Heme Transfer Research Articles

Ligand Induced Allostery in Pseudomonas Aeruginosa Cytoplasmic Heme Binding Protein (Phus) Drives the Protein-Protein Interaction with Heme Oxygenase

Integral to its essential role in the metabolism of extracellular heme, Pseudomonas Aeruginosa's Phus binds incoming heme and tranfers it to the iron-regulated heme oxygenase (HemO). Previous studies have shown that heme binding induces conformational changes required to interact with HemO. Here, we used mass spectrometry and spectroscopic techniques to probe heme binding, characterize the heme-induced conformational changes in Phus and map the holo-Phus-HemO interaction. Hydrogen-Deuterium exchange (HDX-MS) of apo- and holo-PhuS reveals that heme binding results in significant rearrangements of the heme-binding pocket. Notably, C-terminal proximal helices α6/α7/α8 (residues 198-233) are highly labile in the apo-Phus and become largely protected from exchange upon heme binding only to subsequently undergo EX1-type cooperative unfolding within 1hr. Also, N-terminal α1/α2 helices (residues 1-36) undergo cooperative unfolding in apo-Phus which is significantly slowed in holo-Phus. Remarkably, mapping the interface of the holo-PhuS-HemO complex using chemical crosslinking and MALDI-TOF-MS revealed that these same regions, the α7/α8 helices (residues 217-236) and α1 helix (residues 1-29) contact HemO. Together, HDX-MS and crosslinking data is consistent with a heme-binding induced conformational rearrangement of the C-terminal domain driving the initial PhuS-HemO interaction. A binding-competent yet transfer-incompetent mutant, H212R-Phus was used to further interrogate the conformational changes of Phus in response to heme binding. Spectroscopic studies show H212R-Phus binds heme with approximately 2-fold faster kinetics than wt-Phus. In accordance, HDX-MS shows, in the apo form, the C-terminal binding pocket helices α6/α7/α8 of H212R-Phus are slightly but significantly more protected from exchange than wt-Phus. In holo-H212R-Phus, whereas the large conformational rearrangements in the heme binding pocket is still observable, the cooperative unfolding of the C-terminal and N-terminal helices is largely abolished, suggesting a possible role for such cooperative unfolding in heme transfer.

Insight into a Rapid Heme Transfer Reaction between Near Transporter Domains of Staphylococcus Aureus: A Theoretical Study using QM/MM and MD Simulations

Iron is versatile in biological systems and required cofactor in the growth and activity of all living organisms. Some gram-positive pathogens such as Staphylococcus aureus have evolved an iron-regulated surface determinant (Isd) system across its thick cell wall to steal iron from human hemoglobin and heme. Recent studies have demonstrated that heme extracted from hemoglobin is rapidly transferred into cytoplasm through NEAr Transporter (NEAT) domain of the Isd system, despite the high affinity for each domain. These observations suggest the presence of an intermediate NEAT•heme•NEAT complex. In this study, we performed restrained molecular dynamics (MD) simulations to dock the acceptor NEAT domains to the donor NEAT•heme complexes and obtained models where the two NEAT domains were arranged with two-fold pseudo symmetry around the heme molecule. The overall structures of the complex models were stably maintained during subsequent more than 900-ns unrestrained simulations. Although the hydrogen bond between a propionate group of heme and the acceptor domain was completely maintained, the hydrogen bond between the other propionate group of heme and the donor domain was broken, which may facilitate the transition of heme from the donor to the acceptor. Moreover, subsequent structural optimization using the QM/MM method showed that the two tyrosine residues, one from each NEAT domain, were simultaneously coordinated to the ferric heme iron in the intermediate complex, only if they are deprotonated. Therefore, the heme-transfer reaction proceeds by protonation on the coordinating tyrosine residue. Thus, these theoretical studies provide an atomistic insight into the intermediate structure allowing rapid heme transfer between the NEAT domains.

Heme-Hemopexin Scavenging Is Active in the Brain and Associates With Outcome After Subarachnoid Hemorrhage.

