Hemin Affinity Research Articles

Use of plasma induced modification of biomolecules (PLIMB) to evaluate hemin dissociation from fish and bovine methemoglobins

Hemin dissociation occurs much faster from fish methemoglobin (metHb) compared to mammalian metHb yet the mechanism remains poorly understood. This may involve enhanced solvent access to His(E7) of fish metHbs by a protonation mechanism. Plasma induced modification of biomolecules (PLIMB) produces free radicals that covalently modify solvent accessible residues of proteins, and so can provide insight regarding accessibility of hydronium ions to protonate His(E7). PLIMB-induced modifications to heme crevice sites of trout IV and bovine metHb were determined using tandem mass spectrometry after generating peptides with Trypsin/Lys-C. αHis(CE3) was more modified in trout attributable to the more dynamic nature of bovine αHis(CE3) from available crystal structures. Although His(E7) was not found to be more modified in trout, aspects of trout peptides containing His(E7) hampered modification determinations. An existing computational structure-based approach was also used to estimate protonation tendencies, suggesting His(E7) of metHbs with low hemin affinity are more protonatable.

A202 INVESTIGATION OF THE DIRECT EFFECT OF HEMIN-HEMOPEXIN AND HAPTOGLOBIN STIMULATION IN INFLAMMATORY BOWEL DISEASE

Abstract Background Inflammatory bowel disease (IBD) is a global health issue defined by persistent intestinal epithelial inflammation resulting in bleeding, and it is one of the major risk factors of colorectal cancer (CRC). Hemin is an activator of heme-oxygenase and has anti-inflammatory, cytoprotective, and antiapoptotic properties by stimulating hemopexin and haptoglobin production. Hemopexin and haptoglobin are scavenging proteins with high affinity for hemin and hemoglobin, and are activated by interleukin (IL)-22. Purpose To evaluate the protective role of hemin through the stimulation of hemopexin and haptoglobin in experimental acute colitis. Method Mice were preconditioned with hemin by intraperitoneal injection with 5mg/kg body weight of hemin for three days while control mice were treated with PBS. Colitis was induced by giving 2.5-3% dextran sulfate sodium (DSS) in drinking water for 12 days. The disease activity index (DAI) was determined by daily monitoring of body weight, fecal bleeding and stool consistency. At the end of the experiment, liver, feces, serum and colon samples were harvested for the evaluation of fecal levels of hemopexin, fecal haptoglobin, IL-22, IL-22BP, IL-6 and TNF-α. Fecal microbiota composition will be determined using 16S rRNA sequencing. Result(s) We found that hemin treatment resulted in the presence of hemopexin and haptoglobin in fecal samples, where they may exert a protective role and influence the growth of pathobionts able to acquire iron using heme and hemoglobin as sources. Conclusion(s) The findings from this study will contribute to the understanding of the possible protective role of hemopexin and haptoglobin in ulcerative colitis. Please acknowledge all funding agencies by checking the applicable boxes below CIHR Disclosure of Interest None Declared

Depletion assisted hemin affinity (DAsHA) proteomics reveals an expanded landscape of heme-binding proteins in the human proteome.

Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry (MS)-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme-binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin, and finally elution and identification by MS. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in an SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization, and vesicular trafficking, many of which have been associated with heme through complementary studies published recently. Taken together, these results provide support for the emerging role of heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.

Hemin acts as CD36 ligand to activate down-stream signalling to disturb immune responses and cytokine secretion from macrophages

Inflammatory responses to hemin are believed to play an important role in tissue damage and cerebral malaria pathology. Macrophage exposed to hemin exhibits modulation of non-opsonic phagocytosis of aged RBCs, ability to kill bacteria and secretion of cytokines. Immuno-fluorescence study indicates translocation and sequestration of CD36 within the intracellular storage in the hemin treated macrophages. It in-turn modulates the global cytokine secretion from macrophages. CD36 has strong affinity for hemin with a dissociation constant of 1.26±0.24 μM. CD36 has hemin bio-phoric environment involving R292, D372 and Q382. The mutation in biophoric residues significantly reduced the affinity towards hemin. Hemin stimulated MG63 cells (transfected with CD36) showed several folds increment in cytokines TNFα, MCP-1, RANTES and CCL1 and CD36-hemin interaction is crucial for aberrant cytokine secretion. CD-36: Hemin interaction is driving down-stream signalling and subsequent recruitment of adaptor proteins to the cytosolic domain of CD36. Immunoprecipitation of membrane bound CD36 gives Lyn kinase as potential adaptor protein down-stream to CD36: hemin signalling. Interestingly, disruption of Lyn kinase abolishes the hemin mediated dysregulation of immune responses. In summary, hemin-CD36-Lyn kinase signalling axis could be a contribution factor to severe malaria pathology and prognosis.

