure in C2. On titration of C1 and C2 with ATP, the secondary structure is restored. However, the heme ligation pattern and heme exposure change only for C2, but not for C1 on the addition of ATP.

Staphylococcus aureus HrtA Is an ATPase Required for Protection against Heme Toxicity and Prevention of a Transcriptional Heme Stress Response

During systemic infection, Staphylococcus aureus acquires nutrient iron from heme, the cofactor of vertebrate myoglobin and hemoglobin. Upon exposure to heme, S. aureus up-regulates the expression of the heme-regulated transporter, HrtAB. Strains lacking hrtAB exhibit increased sensitivity to heme toxicity, and upon heme exposure they elaborate a secreted protein response that interferes with the recruitment of neutrophils to the site of infection. Taken together, these results have led to the suggestion that hrtAB encodes an efflux system responsible for relieving the toxic effects of accumulated heme. Here we extend these observations by demonstrating that HrtA is the ATPase component of the HrtAB transport system. We show that HrtA is an Mn(2+)/Mg(2+)-dependent ATPase that functions at an optimal pH of 7.5 and exhibits in vitro temperature dependence uncommon to ABC transporter ATPases. Furthermore, we identify conserved residues within HrtA that are required for in vitro ATPase activity and are essential for the functionality of HrtA in vivo. Finally, we show that heme induces an alteration in the gene expression pattern of S. aureus Delta hrtA, implying the presence of a novel transcriptional regulatory mechanism responsible for the previously described immunomodulatory characteristics of hrtA mutants exposed to heme.

The Heme Pocket Geometry of Lucina pectinata Hemoglobin II Restricts Nitric Oxide and Peroxide Entry: Model of Ligand Control for the Design of a Stable Oxygen Carrier,

Blood pressure elevation has been attributed in large part to the consumption of nitric oxide (NO) by extracellular hemoglobin (Hb) therapeutics following infusion in humans. We studied NO and hydrogen peroxide (H2O2) oxidative reaction kinetics of monomeric Hbs isolated from the clam Lucina pectinata to probe the effects of their distinctive heme pocket chemistries on ligand controls and heme oxidative stability. HbI (Phe43(CD1), Gln64(E7), Phe29(B10), and Phe68(E11)) reacted with high avidity with NO (k'(ox,NO) = 91 microM-1 s-1), whereas HbII (Phe44(CD1), Gln65(E7), Tyr30(B10), and Phe69(E11)) reacted at a much slower rate (k'(ox,NO)= 2.8 microM-1 s-1). However, replacing B10 (Phe) by Tyr in recombinant HbI (HbI PheB10Tyr) produced only a 2-fold reduction in the NO-induced oxidation rate (k'(ox,NO)= 49.9 microM-1 s-1). Among the clam Hbs, HbII exhibited the fastest NO dissociation and the slowest NO association with ferrous iron. Autoxidation, H2O2-mediated ferryl iron (FeIV) formation, and the subsequent heme degradation kinetics were much slower in HbII and HbI PheB10Tyr when compared to those of HbI. The Tyr(B10) residue appears to afford a greater heme oxidative stability advantage toward H2O2, whereas the close proximity of this residue together with Gln(E7) to the heme iron contributes largely to the distal control of NO binding. Engineering of second-generation Hb-based oxygen therapeutics that are resistant to NO/H2O2-driven oxidation may ultimately require further optimization of the heme pocket architecture to limit heme exposure to solvent.

Did Bacterial Sensing of Host Environments Evolve from Sensing within Microbial Communities?

Bacteria sense and respond to their environment, enabling adaptation to diverse niches, including multicellular eukaryotes. In this issue of Cell Host & Microbe, Torres et al. describe how the bacterium Staphylococcus aureus responds to heme as a molecular marker of the mammalian host environment. It is likely that mechanisms for sensing such markers evolved from systems that recognized cues present in microbial communities before the emergence of eukaryotes.

A Staphylococcus aureus Regulatory System that Responds to Host Heme and Modulates Virulence

Staphylococcus aureus, a bacterium responsible for tremendous morbidity and mortality, exists as a harmless commensal in approximately 25% of humans. Identifying the molecular machinery activated upon infection is central to understanding staphylococcal pathogenesis. We describe the heme sensor system (HssRS) that responds to heme exposure and activates expression of the heme-regulated transporter (HrtAB). Inactivation of the Hss or Hrt systems leads to increased virulence in a vertebrate infection model, a phenotype that is associated with an inhibited innate immune response. We suggest that the coordinated activity of Hss and Hrt allows S. aureus to sense internal host tissues, resulting in tempered virulence to avoid excessive host tissue damage. Further, genomic analyses have identified orthologous Hss and Hrt systems in Bacillus anthracis, Listeria monocytogenes, and Enterococcus faecalis, suggesting a conserved regulatory system by which Gram-positive pathogens sense heme as a molecular marker of internal host tissue and modulate virulence.