Long-term outcome after subarachnoid hemorrhage (SAH) is potentially linked to cytotoxic heme. Free heme is bound by hemopexin and rapidly scavenged by CD91. We hypothesized that heme scavenging in the brain would be associated with outcome after hemorrhage. Using cerebrospinal fluid and tissue from patients with SAH and control individuals, the activity of the intracranial CD91-hemopexin system was examined using ELISA, ultrahigh performance liquid chromatography, and immunohistochemistry. In control individuals, cerebrospinal fluid hemopexin was mainly synthesized intrathecally. After SAH, cerebrospinal fluid hemopexin was high in one third of cases, and these patients had a higher probability of delayed cerebral ischemia and poorer neurological outcome. The intracranial CD91-hemopexin system was active after SAH because CD91 positively correlated with iron deposition in brain tissue. Heme-hemopexin uptake saturated rapidly after SAH because bound heme accumulated early in the cerebrospinal fluid. When the blood-brain barrier was compromised after SAH, serum hemopexin level was lower, suggesting heme transfer to the circulation for peripheral CD91 scavenging. The CD91-heme-hemopexin scavenging system is important after SAH and merits further study as a potential prognostic marker and therapeutic target.

Rapid Heme Transfer Reactions between NEAr Transporter Domains of Staphylococcus aureus: A Theoretical Study Using QM/MM and MD Simulations.

In vertebrates, most iron is present as heme or is chelated by proteins. Thus, Gram-positive pathogens such as Staphylococcus aureus have evolved an iron-regulated surface determinant (Isd) system that transports heme across thick cell walls into the cytoplasm. Recent studies have demonstrated that heme is rapidly transferred between the NEAr Transporter (NEAT) domains of the Isd system, despite its high affinity toward each domain, suggesting the presence of an intermediate NEAT•heme•NEAT complex. In the present study, we performed short restrained molecular dynamics (MD) simulations to dock the acceptor NEAT domain to the donor NEAT•heme complex and obtained models where the two NEAT domains were arranged with two-fold pseudo symmetry around the heme molecule. After turning off the restraints, complex structures were stably maintained during subsequent unrestrained MD simulations, except for the hydrogen bond between the propionate group of the heme molecule and the donor NEAT domain, potentially facilitating the transition of heme from the donor to the acceptor. Subsequent structural optimization using the quantum mechanics/molecular mechanics (QM/MM) method showed that two tyrosine residues, one from each NEAT domain, were simultaneously coordinated to the ferric heme iron in the intermediate complex only if they were deprotonated. Based on these results, we propose a reaction scheme for heme transfer between NEAT domains.

Different target specificities of haptoglobin and hemopexin define a sequential protection system against vascular hemoglobin toxicity

Free hemoglobin (Hb) triggered vascular damage occurs in many hemolytic diseases, such as sickle cell disease, with an unmet need for specific therapeutic interventions. Based on clinical observations the Hb and heme scavenger proteins haptoglobin (Hp) and hemopexin (Hx) have been characterized as a sequential defense system with Hp as the primary protector and Hx as a backup when all Hp is depleted during more severe intravascular hemolysis. In this study we present a mechanistic rationale for this paradigm based on a combined biochemical and cell biological approach directed at understanding the unique roles of Hp and Hx in Hb detoxification. Using a novel in vitro model of Hb triggered endothelial damage, which recapitulates the well-characterized pathophysiologic sequence of oxyHb(Fe2+) transformation to ferric Hb(Fe3+), free heme transfer from ferric Hb(Fe3+) to lipoprotein and subsequent oxidative reactions in the lipophilic phase. The accumulation of toxic lipid peroxidation products liberated during oxidation reactions ultimately lead to endothelial damage characterized by a specific gene expression pattern with reduced cellular ATP and monolayer disintegration. Quantitative analysis of key chemical and biological parameters allowed us to precisely define the mechanisms and concentrations required for Hp and Hx to prevent this toxicity. In the case of Hp we defined an exponential relationship between Hp availability relative to oxyHb(Fe2+) and related protective activity. This exponential relationship demonstrates that large Hp quantities are required to prevent Hb toxicity. In contrast, the linear relationship between Hx concentration and protection defines a highly efficient backup scavenger system during conditions of large excess of free oxyHb(Fe2+) that occurs when all Hp is consumed. The diverse protective function of Hp and Hx in this model can be explained by the different target specificities of the two proteins.

Heme-albumin: an honorary enzyme.