Studies of Potency and Efficacy of an Optimized Artemisinin-Quinoline Hybrid against Multiple Stages of the Plasmodium Life Cycle.

A recently developed artemisinin-quinoline hybrid, named 163A, has been shown to display potent activity against the asexual blood stage of Plasmodium, the malaria parasite. In this study, we determined its in vitro cytotoxicity to mammalian cells, its potency to suppress P. berghei hepatic infection and to decrease the viability of P. falciparum gametocytes, in addition to determining whether the drug exhibits efficacy of a P. berghei infection in mice. This hybrid compound has a low level of cytotoxicity to mammalian cells and, conversely, a high level of selectivity. It is potent in the prevention of hepatic stage development as well as in killing gametocytes, denoting a potential blockage of malaria transmission. The hybrid presents a potent inhibitory activity for beta-hematin crystal formation, in which subsequent assays revealed that its endoperoxide component undergoes bioactivation by reductive reaction with ferrous heme towards the formation of heme-drug adducts; in parallel, the 7-chloroquinoline component has binding affinity for ferric hemin. Both structural components of the hybrid co-operate to enhance the inhibition of beta-hematin, and this bitopic ligand property is essential for arresting the growth of asexual blood parasites. We demonstrated the in vivo efficacy of the hybrid as an erythrocytic schizonticide agent in comparison to a chloroquine/artemisinin combination therapy. Collectively, the findings suggest that the bitopic property of the hybrid is highly operative on heme detoxification suppression, and this provides compelling evidence for explaining the action of the hybrid on the asexual blood stage. For sporozoite and gametocyte stages, the hybrid conserves the potency typically observed for endoperoxide drugs, and this is possibly achieved due to the redox chemistry of endoperoxide components with ferrous heme.

The major facilitator transporter Str3 is required for low-affinity heme acquisition in Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, acquisition of exogenous heme is largely mediated by the cell membrane-associated Shu1. Here, we report that Str3, a member of the major facilitator superfamily of transporters, promotes cellular heme import. Using a strain that cannot synthesize heme de novo (hem1Δ) and lacks Shu1, we found that the heme-dependent growth deficit of this strain is rescued by hemin supplementation in the presence of Str3. Microscopic analyses of a hem1Δ shu1Δ str3Δ mutant strain in the presence of the heme analog zinc mesoporphyrin IX (ZnMP) revealed that ZnMP fails to accumulate within the mutant cells. In contrast, Str3-expressing hem1Δ shu1Δ cells could take up ZnMP at a 10-μm concentration. The yeast Saccharomyces cerevisiae cannot efficiently transport exogenously supplied hemin. However, heterologous expression of Str3 from S. pombe in S. cerevisiae resulted in ZnMP accumulation within S. cerevisiae cells. Moreover, hemin-agarose pulldown assays revealed that Str3 binds hemin. In contrast, an Str3 mutant in which Tyr and Ser residues of two putative heme-binding motifs (530YX3Y534 and 552SX4Y557) had been replaced with alanines exhibited a loss of affinity for hemin. Furthermore, this Str3 mutant failed to rescue the heme-dependent growth deficit of a hem1Δ shu1Δ str3Δ strain. Further analysis by absorbance spectroscopy disclosed that a predicted extracellular loop region in Str3 containing the two putative heme-binding motifs interacts with hemin, with a KD of 6.6 μm Taken together, these results indicate that Str3 is a second cell-surface membrane protein for acquisition of exogenous heme in S. pombe.