A Conformational Switch to β-Sheet Structure in Cytochrome c Leads to Heme Exposure. Implications for Cardiolipin Peroxidation and Apoptosis

Ultraviolet resonance Raman spectroscopy reveals that, when heated at pH 3, a substantial fraction (30%) of cytochrome c converts to a β-sheet structure, at the expense of turns and helices. β-sheet formation is rapid, exhibiting a 2 μs rise time, following a temperature jump. It is proposed that a short β-sheet segment, comprising residues 37−39 and 58−61, extends itself into the large 37−61 loop when the latter is destabilized by protonation of H26, which forms an anchoring H-bond to loop residue P44. This conformation change ruptures the Met80-Fe bond, as revealed by changes in ligation-sensitive heme-resonant Raman bands. It also induces peroxidase activity with the same temperature profile. This process is suggested to model the apoptotic peroxidation of cardiolipin by cytochrome c.

Functional Roles of the Heme Architecture and Its Environment in Tetraheme Cytochrome c

Cytochromes are involved in a wide variety of redox reactions in living systems. Some of them contain multiple hemes such as Desulfovibrio cytochrome c3 and Shewanella small tetraheme cytochrome c. The significance of c-type tetraheme architectures was discussed. A cyclic heme architecture and its environment regulate the extremely low redox potentials of cytochrome c3 in addition to bis-imidazole coordination and heme exposure. Each heme in cytochrome c3 plays a different role in the electron transport to/from [NiFe] hydrogenase and the specific CO-binding. In contrast, the chain-like heme architecture in Shewanella small tetraheme cytochrome c and soluble fumarate reductase provides a pathway for directional electron transfer. Thus, the tetraheme architectures do not comprise simple heme assemblies but sophisticated devices.

A Heme Export Protein Is Required for Red Cell Differentiation and May Facilitate Iron Homeostasis.

Heme is a component of cytochromes, catalases, glutathione peroxidase, hydroxylases, and nitric oxide synthase, as well as myoglobin and hemoglobin, and thus is critical to all aerobic cells. It is also a transcriptional and translational initiator of globin synthesis. However, free heme is toxic so that cells must balance synthesis with use. We recently determined that the cell surface receptor for Feline Leukemia Virus, subgroup C, (FLVCR), which causes pure red cell aplasia in cats, is a heme export protein (Cell; 118:6, 2004). To understand its physiologic significance, we generated constitutive (Flvcr+/−) and inducible (Flvcrflox/flox;Mx-cre) FLVCR null mice by targeted deletion of exon 3, and confirmed this produced a nonfunctional protein by zinc mesoporphyrin (ZnMP) and 55Fe-heme export studies. Intercrossing Flvcr+/− mice produced 0 null animals among 109 progeny genotyped. FLVCR null embryos die either before E8.5 or at E14.5, suggesting two separate mechanisms for their demise. In situ hybridization studies in normal mouse embryos show highest Flvcr transcript levels in the ectoplacental cone which forms placenta (E8.5), the placenta (E9.5), and high levels in the fetal liver (E12.5). These data plus studies with Flvcrflox/floxx Meox2-Cre mice (Cre inactivates Flvcr in all embryonic tissue, but not extraembryonic visceral endoderm and placenta) suggest that the early lethality of null mice is due to impaired maternal-embryo heme-iron transport. At E14.5, residual null mice were small with deformed limb buds and craniofacial abnormalities resembling the congenital anomalies of Diamond-Blackfan anemia. Flow cytometric analyses of fetal liver cells from deleted animals with Ter119 and CD71 demonstrated a block in erythroid differentiation at the CFU-E/proerythroblast stage, implying that their death results from a stage-specific failure in definitive erythropoiesis. Heterozygous null animals (Flvcr+/−) were viable with normal hematologic parameters. These mice had 30.9% of normal Flvcr mRNA by q RT-PCR, however, they demonstrated normal protein by western blot analysis and normal heme export function by ZnMP studies. Thus, heterozygous null mice compensate at the protein level, confirming FLVCR is critical in vivo. We next utilized our inducible mutant line (Flvcrflox/flox;Mx-cre) to generate a viable null mouse for study. Flvcr deleted mice developed pure red cell aplasia characterized by macrocytic anemia (deleted: HCT 13.8±1.2, MCV 68.35± 1.6; control: HCT 50.24 ± 2.0, MCV 51.3±1.5), reticulocytopenia, and reactive thrombocytosis. Marrow and spleen had excess cells with a proerythroblast morphology and lacked maturing red cells. Flow cytometry confirmed maturation arrest at the CFU-E/proerythroblast stage. CFU-E from FLVCR null mice were undetectable in culture. Deleted mice also demonstrated iron loading of hepatocytes. FLVCR is highly expressed in multiple human tissues with high heme-flux by western blot analyses, including liver, placenta, duodenum, and uterus, in addition to the bone marrow. Our studies prove that FLVCR is required for the survival or differentiation of proerythroblasts (that stage where heme synthesis intensifies). These results, plus additional data not shown, suggest that FLVCR may also protect nonerythroid tissues with high free heme exposure (uterus, duodenum), may mediate maternal-embryo heme-iron transport, and may mediate the transport of heme from hepatocyte to bile which would imply a novel mechanism for iron export from the body.