Human serum albumin displays time-dependent heme-based catalytic properties,1 representing a case for ‘chronosteric effects'.2 In fact, HSA has a pivotal role in heme transfer from high- and low-density lipoproteins to hemopexin. After endocytosis of the hemopexin-heme complex into the hepatic parenchymal cells through the CD91 receptor, hemopexin releases the heme, which undergoes degradation. Then, hemopexin is released intact into the bloodstream.3, 4 The three-domain organization of HSA is at the root of its extraordinary ligand-binding capacity and allosteric control. The most relevant clefts hosting ligands are the so-called fatty acid (FA) binding sites (named FA1 to FA9). Bacterial protein-recognition cleft(s), thyroxine-binding pockets and metal ion-recognition sites also participate to HSA actions.4 Noteworthy, the HSA structure and reactivity is affected not only reversibly by pH and ligands (e.g., heme, FAs and drugs), but also irreversibly by chemical modifications, which in turn confer antigenicity properties.4 Ferrous human serum heme-albumin (HSA-heme-Fe(II)) binds reversibly to NO and CO. Although the heme-Fe atom of HSA-heme-Fe(II) is rapidly oxidized by O2, HSA-heme-Fe(II) mutants bearing residues pivotal for O2 recognition have been proposed not only as red blood cell substitutes, but also as O2-therapeutic agents.1, 4, 5 Moreover, HSA-heme-Fe(II) catalyzes the nitrite conversion to nitrogen monoxide under acidosis and anaerobic conditions,6 HSA-heme-Fe(II)-NO reacts with O2 and peroxynitryte leading to the formation of NO3−,7, 8 and ferric HSA-heme-Fe (HSA-heme-Fe(III)) catalyzes the conversion of peroxynitryte to NO3−,9 and displays weak catalase and peroxidase activities.10 The heme-based catalytic properties of HSA are allosterically modulated by drugs (Figure 1).1 Domains I and II have a major role in the allosteric modulation of ligand-binding and reactivity properties of HSA, the FA1, FA2, FA6 and FA7 sites being functionally linked. Allosteric modulators (e.g., drugs) of the heme-based catalytic properties of HSA-heme affect the coordination state of the heme-Fe atom. In ligand-free active HSA-heme, the heme-Fe atom displays a four- or five-coordinated heme-Fe atom, whereas inactive HSA-heme shows a six-coordinated heme-Fe atom. Upon drug binding to HSA-heme (most probably to the FA2 site), the re-orientation of the Glu131-Arg145 α-helix and the axial coordination of the heme-Fe atom by His146 and Tyr161 occur; as a consequence, the unreactive six-coordinated HSA-heme species becomes predominant.1, 11, 12 Figure 1 Human serum albumin displays time-dependent heme-based catalytic properties, which are allosterically modulated by drugs As a whole, the allosteric modulation of heme-based reactivity properties of HSA-heme by drugs represents a pivotal issue in the pharmacological therapy management, heme-binding switching HSA from a plasmatic carrier to a transient metal-enzyme.

Alzheimer's Disease: A Heme-Aβ Perspective.

Redox active iron is utilized in biology for various electron transfer and catalytic reactions essential for life, yet this same chemistry mediates the formation of partially reduced oxygen species (PROS). Oxidative stress derived from the iron accumulated in the amyloid plaques originating from amyloid β (Aβ) peptides and neurofibrillary tangles derived from hyperphosphorylated tau proteins has been implicated in the pathogenesis of Alzheimer's disease (AD). Altered heme homeostasis leading to dysregulation of expression of heme proteins and heme deposits in the amyloid plaques are characteristic of the AD brain. However, the pathogenic significance of heme in neurodegeneration in AD has been unappreciated due to the lack of detailed understanding of the chemistry of the interaction of heme and Aβ peptides. As a result, the biochemistry and biophysics of heme complexes of Aβ peptides (heme-Aβ) remained largely unexplored. In this Account, we discuss the active site environment of heme bound Aβ complexes, which involves three amino acid residues unique in mammalian Aβ (Arg5, Tyr10, and His13) and missing in Aβ from rodents, which do not get affected by AD. The histidine residue binds heme, while the arginine and the tyrosine act as key second sphere residues of the heme-Aβ active site that play a crucial role in its reactivity. Generation of PROS, enhanced peroxidase activity, and oxidation of neurotransmitters such as serotonin (5-HT) are all found to be catalyzed by heme-Aβ in in vitro assays, and these reactivities can potentially be linked to the observed neuropathologies in AD brain. Association of Cu with heme-Aβ leads to the formation of heme-Cu-Aβ. The heme-Cu-Aβ complex produces a greater amount of PROS than reduced heme-Aβ or Cu-Aβ alone. Nitric oxide (NO), a signaling molecule, is found to ameliorate the detrimental effects of heme-Aβ and Cu bound heme-Aβ complexes by detaching heme from the heme-Aβ complex and releasing it into the environment solution. Heme-Aβ complexes show fast electron transfer with oxidized cytochrome c and rapid heme transfer with apomyoglobin and aponeuroglobin. NO, cytochrome c, and apoglobins can all lead to reduction in PROS generated by reduced heme-Aβ. Synthetic analogues of heme, offering a hydrophobic distal environment, have been used to trap oxygen bound intermediates, which provides insight into the mechanism of PROS generation by reduced heme-Aβ. Artificial constructs of Aβ on nonbiological platforms are used not only to stabilize metastable and physiologically relevant large and small amyloid aggregates but also to monitor the interaction of various drug candidates with heme and Cu bound Aβ aggregates, representing a tractable avenue for testing therapeutic agents targeting metals and cofactors in AD.