In Vitro Studies of the Folding and Assembly Mechanism of Hemoglobin

Deriving the pathway for human hemoglobin assembly is important both for developing treatments for hemoglobinopathies and for designing robust acellular hemoglobin oxygen carriers. The adult human hemoglobin (HbA) comprises two globin heterodimers, (α1β1)(α2β2), with each subunit containing a heme group for coordination of oxygen and having a tertiary structure very similar to the monomeric paralog, myoglobin.In order to determine the complete folding mechanism, we have measured and analyzed guanidinium-induced unfolding curves for both apo- and holo-hemoglobin. This approach expands on previous work done in our lab with myoglobin and has allowed us to deconvolute the globin unfolding pathway and the heme-binding effects for the various folding states. When heme is extracted from human Hb, the resulting apohemoglobin dissociates to an α1β1 heterodimer. Unfolding occurs in two major phases. The first involves melting of the heme pockets to generate a dimeric molten globule, followed by dissociation into almost completely unfolded monomers. Structural identification of these phases was achieved by comparing the unfolding curves for native HbA, recombinant HbF, a N-to-C termini linked α Hb variant, and a series of Hb mutants with large, hydrophobic residues replacing the distal histidine and valine in the α and β heme pockets.Previously, we showed that monomeric apoglobin stability, and not hemin affinity, is the determining factor for myoglobin expression. In the case of hemoglobin, the individual monomers are unstable, but associate strongly to form a stable molten globule dimeric state, which binds hemin and facilitates the overall assembly of the hemoglobin.Supported by the NIH Grant HL110900 and by Grant C-0612 from Robert A. Welch Foundation.

Effect of 4-hydroxy-2-nonenal on myoglobin-mediated lipid oxidation when varying histidine content and hemin affinity.

The compound 4-hydroxy-2-nonenal (HNE) dissolved in water was examined to remove potential effects of using ethanol to solubilize the aldehyde such as altering protein structure or redox properties of myoglobin (Mb). HNE became covalently bound to sperm whale Mb at up to five sites based on ESI-MS analysis. Adducted Mb promoted lipid oxidation in washed muscle more effectively than non-adducted Mb. Alkylation of P88H/Q152HMb with HNE accelerated metMb formation and subsequent lipid oxidation. P88H/Q152HMb exposed to HNE enhanced lipid oxidation compared to wild-type Mb exposed to HNE. Results using H97A Mb suggested that the combination of HNE and low hemin affinity facilitated rapid decomposition of preformed lipid hydroperoxides to secondary lipid oxidation products. HNE and HHE (4-hydroxy-2-hexenal) facilitated Mb-mediated lipid oxidation similarly. The potential mechanisms by which Mb binding of α,β-unsaturated aldehydes affect Mb oxidation and the onset of lipid oxidation are discussed.

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin

Expression levels in animal muscle tissues and in Escherichia coli vary widely for naturally occurring mammalian myoglobins (Mb). To explore this variation, we developed an in vitro transcription and wheat germ extract-based translation assay to examine quantitatively the factors that govern expression of holoMb. We constructed a library of naturally occurring Mbs from two terrestrial and four deep-diving aquatic mammals and three distal histidine mutants designed to enhance apoglobin stability but decrease hemin affinity. A strong linear correlation is observed between cell-free expression levels of holo-metMb variants and their corresponding apoglobin stabilities, which were measured independently by guanidine HCl-induced unfolding titrations using purified proteins. In contrast, there is little dependence of expression on hemin affinity. Our results confirm quantitatively that deep diving mammals have highly stable Mbs that express to higher levels in animal myocytes, E. coli, and the wheat germ cell-free system than Mbs from terrestrial mammals. Our theoretical analyses show that the rate of aggregation of unfolded apoMb is very large, and as a result, the key factor for high level expression of holoMb, and presumably other heme proteins, is an ultra high fraction of folded, native apoglobin that is capable of rapidly binding hemin. This fraction is determined by the overall equilibrium folding constant and not hemin affinity. These results also demonstrate that the cell-free transcription/translation system can be used as a high throughput platform to screen for apoglobin stability without the need to generate large amounts of protein for in vitro unfolding measurements.

Analysis of the binding of bovine and human fibrinogen to ferritin: evidence that fibrinogen is a common ferritin-binding protein in mammals.

Both human and horse fibrinogen are heme-binding proteins, and horse fibrinogen also exhibits heme-mediated ferritin binding. This study found that bovine and human fibrinogen are heme-mediated ferritin-binding proteins and demonstrated direct binding of bovine ferritin to protoporphyrin (PPIX) and its derivatives or to Zn ions. Binding of bovine and human fibrinogen to bovine spleen ferritin coated on microtiter plate wells was detected using an anti-human fibrinogen antibody, and this binding was inhibited in a dose-dependent manner by hemin (iron-PPIX) and also inhibited by Zn-PPIX. PPIX showed less of an inhibitory effect on the binding of bovine and human fibrinogen to bovine ferritin. The inhibitory effect of Sn-PPIX was similar to that of PPIX, but with respect to human fibrinogen, PPIX did not inhibit the binding of human fibrinogen to ferritin. Bovine fibrinogen immobilized on CNBr-activated Sepharose 4B beads showed affinity for hemin, Sn-PPIX, Zn-PPIX, and iron-free PPIX in the order Sn-PPIX < iron-free PPIX < hemin < Zn-PPIX. The fibrinogen beads also directly bound to zinc ions. These results suggest that bovine fibrinogen is a heme- and zinc-binding protein and that binding of circulating mammalian fibrinogen to ferritin is heme mediated.