Self-reduction and Conductivities of Cytochromes

A self-reduction of cytochrome c is found and depends on the pressure of hydrogen, pH, and sorts of buffers and salts. Cytochrome c3 shows the two mechanisms such as the reduction of heme and hydrogen exposure effect.

Oxidation of hemoglobin by lipid hydroperoxide associated with low-density lipoprotein (LDL) and increased cytotoxic effect by LDL oxidation in heme oxygenase-1 (HO-1) deficiency.

Heme-catalyzed oxidation of low-density lipoprotein (LDL) is one of the relevant mechanisms involved in LDL modification. We previously revealed a substantial oxidation of plasma hemoglobin to methemoglobin and a subsequent heme-catalyzed LDL oxidation generating moieties toxic to endothelium in heme oxygenase-1 (HO-1)-deficiency in human. Drawing upon our previous observation we posited a pathway for oxidation of plasma hemoglobin in the HO-1-deficient child involving LDL-associated lipid hydroperoxide. In support, LDL-associated lipid hydroperoxide oxidized ferrohemoglobin to methemoglobin--known to readily release its heme moieties--in a dose-dependent manner. Repeated heme exposure of the child s LDL further increased its lipid hydroperoxide content within min leading to additional cytotoxic effect on endothelium. Both cytotoxicity and HO-1 inducing ability of the oxidized LDL were strongly dependent on its lipid hydroperoxide content. We wondered if cells of the HO-1-deficient patient were prone to oxidative damage arising from heme-mediated oxidation of LDL. Indeed, we found elevated cytotoxicity induced by heme-catalyzed oxidation of LDL in lymphoblastoid cells derived from the HO-1-deficient patient. We conclude that oxidation of hemoglobin to methemoglobin by LDL-associated lipid hydroperoxide and increased sensitivity of cells of the HO-1-deficient child to stress of oxidized LDL might contribute to the vascular disorders reported earlier.

Tuning the reduction potential of engineered cytochrome c-553.

Cytochrome c-553 from Desulfovibrio vulgaris exhibits a highly exposed heme and an unusually low reduction potential with respect to other c-type cytochromes. Solvent heme exposure has been indicated as one of the most important factors in modulating the midpoint potential of the redox center. To test this hypothesis, a unique surface-exposed cysteine has been substituted for either M23 or G51 to produce the corresponding mutants and allow the formation of homodimers through a specific disulfide bridge. The reduction potentials, determined via spectroelectrochemistry, show an increase from +20 +/- 5 mV for the wt to +88 +/- 5 and +105 +/- 5 mV for the M23C-M23C homodimer and G51C-G51C homodimer, respectively. Chemical denaturation of the homodimers leads to parameters related to the hydrophobicity (m) and the number of buried side chains (n(B)), which suggest a decrease of exposure of the heme as a result of dimerization. These results are consistent with the heme-accessible surface area (ASA) calculated from a computer model of the homodimers. The ASA values show a decrease from 73 A(2) for the wt to 66 and 50 A(2) per heme for the M23C-M23C homodimer and G51C-G51C homodimer, respectively. The trend of the m- and n(B)-values, the degree of solvent accessibility, and the midpoint potential observed upon formation of the homodimers indicate a correlation between the reduction potential values and the exclusion of water from the heme surface.

