Structure of the heme/hemoglobin outer membrane receptor ShuA from Shigella dysenteriae: Heme binding by an induced fit mechanism

Shigella dysentriae and other Gram‐negative human pathogens are able to use iron from heme bound to hemoglobin for growing. We solved at 2.6 Å resolution the 3D structure of the TonB‐dependent heme/hemoglobin outer membrane receptor ShuA from S. dysenteriae. ShuA binds to hemoglobin and transports heme across the outer membrane. The structure consists of a C‐terminal domain that folds into a 22‐stranded transmembrane β‐barrel, which is filled by the N‐terminal plug domain. One distal histidine ligand of heme is located at the apex of the plug, exposed to the solvent. His86 is situated 9.86 Å apart from His420, the second histidine involved in the heme binding. His420 is in the extracellular loop L7. The heme coordination by His86 and His420 involves conformational changes. The comparisons with the hemophore receptor HasR of Serratia marcescens bound to HasA‐Heme suggest an extracellular induced fit mechanism for the heme binding. The loop L7 contains hydrophobic residues which could interact with the hydrophobic porphyring ring of heme. The energy required for the transport by ShuA is derived from the proton motive force after interactions between the periplasmic N‐terminal TonB‐box of ShuA and the inner membrane protein, TonB. In ShuA, the TonB‐box is buried and cannot interact with TonB. The structural comparisons with HasR suggest its conformational change upon the heme binding for interacting with TonB. The signaling of the heme binding could involve a hydrogen bond network going from His86 to the TonB‐box. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Functional characterization of the Shigella dysenteriae heme ABC transporter

The heme ATP binding cassette (ABC) transporter, ShuUV, of Shigella dysenteriae has been incorporated into proteoliposomes. Functional characterization of ShuUV revealed that ATP hydrolysis and transport of heme from the periplasmic binding protein, ShuT, to the cytoplasmic binding protein, ShuS, are coupled. Site-directed mutagenesis of ShuT residues proposed to be required for stabilization of the complex abolished heme transport. Furthermore, residues His-252 and His-262, located in the translocation channel of ShuU, were required for the release of heme from ShuT and translocation to ShuS. The initial functional characterization of an in vitro heme uptake system provides a platform for future spectroscopic studies.     

Characterization of the periplasmic heme-binding protein shut from the heme uptake system of Shigella dysenteriae

The heme uptake systems by which bacterial pathogens acquire and utilize heme have recently been described. Such systems may utilize heme directly from the host's hemeproteins or via a hemophore that sequesters and transports heme to an outer membrane receptor and subsequently to the translocating proteins by which heme is further transported into the cell. However, little is known of the heme binding and release mechanisms that facilitate the uptake of heme into the pathogenic organism. As a first step toward elucidating the molecular level events that drive heme binding and release, we have undertaken a spectroscopic and mutational study of the first purified periplasmic heme-binding protein (PBP), ShuT from Shigella dysenteriae. On the basis of sequence identity, the ShuT protein is most closely related to the class of PBPs typified by the vitamin B(12) (BtuF) and iron-hydroxamate (FhuD) PBPs and is a monomeric protein having a molecular mass of 28.5 kDa following proteolytic processing of the periplasmic signaling peptide. ShuT binds one b-type heme per monomer with high affinity and bears no significant homology with other known heme proteins. The resonance Raman, MCD, and UV-visible spectra of WT heme-ShuT are consistent with a five-coordinate high spin heme having an anionic O-bound proximal ligand. Site-directed ShuT mutants of the absolutely conserved Tyr residues, Tyr-94 (Y94A) and Tyr-228 (Y228F), which are found in all putative periplasmic heme-binding proteins, were subjected to UV-visible, resonance Raman, and MCD spectroscopic investigations of heme coordination environment and rates of heme release. The results of these experiments confirmed Tyr-94 as the only axial heme ligand and Tyr-228 as making a significant contribution to the stability of heme-loaded ShuT, albeit without directly interacting with the heme iron.     