Lipid Oxidation in Trout Muscle Is Strongly Inhibited by a Protein That Specifically Binds Hemin Released from Hemoglobin

The recombinant streptococcal protein apoShp can be used as a probe for hemoglobin (Hb) reactivity in fish muscle due to its specific affinity for hemin that is released from Hb at post-mortem pH values. Hemin affinity measurements indicated that apoShp binds hemin released from Hb but not myoglobin (Mb). Hemin affinity of holoShp was higher at pH 5.7 compared to pH 8.0. This may be attributed to enhanced electrostatic interaction of His58 with the heme-7-propionate at lower pH. ApoShp readily acquired hemin that was released from trout IV metHb in the presence of washed cod muscle during 2 °C storage at pH 6.3. This was based on increases in redness in the washed cod matrix, which occurs when apoShp binds hemin that is released from metHb. ApoShp prevented Hb-mediated lipid oxidation in washed cod muscle during 2 °C storage. The prevention of Hb-mediated lipid oxidation by apoShp was likely due to bis-methionyl coordination of hemin that dissociated from metHb. This hexacoordination of hemin appears to prevent peroxide-mediated redox reactions, and there is no component in the matrix capable of dissociating hemin from Shp. ApoShp was also added to minced muscle from rainbow trout ( Oncorhynchus mykiss ) to examine the degree to which Hb contributes to lipid oxidation in trout muscle. Addition of apoShp inhibited approximately 90% of the lipid oxidation that occurred in minced trout muscle during 9 days of 2 °C storage on the basis of lipid peroxide, hexanal, and thiobarituric acid reactive substances (TBARS) values. These results strongly suggest that Hb is the primary promoter of lipid oxidation in trout muscle.

Resolving the effects of albumin glycation using the quartz crystal microbalance

Both lysine and arginine residues are particularly important at receptor sites for binding anionic ligands. These receptor sites may become compromised via non-enzymatic glycation. While lysine residues are glycated in the presence of glucose, arginine residues are predominantly glycated by α-oxoaldehydes like glyoxal. This study used a quartz crystal microbalance with dissipation monitoring (QCM-D) to examine the binding affinity of surface immobilized human serum albumin (HSA) to hemin after the HSA was pre-incubated with glucose or glyoxal. We found it necessary to pre-expose the HSA functionalized crystal surface to hemin to block irreversible unintended interactions. Glycation with glucose showed little affect on HSA’s affinity for hemin, however, modification with glyoxal showed diminished hemin binding capacity. Despite the hemin-blocking step, we were unable to obtain Kd values consistent with those in literature, which we attribute to other unaccounted for nonspecific interactions. This study highlights the need for a kinetic QCM-D analysis method that accounts for unintended interactions at the sensor surface so that the hemin-blocking step may be eliminated.

Ferriprotoporphyrin IX-Combretum imberbe crude extracts interactions: Implication for malaria treatment

Leaves from Combretum imberbe traditionally used to treat symptoms of malaria in most parts of Southern Africa were studied for interaction of its crude methanolic, ethyl acetate and hexane extracts with hemin a product of hemoglobin degradation in 40% water-dimethylsulphoxide at pH 9, 7.4 and 5 using a spectrophotometric method. It has been established that hemin is the target of antimalaria quinolines thus, interaction of extracts with hemin may represent a crucial initial screening test to define efficacy. Observations were compared to results of interaction of hemin with quinine and artemisia as standards. The present results indicate that hemin complexed more strongly with methanolic extracts than with ethyl acetate and hexane extracts. The binding constants were pH-dependent. The present results are interesting because the crude extracts share a similar mode of action with quinine and artemisia with an isosbetic point at 352 nm. Methanolic extract showed better affinity for hemin than artemisia with complexation constants (K) of 1.011 × 104 and 0.612 × 104 M, respectively. However, quinine showed better affinity than methanolic extract with K of 1.101 × 104 M. Key words: Ferriprotoporphyrin IX, Combretum imberbe, crude extracts, hemin.