The Effect of Water on the Fe3+/Fe2+ Reduction Potential of Heme

Hemeproteins can act as catalysts, oxygen carriers or electron conductors. The ferric/ferrous reduction potential Em7 of iron in the center of the prosthetic group ranges from negative values for peroxidases to an extreme positive value for cytochrome a3 with Hb and Mb in the middle [1]. Proteins exercise their influence on Em7 in several ways: via substituents at the periphery of the chelate structure, via the proximal ligand, and via interaction with the surrounding medium, amino acid side chains, or polar solvents. Work on recombined proteins and 2,4-substituted free hemes documented that the first two effects are additive [2]. For the third effect, models of the dielectric media on a molecular level have been successfully applied [3–5]. Em7 has also been empirically correlated to the degree of heme exposure to water [6–8]. The apoprotein/porphyrin and water/porphyrin interfaces are complementary since water molecules fill any empty space in the crevice and surround any pertinent part of heme outside the protein boundary. The present work links to this idea by a combination of statistical mechanics simulations and quantum mechanical calculations comparing heme in water with heme in an apolar environment. Our results show that polarization of the porphyrin π-electron cloud by the field from water dipoles influences Em7. The dominant effect of this and other determinates of iron electron availability is perturbations of delocalized electron density in the porphyrin chelate, reproduced by a model where the prosthetic group is treated as a disc of uniform electron density. The present work is also of interest since the interfacial energy constitutes the main barrier for heme-protein separation [9–11].

Heme exposure does not sensitize human endothelial cells in culture to the cytoxic effect of H 2O 2: [formula omitted], Caroline M. Woods, George P. Biro. Hemosol Inc., Toronto, Ontario, Canada, M9W 4Z7

Heme exposure does not sensitize human endothelial cells in culture to the cytoxic effect of H 2O 2: [formula omitted], Caroline M. Woods, George P. Biro. Hemosol Inc., Toronto, Ontario, Canada, M9W 4Z7

Heme induces the expression of adhesion molecules ICAM-1, VCAM-1, and E selectin in vascular endothelial cells.

Heme is an important immunostimulating agent and oxidative factor contributing to endothelial cell activation. To investigate the mechanism of heme-induced endothelial cell activation, we analyzed the effect of heme and the inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), on the expression of the heme-degrading stress protein, heme oxygenase (HO), and adhesion molecules in human umbilical vein endothelial cells (HUVEC). Indirect immunofluorescence double labeling studies demonstrated a simultaneous increase of ICAM-1 and HO-1 after exposure of cells to heme for 24 hr. Co-expression of HO-1 and ICAM-1 was also demonstrated in TNF-alpha-exposed cells. Dot blot immunoassay and quantitative analysis by ELISA demonstrated that heme treatment for 24 hr caused a 2-fold increase in ICAM-1 expression (P < 0.002) compared with quiescent cells, while in cells stimulated by TNF-alpha for 24 hr ICAM-1 gene expression increased by 5-fold. Moreover, heme exposure also resulted in a marked increase in VCAM-1 and E selectin expression (three and four times over control levels, respectively). On the other hand, TNF-alpha treatment showed similar expression levels for VCAM-1 and E selectin, compared with stimulation by heme (100 microM). The level of HO activity in endothelial cells exposed to heme or TNF-alpha was increased from 24.7 +/- 5.7 pmol bilirubin/mg protein/min in control to 70.0 +/- 9.5 and 36.7 +/- 3.1 pmol bilirubin/mg protein/min in heme- and TNF-alpha-stimulated cells, respectively. These results suggest that upregulation of ICAM-1, VCAM-1, and E selectin expression is associated with oxidative stress induced by hemoglobin/heme and that HO-1 may play a modulating role via its ability to degrade heme to a substance with antioxidant properties.

Structure of the tetraheme cytochrome from Desulfovibrio desulfuricans ATCC 27774: X-ray diffraction and electron paramagnetic resonance studies.