The crystal structure of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt): Roles of the axial ligand Tyr75 and two distal arginines in heme binding

Some Gram-negative pathogens utilize an extracellular heme-binding protein called hemophore to satisfy their needs for iron, a metal element essential for most living things. We report here crystal structures of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt) and its Y75A mutant. The wild-type HasAypt structure revealed that the heme iron is coordinated with Tyr75 and a water molecule. The heme-bound water molecule makes extensive hydrogen bond network that includes Arg40 and Arg144 on the distal heme pocket. Arg40, highly conserved for HasAs from Yersinia species, forms a salt bridge with the propionate side chain of heme, and makes π–π stacking and hydrophobic interactions with porphyrin plane. Interestingly, similar Arg–heme interactions are also found for periplasmic heme transporter, PhuT, suggesting that this is an example of a convergent evolution and one of the important interactions for bacterial heme transportation. Heme titration, heme binding kinetics, and the crystal structures of wild-type and Y75A proteins show that, although Tyr75 is primarily important for heme capturing, other interactions with Arg40, Arg144, and hydrophobic residues also contribute for heme acquisition. We also found that HasAypt can form a dimer in solution. The structure of the domain-swapped Y75A HasAypt dimer shows the presence of two low-spin heme molecules coordinated with His84 and His140, and displacement of the Arg40 loop of dimeric Y75A HasAypt results in deformation of the heme-binding pocket. A similar rearrangement of the distal heme loop might occur in heme transfer from HasAypt to HasRypt.

Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease

AbstractIntravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD.

A Genetically Encoded FRET Sensor for Intracellular Heme.

Heme plays pivotal roles in various cellular processes as well as in iron homeostasis in living systems. Here, we report a genetically encoded fluorescence resonance energy transfer (FRET) sensor for selective heme imaging by employing a pair of bacterial heme transfer chaperones as the sensory components. This heme-specific probe allows spatial-temporal visualization of intracellular heme distribution within living cells.

Bacterial Cytochrome c Peroxidases: Insight into the Structure‐Function Relationship

Understanding how a sequence of amino acids serves as a functional, catalytic unit is a fundamental question in enzymology. The Elliott group studies the structure‐function relationship using bacterial cytochrome c peroxidases (bCCPs) as a model system for probing the interplay between structure, redox chemistry, and catalysis. bCCPs are diheme periplasmic enzymes that reduce hydrogen peroxide to water, requiring two c‐type heme cofactors and two electrons from the cytochrome c pool. Peroxide detoxification is an essential bacterial defense mechanism. A hallmark of bCCPs lies in the potentials of their hemes: a low potential peroxidatic heme (FeL) and a high potential electron transfer heme (FeH). Interestingly, the bCCPs from Shewanella oneidensis (SoCCP) and Nitrosomonas europaea (NeCCP) feature a high sequence identity (60%), yet have key catalytic differences: SoCCP requires reductive activation of FeH, where NeCCP does not. Further, the reduction potentials governing respective FeH are very different: where SoCCP (+245 mV vs NHE) is average for bCCPs, and NeCCP (>400 mV vs NHE) is unusually high. Previous experiments in the Elliott group have found that point mutants in one heme environment can globally affect the redox properties. Specifically, mutating a key glutamate to lysine at FeL, which is expected to directly hinder activity, has unexpectedly altered the reduction potentials of FeH as well as FeL. The high sequence and structural similarities, yet differences in electrochemical properties, present a robust model to probe the influence of nearby residues on redox potentials and to understand bacterial defense mechanisms against damaging oxygen species.