Effects of hemopexin on hemin and hemoglobin-mediated lipid oxidation in washed fish muscle

Hemopexin (Hx) was isolated from pig blood using hemin-agarose chromatography. The effect of addition of Hx on hemin and hemoglobin (Hb) mediated lipid oxidation in washed cod muscle was investigated during iced storage at pH 5.5. At a 1:1 ratio of Hx to hemin, lipid oxidation as measured by development of thiobarbituric acid reactive substances (TBARS) was not significantly inhibited (p = 0.12). This may be attributed to a lack of hemin binding by Hx due to influence of pH and hemin precipitation. At a 1:2 ratio of Hx to Hb on a heme basis, TBARS development was significantly inhibited (p < 0.01) but not prevented. Equimolar addition of bovine serum albumin (BSA) in place of Hx did not inhibit TBARS development (p = 0.43). Hemin release from porphyrin-containing Hx, i.e. holoHx, and ferric sperm whale myoglobin (Mb) was measured at 37 °C, pH 5.7. Hemin release rates of 0.27 h−1 and 0.05 h−1, were calculated for holoHx and Mb, respectively. While the hemin affinity of Hx was greater than published values for trout Hb, the relatively low value measured for Hx compared to Mb may be caused by a combination of low pH and the absence of NaCl in the assays.

Delineation of the Pasteurellaceae-specific GbpA-family of glutathione-binding proteins

BackgroundThe Gram-negative bacterium Haemophilus influenzae is a glutathione auxotroph and acquires the redox-active tripeptide by import. The dedicated glutathione transporter belongs to the ATP-binding cassette (ABC)-transporter superfamily and displays more than 60% overall sequence identity with the well-studied dipeptide (Dpp) permease of Escherichia coli. The solute binding protein (SBP) that mediates glutathione transport in H. influenzae is a lipoprotein termed GbpA and is 54% identical to E. coli DppA, a well-studied member of family 5 SBP's. The discovery linking GbpA to glutathione import came rather unexpectedly as this import-priming SBP was previously annotated as a heme-binding protein (HbpA), and was thought to mediate heme acquisition. Nonetheless, although many SBP's have been implicated in more than one function, a prominent physiological role for GbpA and its partner permease in heme acquisition appears to be very unlikely. Here, we sought to characterize five representative GbpA homologs in an effort to delineate the novel GbpA-family of glutathione-specific family 5 SBPs and to further clarify their functional role in terms of ligand preferences.ResultsLipoprotein and non-lipoprotein GbpA homologs were expressed in soluble form and substrate specificity was evaluated via a number of ligand binding assays. A physiologically insignificant affinity for hemin was observed for all five GbpA homologous test proteins. Three out of five test proteins were found to bind glutathione and some of its physiologically relevant derivatives with low- or submicromolar affinity. None of the tested SBP family 5 allocrites interacted with the remaining two GbpA test proteins. Structure-based sequence alignments and phylogenetic analysis show that the two binding-inert GbpA homologs clearly form a separate phylogenetic cluster. To elucidate a structure-function rationale for this phylogenetic differentiation, we determined the crystal structure of one of the GbpA family outliers from H. parasuis. Comparisons thereof with the previously determined structure of GbpA in complex with oxidized glutathione reveals the structural basis for the lack of allocrite binding capacity, thereby explaining the outlier behavior.ConclusionsTaken together, our studies provide for the first time a collective functional look on a novel, Pasteurellaceae-specific, SBP subfamily of glutathione binding proteins, which we now term GbpA proteins. Our studies strongly implicate GbpA family SBPs in the priming step of ABC-transporter-mediated translocation of useful forms of glutathione across the inner membrane, and rule out a general role for GbpA proteins in heme acquisition.

Role of Heme in the Unfolding and Assembly of Myoglobin

The unfolding of wild-type holomyoglobin in the ferric state (metMb) appears to be a simple two-state process, even though hemichrome spectra are often observed and apoMb denaturation involves an intermediate. To resolve these discrepancies, we measured GuHCl-induced, equilibrium unfolding of five sperm whale metMb variants, which were selected to examine the relative importance of apoglobin stability and hemin affinity. Combined analysis of CD, Trp fluorescence, and Soret absorbance titration curves for all the variants requires a six-state mechanism containing native (N), intermediate (I), and unfolded (U) states of apoMb and their hemin-bound counterparts, NH (holoMb), IH, and UH, respectively. The unfolding parameters for the apoMbs were obtained in independent experiments and then fixed in the analysis of the holoprotein data, where only the affinities of the apoglobin states for hemin were allowed to vary. This cofactor binding analysis applies generally to all globins and led to three specific conclusions. (1) The stability of holo-metMb is determined primarily by the high affinity (K(d) approximately 10(-13) M) of native apoMb (N) for hemin. (2) The partially unfolded intermediate with hemin bound (IH) has a hemichrome spectrum indicative of a bis-histidyl axial coordination and is seen clearly when the stability of the N state or its affinity for hemin is reduced. (3) Although the affinity of the intermediate for hemin (K(d) approximately 10(-11) M) is approximately 100-fold lower than that for the native state, free hemin can bind to it and promote the assembly of the holoprotein.