The three-dimensional X-ray structure of cytochrome c3 from a sulfate reducing bacterium, Desulfovibrio desulfuricans ATCC 27774 (107 residues, 4 heme groups), has been determined by the method of molecular replacement [Frazão et al. (1994) Acta Crystallogr. D50, 233-236] and refined at 1.75 A to an R-factor of 17.8%. When compared with the homologous proteins isolated from Desulfovibrio gigas, Desulfovibrio vulgaris Hildenborough, Desulfovibrio vulgaris Miyazaki F, and Desulfomicrobium baculatus, the general outlines of the structure are essentialy kept [heme-heme distances, heme-heme angles, His-His (axial heme ligands) dihedral angles, and the geometry of the conserved aromatic residues]. The three-dimensional structure of D. desulfuricans ATCC 27774 cytochrome c3Dd was modeled on the basis of the crystal structures available and amino acid sequence comparisons within this homologous family of multiheme cytochromes [Palma et al. (1994) Biochemistry 33, 6394-6407]. This model is compared with the refined crystal structure now reported, in order to discuss the validity of structure prediction methods and critically evaluate the steps used to predict protein structures by homology modeling. The four heme midpoint redox potentials were determined by using deconvoluted electron paramagnetic resonance (EPR) redox titrations. Structural criteria (electrostatic potentials, heme ligand orientation, EPR g values, heme exposure, data from protein-protein interaction studies) are invoked to assign the redox potentials corresponding to each specific heme in the three-dimensional structure.

Unfolding and release of heme from human hemoglobins A, S and F

1. 1. Analysis of the Soret spectra of hemoglobins A, S and F has been used to determine the extent of heme exposure and release from these hemoglobins in the presence of several solvent perturbants. 2. 2. Oxyhemoglobin S unfolding in the presence of either urea or propyl urea resulted in greater heme exposure and release than either oxyhemoglobins A or F. 3. 3. Methemoglobin formation resulted in lower denaturation midpoints for each hemoglobin compared to the reduced oxyhemoglobin state; methemoglobin F had the lowest denaturation midpoint under isothermal denaturing conditions. 4. 4. Rate of heme exposure was greater for oxyhemoglobin S than oxyhemoglobin A in the presence of 200 μM the anionic detergent sodium dodecyl sulfate. 5. 5. Evidence for increased levels of heme release in hemoglobin S may be related to the greater tendency of sickled red cell membranes to undergo lipid oxidation.

Brownian simulation of cytochrome c551 association and electron self-exchange

In this paper we describe the results of a theoretical study of the dynamics of electron self-exchange in Pseudomanas aeruginosa cytochrome c551 using the Brownian Dynamics computer simulation method. The proteins are treated as spheres with imbedded monopole/dipole charge arrangements based on an electrostatic analysis of the crystallographic coordinate sets. For the first time, the electron transfer event is explicitly coupled to the Brownian association dynamics. The intrinsic electron transfer rate constant is modelled as an exponential function ∼exp(-αr) of distance r between porphyrin edges. Reasonable agreement of this simple theory with kinetic data from NMR line-broadening experiments is obtained. The role of factors such as solvent electrolyte screening effects, protein-protein electrostatic interactions, heme exposure and overall protein size are elucidated. An estimate is made of the electron transfer exponential distance factor α which agrees quite well with various experiments.

Plastocyanin cytochrome f interaction.

Spinach plastocyanin and turnip cytochrome f have been covalently linked by using a water-soluble carbodiimide to yield an adduct of the two proteins. The redox potential of cytochrome f in the adduct was shifted by -20 mV relative to that of free cytochrome f, while the redox potential of plastocyanin in the adduct was the same as that of free plastocyanin. Solvent perturbation studies showed the degree of heme exposure in the adduct to be less than in free cytochrome f, indicating that plastocyanin was linked in such a way as to bury the exposed heme edge. Small changes were also observed when the resonance Raman spectrum of the adduct was compared to that of free cytochrome f. The adduct was incapable of interacting with or donating electrons to photosystem I. Peptide mapping and sequencing studies revealed two sites of linkage between the two proteins. In one site of linkage, Asp-44 of plastocyanin is covalently linked to Lys-187 of cytochrome f. This represents the first identification of a group on cytochrome f that is involved in the interaction with plastocyanin. The other site of linkage involves Glu-59 and/or Glu-60 of plastocyanin to as yet unidentified amino groups on cytochrome f. Euglena cytochrome c-552 could also be covalently linked to turnip cytochrome f, although with a lower efficiency than spinach plastocyanin. In contrast, a variety of cyanobacterial cytochrome c-553's and a cyanobacterial plastocyanin could not be covalently linked to turnip cytochrome f.