Interaction of apoNeuroglobin with heme-Aβ complexes relevant to Alzheimer's disease.

Heme-Aβ complexes are known to produce toxic partially reduced oxygen species (PROS), catalyze oxidation of neurotransmitters and have been associated with Alzheimer's disease (AD). Neuroglobin (Ngb) play a crucial neuroprotective role against oxidative damage, hypoxic injuries, stroke and apoptosis of neuronal cells. In this study, the interaction of heme-Aβ with apoNeuroglobin (apoNgb) has been investigated using a combination of spectroscopic techniques. Absorption and resonance Raman data confirm that apoNgb can uptake heme from heme-Aβ and constitute a six-coordinate low-spin ferric heme-active site identical to that of Ngb. ApoNgb can also uptake heme from reduced heme-Aβ resulting in the formation of ferrous Ngb. The rate of the heme transfer reaction has been found to be of the order of 10(6) M(-1) s(-1). The reaction is faster for oxidized heme-Aβ than the reduced form. The amount of PROS formation by heme-Aβ complexes has been found to diminish drastically after reaction with apoNgb. ApoNgb can also sequester ligand-bound heme from heme-Aβ, e.g., the CO-bound heme from heme-Aβ-CO complex resulting in the formation of Ngb-CO complex. Additionally, ApoNgb can sequester heme from self-assembled monolayer (SAM) of surface-bound heme-Aβ formed over Au surface. This heme sequestration by apoNgb from heme-Aβ not only diminishes heme-induced toxicity but more significantly it produces Ngb which has well-documented neuroprotective role and can thereby potentially reduce risks associated with AD.

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification.

Correlation of heme binding affinity and enzyme kinetics of dehaloperoxidase.

Chemical and thermal denaturation of dehaloperoxidase-hemoglobin (DHP) was investigated to test the relative stability of isoforms DHP A and DHP B and the H55V mutant of DHP A with respect to heme loss. In thermal denaturation experiments, heme loss was observed at temperatures of 54, 46, and 61 °C in DHP A, DHP B, and H55V, respectively. Guanidinium hydrochloride (GdnHCl)- and urea-induced denaturation was observed at respective concentrations of 1.15 ± 0.01 M DHP A and 1.09 ± 0.02 M DHP B, and 5.19 ± 0.05 M DHP A and 4.12 ± 0.14 M DHP B, respectively. The binding affinity of heme appears to be significantly smaller in both isoforms of DHP than in myoglobins. This observation was corroborated by heme transfer experiments, in which heme was observed to transfer for DHP A and B to horse skeletal muscle myoglobin (HSMb). GdnHCl-induced denaturation suggests a threshold of 1 mM for stabilization by binding of the inhibitor 4-bromophenol (4-BP). Concentrations of 4-BP greater than 1 mM caused destabilization. Urea-induced denaturation showed only destabilizing effects from phenolic ligand binding. Heme transfer experiments from DHP to HSMb further support the hypothesis that the binding of halophenols to DHP facilitates the removal of the heme. Thermal denaturation assessed via UV-visible spectroscopy and that assessed by differential scanning calorimetry (DSC) are both in agreement with chemical denaturation experiments and show that the denaturing abilities of the halophenols improve with the size of the para halogen atom in 4-XP, where X = iodo, bromo, chloro, or fluoro (4-IP > 4-BP > 4-CP > 4-FP), and the number of halo substituents as in 2,4,6-tribromophenol (2,4,6-TBP > 4-BP). DHP B, which differs in five amino acids, is less stable than DHP A with ΔHcal and Tm values of 165.1 kJ/mol and 47.5 °C compared to values of 183.3 kJ/mol and 50.4 °C for DHP B and DHP A, respectively. Kinetic studies verified that DHP B has a catalytic efficiency (kcat/Km) ∼5-6 times greater than that of DHP A but showed an increased level of substrate inhibition in DHP B for both 2,4,6-TCP and 2,4,6-TBP. An inverse correlation between protein stability with respect to heme loss and catalytic efficiency is suggested on the basis of the fact that the heme in DHP B has a stability lower than that of DHP A but a catalytic efficiency higher than that of DHP A.

A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin.

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7−/− mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.

Can gas replace protein function? CO abrogates the oxidative toxicity of myoglobin.

Outside their cellular environments, hemoglobin (Hb) and myoglobin (Mb) are known to wreak oxidative damage. Using haptoglobin (Hp) and hemopexin (Hx) the body defends itself against cell-free Hb, yet mechanisms of protection against oxidative harm from Mb are unclear. Mb may be implicated in oxidative damage both within the myocyte and in circulation following rhabdomyolysis. Data from the literature correlate rhabdomyolysis with the induction of Heme Oxygenase-1 (HO-1), suggesting that either the enzyme or its reaction products are involved in oxidative protection. We hypothesized that carbon monoxide (CO), a product, might attenuate Mb damage, especially since CO is a specific ligand for heme iron. Low density lipoprotein (LDL) was chosen as a substrate in circulation and myosin (My) as a myocyte component. Using oxidation targets, LDL and My, the study compared the antioxidant potential of CO in Mb-mediated oxidation with the antioxidant potential of Hp in Hb-mediated oxidation. The main cause of LDL oxidation by Hb was found to be hemin which readily transfers from Hb to LDL. Hp prevented heme transfer by sequestering hemin within the Hp-Hb complex. Hemin barely transferred from Mb to LDL, and oxidation appeared to stem from heme iron redox in the intact Mb. My underwent oxidative crosslinking by Mb both in air and under N2. These reactions were fully arrested by CO. The data are interpreted to suit several circumstances, some physiological, such as high muscle activity, and some pathological, such as rhabdomyolysis, ischemia/reperfusion and skeletal muscle disuse atrophy. It appear that CO from HO-1 attenuates damage by temporarily binding to deoxy-Mb, until free oxygen exchanges with CO to restore the equilibrium.

Non-heme-binding domains and segments of the Staphylococcus aureus IsdB protein critically contribute to the kinetics and equilibrium of heme acquisition from methemoglobin.

The hemoglobin receptor IsdB rapidly acquires heme from methemoglobin (metHb) in the heme acquisition pathway of Staphylococcus aureus. IsdB consists of N-terminal segment (NS), NEAT1 (N1), middle (MD), and heme binding NEAT2 (N2) domains, and C-terminal segment (CS). This study aims to elucidate the roles of these domains or segments in the metHb/IsdB reaction. Deletion of CS does not alter the kinetics and equilibrium of the reaction. Sequential deletions of NS and N1 in NS-N1-MD-N2 progressively reduce heme transfer rates and change the kinetic pattern from one to two phases, but have no effect on the equilibrium of the heme transfer reaction, whereas further deletion of MD reduces the percentage of transferred metHb heme. MD-N2 has higher affinity for heme than N2. MD in trans reduces rates of heme dissociation from holo-N2 and increases the percentage of metHb heme captured by N2 by 4.5 fold. NS-N1-MD and N2, but not NS-N1, MD, and N2, reconstitute the rapid metHb/IsdB reaction. NS-N1-MD-NIsdC, a fusion protein of NS-N1-MD and the NEAT domain of IsdC, slowly acquires heme from metHb by itself but together with N2 results in rapid heme loss from metHb. Thus, NS-N1 and MD domains specifically and critically contribute to the kinetics and equilibrium of the metHb/IsdB reaction, respectively. These findings support a mechanism of direct heme acquisition by IsdB in which MD enhances the affinity of N2 for heme to thermodynamically drive heme transfer from metHb to IsdB and in which NS is required for the rapid and single phase kinetics of the metHb/IsdB reaction.

Solution structure and molecular determinants of hemoglobin binding of the first NEAT domain of IsdB in Staphylococcus aureus.

The human pathogen Staphylococcus aureus acquires heme iron from hemoglobin (Hb) via the action of a series of iron-regulated surface determinant (Isd) proteins. The cell wall anchored IsdB protein is recognized as the predominant Hb receptor, and is comprised of two NEAr transporter (NEAT) domains that act in concert to bind, extract, and transfer heme from Hb to downstream Isd proteins. Structural details of the NEAT 2 domain of IsdB have been investigated, but the molecular coordination between NEAT 2 and NEAT 1 to extract heme from hemoglobin has yet to be characterized. To obtain a more complete understanding of IsdB structure and function, we have solved the 3D solution structure of the NEAT 1 domain of IsdB (IsdBN1) spanning residues 125–272 of the full-length protein by NMR. The structure reveals a canonical NEAT domain fold and has particular structural similarity to the NEAT 1 and NEAT 2 domains of IsdH, which also interact with Hb. IsdBN1 is also comprised of a short N-terminal helix, which has not been previously observed in other NEAT domain structures. Interestingly, the Hb binding region (loop 2 of IsdBN1) is disordered in solution. Analysis of Hb binding demonstrates that IsdBN1 can bind metHb weakly and the affinity of this interaction is further increased by the presence of IsdB linker domain. IsdBN1 loop 2 variants reveal that phenylalanine 164 (F164) of IsdB is necessary for Hb binding and rapid heme transfer from metHb to IsdB. Together, these findings provide a structural role for IsdBN1 in enhancing the rate of extraction of metHb heme by the IsdB NEAT 2 domain.

Investigations of the low frequency modes of ferric cytochrome c using vibrational coherence spectroscopy.

Femtosecond vibrational coherence spectroscopy is used to investigate the low frequency vibrational dynamics of the electron transfer heme protein, cytochrome c (cyt c). The vibrational coherence spectra of ferric cyt c have been measured as a function of excitation wavelength within the Soret band. Vibrational coherence spectra obtained with excitation between 412 and 421 nm display a strong mode at ∼44 cm–1 that has been assigned to have a significant contribution from heme ruffling motion in the electronic ground state. This assignment is based partially on the presence of a large heme ruffling distortion in the normal coordinate structural decomposition (NSD) analysis of the X-ray crystal structures. When the excitation wavelength is moved into the ∼421–435 nm region, the transient absorption increases along with the relative intensity of two modes near ∼55 and 30 cm–1. The intensity of the mode near 44 cm–1 appears to minimize in this region and then recover (but with an opposite phase compared to the blue excitation) when the laser is tuned to 443 nm. These observations are consistent with the superposition of both ground and excited state coherence in the 421–435 nm region due to the excitation of a weak porphyrin-to-iron charge transfer (CT) state, which has a lifetime long enough to observe vibrational coherence. The mode near 55 cm–1 is suggested to arise from ruffling in a transient CT state that has a less ruffled heme due to its iron d6 configuration.

Spectroscopic studies on HasA from Yersinia pseudotuberculosis

Heme acquisition system A (HasA) is known as a hemophore in Gram-negative pathogens. The ferric heme iron is coordinated by Tyr-75 and His-32 in holo-HasA from Pseudomonas aeruginosa (HasApa). In contrast, in holo-HasA from Yersinia pseudotuberculosis (HasAyp), our spectroscopic studies suggest that only Tyr-75 coordinates to the ferric heme iron. The substitution of Gln-32 with alanine in HasAyp does not alter the spectroscopic properties, indicating that Gln-32 is not an axial ligand for the heme iron. Somewhat surprisingly, the Y75A mutant of HasAyp can capture a free hemin molecule but the rate of hemin uptake is slower than that of wild type, suggesting that the hydrophobic interaction in the heme pocket may also play a role in heme acquisition. Unlike in wild type apoprotein, ferric heme transfer from Hb to Y75A apo-HasAyp has not been observed. These results imply that coordination (bonding/interaction) between Tyr-75 and the heme iron is important for heme transfer from Hb. Interestingly, HasAyp differs from HasApa in its ability to bind the ferrous heme iron. Apo-HasAyp can capture ferrous heme and resonance Raman spectra of ferrous-carbon monoxide holo-HasAyp suggest that Tyr-75 is protonated when the heme iron is in the ferrous state. The ability of HasAyp to acquire the ferrous heme iron might be beneficial to Y. pseudotuberculosis, a facultative anaerobe in the Enterobacteriaceae family.