The 2-Cys Peroxiredoxin Alkyl Hydroperoxide Reductase C Binds Heme and Participates in Its Intracellular Availability in Streptococcus agalactiae

Heme is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular heme partners were identified to date and are not well conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (group B Streptococcus) is a heme auxotroph, which acquires exogenous heme to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a heme-binding protein. AhpC binds hemin with a K(d) of 0.5 microm and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo. A group B Streptococcus ahpC mutant displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in heme intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a heme-binding protein.

General Mechanisms for the Folding and Assembly of Myoglobins and Hemoglobins

Mammalian myoglobin has served as the archetype globin for understanding the folding properties of single domain globins with the 3 on 3 helical fold. After removal of heme, the resultant apo-Mb shows a loss of structure in the proximal F helix and adjacent loops, and during acid or GdmCl-induced denaturation, apo-Mb populates at least one intermediate. In contrast, unfolding of holo-Mb appears to be a simple two-state process with little protein concentration dependence but the underlying mechanism is much more complex. The lack of protein concentration dependence implies that heme either interacts with the unfolded polypeptide, self-associates, or both. The observed steepness of the unfolding curves for holo met-Mb requires that the affinity of hemin for the intermediate and completely unfolded states must be at least be 1000 fold weaker than that for the native apo-state, and as a result, unfolding of holo met-Mb is governed primarily by the affinity of the folded native apo-state for hemin. The generality of this conclusion for holo-Mb has been tested in several other monomeric hemoglobins, including the miniglobin from Cerebratulus lacteus and the thermoglobin from Aquifex aeolicus.Human hemoglobin unfolding is even more complex due to association of the α and β subunits into dimers and tetramers. Removal of hemin leads to formation of an apo-α1β1 dimer and its unfolding appears to involve an intermediate whose stability is dependent on protein concentration. This dependence suggests the formation of a dimer intermediate with partially folded subunits still attached to each other through the α1β1 interface. Folding and assembly of holo-Hb is even more complex because there are significant differences in hemin affinity between the α and β subunits, and between tetramers, dimers and monomers.

Phenylalanine Substitution at Site B10 (L29F) Inhibits Metmyoglobin Formation and Myoglobin-Mediated Lipid Oxidation in Washed Fish Muscle: Mechanistic Implications

Wild-type sperm whale myoglobin (WT Mb) and L29F mutant were used to examine the effect of metMb formation rate on Mb-mediated lipid oxidation in washed cod muscle. MetMb formation was 15-fold slower in L29F compared to WT Mb at 2 degrees C (pH 5.7). The electrostatic interaction of bound O(2) and the partial positive edge of the phenyl ring of phenylalanine(29) inhibits deoxyMb-mediated metMb formation and may displace protons that promote oxyMb oxidation. Ferrous L29F was a poor promoter of lipid oxidation in washed cod during extended storage, whereas ferrous WT Mb oxidized the substrate readily. This diminishes a role for free radicals produced by the reaction of oxyMb with peroxides. MetL29F was an effective promoter of lipid oxidation. Mb mutants with low hemin affinity (H97A) were better promoters of microsomal lipid oxidation than mutants with higher hemin affinity (WT Mb and V68T). The much higher heme affinity of oxyMb compared to metMb partly explains the poor ability of ferrous L29F to oxidize lipids in washed cod at post-mortem pH during 2 degrees C storage. The relative roles of cross-linked Mb and hypervalent Mb species to promote lipid oxidation in washed cod at post-mortem pH are discussed.

Direct Hemin Transfer from IsdA to IsdC in the Iron-regulated Surface Determinant (Isd) Heme Acquisition System of Staphylococcus aureus

The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 +/- 5 at 22 degrees C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 +/- 1.8 s(-1) at 25 degrees C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holo-IsdA into solvent (k transfer = 54.3 s(-1) versus k -hemin = 0.00076 s(-1)). The standard free energy change, Delta G 0, is -27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holo-IsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems.