The surface protein Shr of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> binds heme and transfers it to the streptococcal heme-binding protein Shp

Background  

The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established.     

Kinetics of heme transfer by the Shr NEAT domains of Group A Streptococcus

The hemolytic Group A Streptococcus (GAS) is a notorious human pathogen. Shr protein of GAS participates in iron acquisition by obtaining heme from host hemoglobin and delivering it to the adjacent receptor on the surface, Shp. Heme is then conveyed to the SiaABC proteins for transport across the membrane. Using rapid kinetic studies, we investigated the role of the two heme binding NEAT modules of Shr. Stopped-flow analysis showed that holoNEAT1 quickly delivered heme to apoShp. HoloNEAT2 did not exhibit such activity; only little and slow transfer of heme from NEAT2 to apoShp was seen, suggesting that Shr NEAT domains have distinctive roles in heme transport. HoloNEAT1 also provided heme to apoNEAT2, by a fast and reversible process. To the best of our knowledge this is the first transfer observed between isolated NEAT domains of the same receptor. Sequence alignment revealed that Shr NEAT domains belong to two families of NEAT domains that are conserved in Shr orthologs from several species. Based on the heme transfer kinetics, we propose that Shr proteins modulate heme uptake according to heme availability by a mechanism where NEAT1 facilitates fast heme delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface.     

Bis-methionyl coordination in the crystal structure of the heme-binding domain of the streptococcal cell surface protein Shp

Surface proteins Shr, Shp, and the ATP-binding cassette (ABC) transporter HtsABC are believed to make up the machinery for heme uptake in Streptococcus pyogenes. Shp transfers its heme to HtsA, the lipoprotein component of HtsABC, providing the only experimentally demonstrated example of direct heme transfer from a surface protein to an ABC transporter in Gram-positive bacteria. To understand the structural basis of heme transfer in this system, the heme-binding domain of Shp (Shp¹⁸⁰) was crystallized, and its structure determined to a resolution of 2.1 Å. Shp¹⁸⁰ exhibits an immunoglobulin-like β-sandwich fold that has been recently found in other pathogenic bacterial cell surface heme-binding proteins, suggesting that the mechanisms of heme acquisition are conserved. Shp shows minimal amino acid sequence identity to these heme-binding proteins and the structure of Shp¹⁸⁰ reveals a unique heme-iron coordination with the axial ligands being two methionine residues from the same Shp molecule. A negative electrostatic surface of protein structure surrounding the heme pocket may serve as a docking interface for heme transfer from the more basic outer cell wall heme receptor protein Shr. The crystal structure of Shp¹⁸⁰ reveals two exogenous, weakly bound hemins, which form a large interface between the two Shp¹⁸⁰ molecules in the asymmetric unit. These “extra” hemins form a stacked pair with a structure similar to that observed previously for free hemin dimers in aqueous solution. The propionates of the protein-bound heme coordinate to the iron atoms of the exogenous hemin dimer, contributing to the stability of the protein interface. Gel filtration and analytical ultracentrifugation studies indicate that both full-length Shp and Shp¹⁸⁰ are monomeric in dilute aqueous solution.     

The mechanism of direct heme transfer from the streptococcal cell surface protein Shp to HtsA of the HtsABC transporter

The heme-binding proteins Shp and HtsA are part of the heme acquisition machinery found in Streptococcus pyogenes. The hexacoordinate heme (Fe(II)-protoporphyrin IX) or hemochrome form of holoShp (hemoShp) is stable in air in Tris-HCl buffer, pH 8.0, binds to apoHtsA with a K(d) of 120 +/- 18 microm, and transfers its heme to apoHtsA with a rate constant of 28 +/- 6s(-1) at 25 degrees C, pH 8.0. The hemoHtsA product then autoxidizes to the hexacoordinate hemin (Fe(III)-protoporphyrin IX) or hemichrome form (hemiHtsA) with an apparent rate constant of 0.017 +/- 0.002 s(-1). HemiShp also rapidly transfers hemin to apoHtsA through a hemiShp.apoHtsA complex (K(d) = 48 +/- 7 microM) at a rate approximately 40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent (k(transfer) = 43 +/- 3s(-1) versus k(-hemin) = 0.0003 +/- 0.00006 s(-1)). The rate constants for hemin binding to and dissociation from HtsA (k'(hemin) approximately 80 microm(-1) s(-1), k(-hemin) = 0.0026 +/- 0.0002 s(-1)) are 50- and 10-fold greater than the corresponding rate constants for Shp (k(hemin) approximately 1.6 microM(-1) s(-1), k(-hemin) = 0.0003 s(-1)), which implies that HtsA has a more accessible active site. However, the affinity of apoHtsA for hemin (k(hemin) approximately 31,000 microm(-1)) is roughly 5-fold greater than that of apoShp (k(hemin) approximately 5,300 microM(-1)), accounting for the net transfer from Shp to HstA. These results support a direct, rapid, and affinity-driven mechanism of heme and hemin transfer from the cell surface receptor Shp to the ATP-binding cassette transporter system.     

Pathway for heme uptake from human methemoglobin by the iron-regulated surface determinants system of Staphylococcus aureus

The iron-regulated surface proteins IsdA, IsdB, and IsdC and transporter IsdDEF of Staphylococcus aureus are involved in heme acquisition. To establish an experimental model of heme acquisition by this system, we have investigated hemin transfer between the various couples of human methemoglobin (metHb), IsdA, IsdB, IsdC, and IsdE by spectroscopic and kinetic analyses. The efficiencies of hemin transfer from hemin-containing donors (holo-protein) to different hemin-free acceptors (apo-protein) were examined, and the rates of the transfer reactions were compared with that of indirect loss of hemin from the relevant donor to H64Y/V68F apomyoglobin. The efficiencies, spectral changes, and kinetics of the transfer reactions demonstrate that: 1) metHb directly transfers hemin to apo-IsdB, but not to apo-IsdA, apo-IsdC, and apo-IsdE; 2) holo-IsdB directly transfers hemin to apo-IsdA and apo-IsdC, but not to apo-IsdE; 3) apo-IsdE directly acquires hemin from holo-IsdC, but not from holo-IsdB and holo-IsdA; and 4) IsdB and IsdC enhance hemin transfer from metHb to apo-IsdC and from holo-IsdB to apo-IsdE, respectively. Taken together with our recent finding that holo-IsdA directly transfers its hemin to apo-IsdC, these results provide direct experimental evidence for a model in which IsdB acquires hemin from metHb and transfers it directly or through IsdA to IsdC. Hemin is then relayed to IsdE, the lipoprotein component of the IsdDEF transporter.     

Bis-methionine ligation to heme iron in the streptococcal cell surface protein Shp facilitates rapid hemin transfer to HtsA of the HtsABC transporter

The surface protein Shp of Streptococcus pyogenes rapidly transfers its hemin to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. The structural basis and molecular mechanism of this hemin transfer have been explored by mutagenesis and truncation of Shp. The heme-binding domain of Shp is in the amino-terminal region and is functionally active by itself, although inclusion of the COOH-terminal domain speeds up the process approximately 10-fold. Single alanine replacements of the axial methionine 66 and 153 ligands (Shp(M66A) and Shp(M153A)) cause formation of pentacoordinate hemin-Met complexes. The association equilibrium constants for hemin binding to wild-type, M66A, and M153A Shp are 5,300, 22,000, and 38 microM(-1), respectively, showing that the Met(153)-Fe bond is critical for high affinity binding and that Met(66) destabilizes hemin binding to facilitate its rapid transfer. Shp(M66A) and Shp(M153A) rapidly bind to hemin-free HtsA (apoHtsA), forming stable transfer intermediates. These intermediates appear to be Shp-hemin-HtsA complexes with one axial ligand from each protein and decay to the products with rate constants of 0.4-3 s(-1). Thus, the M66A and M153A replacements alter the kinetic mechanism and unexpectedly slow down hemin transfer by stabilizing the intermediates. These results, in combination with the structure of the Shp heme-binding domain, allow us to propose a "plug-in" mechanism in which side chains from apoHtsA are inserted into the axial positions of hemin in Shp to extract it from the surface protein and pull it into the transporter active site.     

Benfang Lei's research on heme acquisition in Gram-positive pathogens and bacterial pathogenesis

Benfang Lei's laboratory conducts research on pathogenesis of human pathogen Group A Streptococcus (GAS) and horse pathogen Streptococcus equi (S. equi). His current research focuses on heme acquisition in Gram-positive pathogens and molecular mechanism of GAS and S. equi pathogenesis. Heme is an important source of essential iron for bacterial pathogens. Benfang Lei and colleagues identified the first cell surface heme-binding protein in Gram-positive pathogens and the heme acquisition system in GAS, demonstrated direct heme transfer from one protein to another, demonstrated an experimental pathway of heme acquisition by the Staphylococcus aureus Isd system, elucidated the activated heme transfer mechanism, and obtained evidence for a chemical mechanism of direct axial ligand displacement during the Shp-to-HtsA heme transfer reaction. These findings have considerably contributed to the progress that has been made over recent years in understanding the heme acquisition process in Gram-positive pathogens. Pathogenesis of GAS is mediated by an abundance of extracellular proteins, and pathogenic role and functional mechanism are not known for many of these virulence factors. Lei laboratory identified a secreted protein of GAS as a CovRS-regulated virulence factor that is a protective antigen and is critical for GAS spreading in the skin and systemic dissemination. These studies may lead to development of novel strategies to prevent and treat GAS infections.     

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.     

Haptoglobin phenotypes differ in their ability to inhibit heme transfer from hemoglobin to LDL

LDL oxidation plays a pivotal role in atherosclerosis. Excellular hemoglobin (Hb) is a trigger of LDL oxidation. By virtue of its ability to bind hemoglobin, haptoglobin (Hp) serves as an antioxidant. Oxidation of LDL by hemoglobin was analyzed to occur by heme displacement from methemoglobin lodged in LDL. The LDL-associated heme is disintegrated, and iron inserted this way in LDL triggers formation of lipid peroxides. The genetic polymorphism of haptoglobin was found to be a risk factor in the pathogenesis of atherosclerosis. Individuals with Hp2-2 have more vascular incidences as compared to those with Hp1-1. In the current study, oxidation of LDL by metHb was carried out at physiological pH without addition of external peroxides. Hb-derived oxidation of lipids and protein was found to be practically inhibited by Hp1-1 but only partially by Hp2-2. Heme transfer from metHb to LDL was almost completely omitted by Hp1-1 and only partially by Hp2-2. We concluded that partial heme transfer from the Hb-Hp2-2 complex to LDL is the reason for oxidation of LDL lipids as well as protein. These findings provide a molecular basis for Hp2-2 atherogenic properties.     

Shr of group A streptococcus is a new type of composite NEAT protein involved in sequestering haem from methaemoglobin

A growing body of evidence suggests that surface or secreted proteins with NEAr Transporter (NEAT) domains play a central role in haem acquisition and trafficking across the cell envelope of Gram‐positive bacteria. Group A streptococcus (GAS), a β‐haemolytic human pathogen, expresses a NEAT protein, Shr, which binds several haemoproteins and extracellular matrix (ECM) components. Shr is a complex, membrane‐anchored protein, with a unique N‐terminal domain (NTD) and two NEAT domains separated by a central leucine‐rich repeat region. In this study we have carried out an analysis of the functional domains in Shr. We show that Shr obtains haem in solution and furthermore reduces the haem iron; this is the first report of haem reduction by a NEAT protein. More specifically, we demonstrate that both of the constituent NEAT domains of Shr are responsible for binding haem, although they are missing a critical tyrosine residue found in the ligand‐binding pocket of other haem‐binding NEAT domains. Further investigations show that a previously undescribed region within the Shr NTD interacts with methaemoglobin. Shr NEAT domains, however, do not contribute significantly to the binding of methaemoglobin but mediate binding to the ECM components fibronectin and laminin. A protein fragment containing the NTD plus the first NEAT domain was found to be sufficient to sequester haem directly from methaemoglobin. Correlating these in vitro findings to in vivo biological function, mutants analysis establishes the role of Shr in GAS growth with methaemoglobin as a sole source of iron, and indicates that at least one NEAT domain is necessary for the utilization of methaemoglobin. We suggest that Shr is the prototype of a new group of NEAT composite proteins involved in haem uptake found in pyogenic streptococci and Clostridium novyi.     

Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus

In this study, we report experimental results that provide the first complete challenge of a proposed model for heme acquisition by Staphylococcus aureus via the Isd pathway first put forth by Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M., Gornicki, P., Jelenska, J., Joachmiak, A., Missiakas, D. M., and Schneewind, O. (2003) Science 299, 906-909. The heme-binding NEAT domains of Isd proteins IsdA, IsdB (domain 2), IsdC, and HarA/IsdH (domain 3), and the heme-binding IsdE protein, were overexpressed and purified in apo (heme-free) form. Absorption and magnetic circular dichroism spectral data, together with electrospray ionization mass spectrometry were used to unambiguously identify that heme transfers from NEAT-A through NEAT-C to IsdE. Heme transfer was demonstrated to occur in a unidirectional fashion in the sequence NEAT-B2 --> NEAT-A --> NEAT-C --> IsdE or, alternatively, initiating from NEAT-H3 instead of NEAT-B2: NEAT-H3 --> NEAT-A --> NEAT-C --> IsdE. Under the conditions of our experiments, only NEAT-H3 and NEAT-B2 could transfer bidirectionally, which is in the reverse direction as well, and only with each other. Whereas apo-IsdE readily accepted heme from holo-NEAT-C, it would not accept heme from holo-NEAT-A. Heme transfer to IsdE requires the presence of holo-NEAT-C, in agreement with the proposal that IsdC serves as the central conduit of the heme transfer pathway. These experimental findings corroborate the heme transfer model first proposed by the Schneewind group. Our data show that heme transport from the wall-anchored IsdH/IsdB proteins proceeds directly to IsdE at the membrane and, for this to occur, we propose that specific protein-protein interactions must take place.     

Reaction of hydrogen peroxide with ferrylhemoglobin: superoxide production and heme degradation

The reaction of Fe(II) hemoglobin (Hb) but not Fe(III) hemoglobin (metHb) with hydrogen peroxide results in degradation of the heme moiety. The observation that heme degradation was inhibited by compounds, which react with ferrylHb such as sodium sulfide, and peroxidase substrates (ABTS and o-dianisidine), demonstrates that ferrylHb formation is required for heme degradation. A reaction involving hydrogen peroxide and ferrylHb was demonstrated by the finding that heme degradation was inihibited by the addition of catalase which removed hydrogen peroxide even after the maximal level of ferrylHb was reached. The reaction of hydrogen peroxide with ferrylHb to produce heme degradation products was shown by electron paramagnetic resonance to involve the one-electron oxidation of hydrogen peroxide to the oxygen free radical, superoxide. The inhibition by sodium sulfide of both superoxide production and the formation of fluorescent heme degradation products links superoxide production with heme degradation. The inability to produce heme degradation products by the reaction of metHb with hydrogen peroxide was explained by the fact that hydrogen peroxide reacting with oxoferrylHb undergoes a two-electron oxidation, producing oxygen instead of superoxide. This reaction does not produce heme degradation, but is responsible for the catalytic removal of hydrogen peroxide. The rapid consumption of hydrogen peroxide as a result of the metHb formed as an intermediate during the reaction of reduced hemoglobin with hydrogen peroxide was shown to limit the extent of heme degradation.     

A bacterial cytochrome c heme lyase. CcmF forms a complex with the heme chaperone CcmE and CcmH but not with apocytochrome c.

Biogenesis of c-type cytochromes in Escherichia coli involves a number of membrane proteins (CcmA-H), which are required for the transfer of heme to the periplasmically located apocytochrome c. The pathway includes (i) covalent, transient binding of heme to the periplasmic domain of the heme chaperone CcmE; (ii) the subsequent release of heme; and (iii) transfer and covalent attachment of heme to apocytochrome c. Here, we report that CcmF is a key player in the late steps of cytochrome c maturation. We demonstrate that the conserved histidines His-173, His-261, His-303, and His-491 and the tryptophan-rich signature motif of the CcmF protein family are functionally required. Co-immunoprecipitation experiments revealed that CcmF interacts directly with the heme donor CcmE and with CcmH but not with apocytochrome c. We propose that CcmFH forms a bacterial heme lyase complex for the transfer of heme from CcmE to apocytochrome c.     

Tyrosine as a redox-active center in electron transfer to ferryl heme in globins

A wide range of organic reductants, including many iron chelators, reduce ferryl myoglobin to its ferric states in exponential time courses whose rate constants display double hyperbolic dependencies on the reductant concentration. This concentration dependence is consistent with a mechanism in which electron transfer to the heme takes place at two independent sites where reductants appear to bind. We propose that the low-affinity site is located close to the heme edge, within the heme pocket; the maximum rate of electron transfer is highly variable depending on the nature of the reductant (0.005 to >10 s(-1)). The other site has higher apparent affinity (K(D) 0.2-50 microM) but a low maximum rate of electron transfer (0.005 to 0.01 s(-1)). By examining native and engineered proteins we have determined that the high-affinity pathway represents a through-protein electron transfer pathway that involves a specific tyrosine residue. The low apparent rate constant for electron transfer from the tyrosine to the heme (approximately 5 A) is accounted for by proposing that electron transfer occurs only in a very poorly populated protonated state of ferryl heme and tyrosine. Hemoglobin shows similar kinetics but only one subunit exhibits double rectangular hyperbolic concentration dependency. The consequence of a high-affinity through-protein electron transfer pathway to the cytotoxicity of ferryl heme is discussed.     

Internal electron transfer between hemes and Cu(II) bound at cysteine beta93 promotes methemoglobin reduction by carbon monoxide

Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.     

The Near-iron Transporter (NEAT) Domains of the Anthrax Hemophore IsdX2 Require a Critical Glutamine to Extract Heme from Methemoglobin

Several Gram-positive pathogenic bacteria employ near-iron transporter (NEAT) domains to acquire heme from hemoglobin during infection. However, the structural requirements and mechanism of action for NEAT-mediated heme extraction remains unknown. Bacillus anthracis exhibits a rapid growth rate during systemic infection, suggesting that the bacterium expresses efficient iron acquisition systems. To understand how B. anthracis acquires iron from heme sources, which account for 80% of mammalian iron stores, we investigated the properties of the five-NEAT domain hemophore IsdX2. Using a combination of bioinformatics and site-directed mutagenesis, we determined that the heme extraction properties of IsdX2 are dependent on an amino acid with an amide side chain within the 3₁₀-helix of the NEAT domain. Additionally, we used a spectroscopic analysis to show that IsdX2 NEAT domains only scavenge heme from methemoglobin (metHb) and that autoxidation of oxyhemoglobin to metHb must occur prior to extraction. We also report the crystal structures of NEAT5 wild type and a Q29T mutant and present surface plasmon resonance data that indicate that the loss of this amide side chain reduces the affinity of the NEAT domain for metHb. We propose a model whereby the amide side chain is first required to drive an interaction with metHb that destabilizes heme, which is subsequently extracted and coordinated in the aliphatic heme-binding environment of the NEAT domain. Because an amino acid with an amide side chain in this position is observed in NEAT domains of several genera of Gram-positive pathogenic bacteria, these results suggest that specific targeting of this or nearby residues may be an entry point for inhibitor development aimed at blocking bacterial iron acquisition during infection.     

The mechanism of heme transfer from the cytoplasmic heme binding protein PhuS to the delta-regioselective heme oxygenase of Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa has evolved two outer membrane receptor-mediated uptake systems (encoded by the phu and has operons) by which it can utilize the hosts heme and hemeproteins as a source of iron. PhuS is a cytoplasmic heme binding protein encoded within the phu operon and has previously been shown to function in the trafficking of heme to the iron-regulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (approximately 10(5) s(-1)) was observed, in keeping with a function in heme-trafficking. Additionally, the transfer of heme from PhuS to pa-HO was shown to be specific and unidirectional when compared to transfer to the non-iron regulated heme oxygenase (BphO), in which heme distribution between the two proteins merely reflects their relative intrinsic affinities for heme. Furthermore, the rate of transfer of heme from holo-PhuS to pa-HO of 0.11 +/- 0.01 s(-1) is 30-fold faster than that to apo-myoglobin, despite the significant higher binding affinity of apo-myoglobin for heme (kH = 1.3 x 10(-8) microM) than that of PhuS (0.2 microM). This data suggests that heme transfer to pa-HO is independent of heme affinity and is consistent with temperature dependence studies which indicate the reaction is driven by a negative entropic contribution, typical of an ordered transition state, and supports the notion that heme transfer from PhuS to pa-HO is mediated via a specific protein-protein interaction. In addition, pH studies, and reactions conducted in the presence of cyanide, suggest the involvement of spin transition during the heme transfer process, whereby the heme undergoes spin change from 6-c LS to 6-c HS either in PhuS or pa-HO. On the basis of the magnitudes of the activation parameters obtained in the presence of cyanide, whereby both complexes are maintained in a 6-c LS state, and the biphasic kinetics of heme transfer from holo-PhuS to pa-HO-wt, supports the notion that the spin-state crossover occur within holo-PhuS prior to the heme transfer step. Alternatively, the lack of the biphasic kinetic with pa-HO-G125V, 6-c LS, and with comparable rate of heme transfer as pa-HO is supportive of a mechanism in which the spin-change could occur within pa-HO. The present data suggests either or both of the two pathways proposed for heme transfer may occur under the present experimental conditions. The dissection of which pathway is physiologically relevant is the focus of ongoing studies.     

Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system

Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The lipoprotein-anchored heme binding protein (HBP) of this transporter is SiaA (HtsA). In the current study, resonance Raman (rR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopies were used to determine the coordination state and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggest that the heme is six-coordinate and low-spin in both oxidation states of the protein, with methionine and histidine as axial ligands. SiaA has a pKa of 9.7 +/- 0.1, attributed to deprotonation of the axial histidine. Guanidinium titration studies show that the ferric state is less stable than the ferrous state, with DeltaG(H2O) values for the oxidized and reduced proteins of 7.3 +/- 0.8 and 16.0 +/- 3.6 kcal mol-1, respectively. The reductive and oxidative midpoint potentials determined via spectroelectrochemistry are 83 +/- 3 and 64 +/- 3 mV, respectively; the irreversibility of heme reduction suggests that redox cycling of the heme is coupled to a kinetically sluggish change in structure or conformation. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.     

Neuronal nitric-oxide synthase mutant (Ser-1412 --> Asp) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis

Rat neuronal NO synthase (nNOS) contains an Akt-dependent phosphorylation motif in its reductase domain. We mutated a target residue in that site (Ser-1412 to Asp) to mimic phosphorylation and then characterized the mutant using conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but displayed slower steady-state NO synthesis with greater uncoupling of NADPH oxidation. Paradoxically, the mutant had faster heme reduction, faster heme-NO complex formation, and greater heme-NO complex accumulation at steady state. To understand how these behaviors related to flavin and heme reduction rates, we utilized three soybean calmodulins (CaMs) that supported a range of slower flavin and heme reduction rates in mutant and wild-type nNOS. Reductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reduction. In contrast, steady-state NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domain. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regarding NO release during steady state. The unique behavior of S1412D nNOS reveals the importance of heme reduction rate in controlling steady-state activity and suggests that nNOS already has a near-optimal rate of heme reduction.     

Insight into blocking heme transfer by exploiting molecular interactions in the core Isd heme transporters IsdA-NEAT, IsdC-NEAT, and IsdE of Staphylococcus aureus

The pathogenic bacterium Staphylococcus aureus has adopted specialized mechanisms for scavenging iron from its host. The nine cell wall and membrane-associated iron regulated surface determinant (Isd) proteins (IsdH, IsdB, IsdA, IsdC, IsdDEF, IsdG and IsdI) allow Staphylococcus aureus to scavenge iron from the heme in hemoglobin and haptoglobin-hemoglobin. Of these, it is IsdE that chaperones the heme to the ATP binding cassette-type transmembrane transporter (IsdF). IsdH, IsdB, IsdA and IsdC contain at least one heme binding Near Transporter (NEAT) domain. Previous studies have shown that ferric heme is transferred unidirectionally in the sequence IsdA-NEAT (Tyr - proximal amino acid) → IsdC-NEAT (Tyr) → IsdE (His). IsdA-NEAT does not transfer heme directly to IsdE. In this paper we investigated PPIX transfer through the core cell wall proteins of the Isd system (IsdA-NEAT, IsdC-NEAT and IsdE) with FePPIX-dimethylester, and the metal substituted CoPPIX and MnPPIX using ESI-MS, UV-visible absorption and MCD spectroscopy. IsdA binds each of the rings but the subsequent transfer properties to IsdC-N or IsdE are not the same as found with heme. FePPIX-DME transfers from IsdA-N to IsdC-N but neither protein transfers the ring to IsdE. IsdA-N does not transfer CoPPIX to IsdC-N or IsdE. IsdA-N does transfer MnPPIX to both IsdC-N and IsdE. Significantly, it is possible that since CoPPIX and FePPIX-DME bind to IsdA-N, the lack of transfer to IsdC-N and subsequently to IsdE for CoPPIX could prove to be used as a potential disruption agent to the S. aureus heme transfer system and may identify a possible anti-microbial.