Purification and biochemical characterization of a heme containing peroxidase from the human parasite P. falciparum

A peroxidase (30 kDa) has been purified from the human malaria parasite Plasmodium falciparum to its homogeneity. The protein is a dimer of 15 kDa subunit as evident from SDS-PAGE and MALDI-TOF mass analysis. The antibodies developed against the purified protein cross-react selectively with this protein present in parasite lysate. It is a heme containing peroxidase [R/Z value (A408/A278)=2.33] showing characteristic heme spectra with Soret peak at 408 nm and visible peaks at 536 and 572 nm. Analysis of Soret spectra in presence or absence of cyanide or azide reveals that iron of heme is in Fe-III state. Circular dichroism spectral analysis establishes that this protein contains mainly alpha-helix (60-70%). H2O2 interacts with the heme moiety of the enzyme as evidenced by optical difference spectroscopy and spectral studies indicate the formation of catalytically active peroxidase-H2O2 complex (Soret peak at 413 nm) to exhibit peroxidase activity. During the erythrocytic stages of its life cycle, the parasite is exposed to oxidative stress. As the parasite is susceptible to oxidative stress, this peroxidase may offer antioxidant role by scavenging endogenous H2O2.     

Genetics and regulation of heme iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7.

Shigella species can use heme as the sole source of iron. In this work, the heme utilization locus of Shigella dysenteriae was cloned and characterized. A cosmid bank of S. dysenteriae serotype 1 DNA was constructed in an Escherichia coli siderophore synthesis mutant incapable of heme transport. A recombinant clone, pSHU12, carrying the heme utilization system of S. dysenteriae was isolated by screening on iron-poor medium supplemented with hemin. Transposon insertional mutagenesis and subcloning identified the region of DNA in pSHU12 responsible for the phenotype of heme utilization. Minicell analysis indicated that a 70-kDa protein encoded by this region was sufficient to allow heme utilization in E. coli. Synthesis of this protein, designated Shu (Shigella heme uptake), was induced by iron limitation. The 70-kDa protein is located in the outer membrane and binds heme, suggesting it is the S. dysenteriae heme receptor. Heme iron uptake was found to be TonB dependent in E. coli. Transformation of an E. coli hemA mutant with the heme utilization subclone, pSHU262, showed that heme could serve as a source of porphyrin as well as iron, indicating that the entire heme molecule is transported into the bacterial cell. DNA sequences homologous to shu were detected in strains of S. dysenteriae serotype 1 and E. coli O157:H7.     

Effects of heme ligand mutations including a pathogenic variant, H65R, on the properties of human cystathionine beta-synthase

Human cystathionine beta-synthase is a hemeprotein that catalyzes a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine into cystathionine. Biophysical characterization of this enzyme has led to the assignment of the heme ligands as histidine and cysteinate, respectively, which has recently been confirmed by crystal structure determination of the catalytic core of the protein. Using site-directed mutagenesis, we confirm that C52 and H65 represent the thiolate and histidine ligands to the heme. Conversion of C52 to alanine or serine results in spectral properties of the resulting hemeprotein that are consistent with the loss of a thiolate ligand. Thus, the Soret peak blue-shifts from 428 to 415 and 417 nm in the ferric forms of the C52S and C52A mutants, respectively, and from 450 to 423 nm in the ferrous states of both mutants. Addition of CO to the dithionite-reduced ferrous C52 mutants results in spectra with Soret peaks at 420 nm. EPR spectroscopy of the ferric C52 variants reveals the predominance of a high-spin species. The H65R mutant, a variant described in a homocystinuric patient, has Soret peaks at 424, 421, and 420 nm in the ferric, ferrous, and ferrous CO states, respectively. EPR spectroscopy reveals predominance of the low-spin species. Both C52A and C52S mutations lead to protein with substoichiometric heme (19% with respect to wild type); however, the PLP content is comparable to that of wild-type enzyme. The heme and PLP contents of the H65R mutant are 40% and 75% that of wild-type enzyme. These results indicate that heme saturation does not dictate PLP saturation in these mutant enzymes. Both H65 and C52 variants display low catalytic activity, revealing that changes in the heme binding domain modulate activity, consistent with a regulatory role for this cofactor.     

Faster heme loss from hemoglobin E than HbS,in acidic pH: Effect of aminophospholipids

We report studies on loss of heme at or below pH 3.0 from two clinically important hemoglobin variants, HbE and HbS, in the presence and absence of phopholipid membranes. The kinetics of heme loss has been studied at pH 3.0 to simulate the same at a faster rate than at physiological pH, for spectroscopic investigation. Results obtained from the study clearly establish the probable fate of the lost heme to partition into the phospholipid bilayer independent of the pH range. This is also of particular importance to membranes containing the aminophospholipid and cholesterol which are predominantly localized in the inner leaflet of erythrocytes. Absorption measurements indicated such loss of heme when the Soret peak at 415 nm blue-shifted to 380 nm at pH 3.0. The extent of this blue shift decreased from 35 nm to ~15 nm in the presence of small unilammelar vesicles of both dimyristoyl- and dioleoyl-based phosphatidylcholine and phosphatidylethanolamine, indicating partitioning of the released heme in the membrane bilayer. The kinetics of heme loss was faster from HbE than HbA and HbS, obeying first-order reaction kinetics. Released heme could be involved in the premature destruction of erythrocytes in hemoglobin disorders.     

Flavocytochrome P450 BM3 mutant A264E undergoes substrate-dependent formation of a novel heme iron ligand set

A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala264. The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at approximately 420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formate-ligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substrate-free enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M. G., Girvan, H. M., Munro, A. W., and Leys, D. (2004) J. Biol. Chem. 279, 23287-23293).     

Recombinant expression and initial characterization of the putative human enteric ferric reductase Dcytb

Recent studies of mutant mice with compromised ability to absorb dietary iron have identified involvement of two integral membrane proteins in the intestinal epithelial lining in iron uptake, a divalent metal ion transporter and a ferric reductase. The current study concerns the recombinant expression, purification, and initial spectroscopic characterization of a recombinant form of the human ferric reductase that was expressed and purified as the apoprotein from Escherichia coli. Reconstitution of the recombinant protein with ferriprotoporphyrin IX produced a red product with Soret (Fe3+, lambdamax 413.5 nm; Fe2+, lambdamax = 426 nm) and visible absorption maxima indicative of bisimidazole axial coordination. This observation was confirmed by electron paramagnetic resonance and magnetic circular dichroism spectroscopy. Titration of apo-Dcytb with ferriprotoporphyrin IX was consistent with the binding of two heme groups to the protein as predicted by the phylogenetic relationship of this protein to the cytochrome b561 family. Similar titrations and spectroscopic studies of two double variants of Dcytb, each lacking a pair of histidyl residues (H50 and H120 or H86 and H159) proposed on the basis of sequence alignment with other members of the cytochrome b561 family to provide axial ligands to bound heme, indicated that these variants were able to bind just one heme group each.     

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution studies on heme proteins. Circular dichroism and optical rotation of Glycera dibranchiata hemoglobins

Circular dichroism (CD) and optical rotatory dispersion (ORD) spectra of several liganded derivatives of the monomer and polymer hemoglobin components of the marine annelid, Glycera dibranchiata were measured over the wavelength range 650--195 nm. The differences observed between the monomer and polymer components for the heme dichroic bands in the visible, Soret and ultraviolet wavelength regions seem to result from changes in the heme environment, geometry and coordination state of the central heme iron in these proteins. Within the Soret region, the liganded derivatives of the monomer hemoglobin exhibit predominantly negative circular dichroic bands. The heme band at 260 nm is also absent for the monomer hemoglobin. The ORD and CD spectra in the far-ultraviolet, peptide absorbing region suggest also differences in the alpha-helix content of the monomer and polymer hemoglobins. The values for the single-chain G. dibranchiata hemoglobin are in the expected range (about 70% alpha-helix) as predicted by the X-ray structure of this protein. The lower estimates of the alpha-helix content for the polymer hemoglobin (approx. 50%), may reflect the differences in amino acid composition, primary structure and polypeptide chain foldings. Changes in oxidation state and ligand binding appears to have no pronounced effect on the helicity of either the monomer or polymer hemoglobins. The removal of the heme moiety from the monomer hemoglobin did result in a major decrease in its helix content similar to the loss of heme from myoglobin.     

Structure of the Escherichia coli O157:H7 heme oxygenase ChuS in complex with heme and enzymatic inactivation by mutation of the heme coordinating residue His-193

Heme oxygenases catalyze the oxidation of heme to biliverdin, CO, and free iron. For pathogenic microorganisms, heme uptake and degradation are critical mechanisms for iron acquisition that enable multiplication and survival within hosts they invade. Here we report the first crystal structure of the pathogenic Escherichia coli O157:H7 heme oxygenase ChuS in complex with heme at 1.45 A resolution. When compared with other heme oxygenases, ChuS has a unique fold, including structural repeats and a beta-sheet core. Not surprisingly, the mode of heme coordination by ChuS is also distinct, whereby heme is largely stabilized by residues from the C-terminal domain, assisted by a distant arginine from the N-terminal domain. Upon heme binding, there is no large conformational change beyond the fine tuning of a key histidine (His-193) residue. Most intriguingly, in contrast to other heme oxygenases, the propionic side chains of heme are orientated toward the protein core, exposing the alpha-meso carbon position where O(2) is added during heme degradation. This unique orientation may facilitate presentation to an electron donor, explaining the significantly reduced concentration of ascorbic acid needed for the reaction. Based on the ChuS-heme structure, we converted the histidine residue responsible for axial coordination of the heme group to an asparagine residue (H193N), as well as converting a second histidine to an alanine residue (H73A) for comparison purposes. We employed spectral analysis and CO measurement by gas chromatography to analyze catalysis by ChuS, H193N, and H73A, demonstrating that His-193 is the key residue for the heme-degrading activity of ChuS.     

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.     

Electron structure of the heme of reduced cytochrome P450 and P420 from the data of low-temperature magnetic circular dichroism

Magnetic circular dichroism spectra (MCD) of reduced cytochromes P450 and P420 from rabbit liver microsomes have been recorded and analyzed for the 350-600 nm spectral region in the temperature interval from 2 to 290 K. The shape, intensity and temperature dependence of the MCD of reduced P450 in the Soret region are quite different from that of other high-spin ferrous hemoproteins, whose heme iron is coordinated to the imidazole of histidine (deoxymyoglobin, deoxyhemoglobin, reduced peroxidase and cytochrome c oxidase). Assuming that in the reduced P450 as well as in its CO-complex the protein-derived ligand is mercaptide (RS-) the differences can be explained by the existence of two electronic transitions in the Soret region: the common for hemoproteins pi----pi porphyrin transition and sulfur to porphyrin charge-transfer transition, p+(Sp)----eg (pi). The unusual spectral characteristics of the CO-complex of P450 have been ascribed earlier to strong configurational interaction of these two transitions. From the similarities of the Soret MCD and their temperature dependences for the reduced P420 and for other high-spin ferrous hemoproteins one can conclude that heme iron of the reduced P420 is high-spin and is coordinated to the imidazole of histidine. The zero-field splitting parameter D of the spin Hamiltonian has been estimated from the MCD temperature dependences. The obtained splitting of approximately 30 cm-1 for P450 and of approximately 10 cm-1 for P420 exceeds that for myoglobin and hemoglobin (approximately 5 cm-1).     

Nonionic amphiphilic polymers derived from Tris(hydroxymethyl)-acrylamidomethane keep membrane proteins soluble and native in the absence of detergent

The UV-visible, circular dichroism (CD), and resonance Raman (RR) spectra of the wild type yeast iso-1-cytochrome c (WT) and its mutant F82H in which phenylalanine-82 (Phe-82) is substituted with His are measured and compared for oxidized and reduced forms. The CD spectra in the intrinsic and Soret spectral region, as well as RR spectra in high, middle, and low frequency regions, are discussed. From the analysis of the spectra, it is determined that in the oxidized F82H the two axial ligands to the heme iron are His-18 and His-82 whereas in the reduced form the sixth ligand switches from His-82 to Met-80 providing the coordination geometry similar to that of WT. Based on the spectroscopic data, the conclusion is that the porphyrin macrocycle is less distorted in the oxidized F82H compared to the oxidized WT. Similar distortions are present in the reduced form of the proteins. Frequency shifts of Raman bands, as well as the decrease of the alpha-helix content in the CD spectra, indicate more open conformation of the protein around the heme.     

Spectroscopic evidence for a 5-coordinate oxygenic ligated high spin ferric heme moiety in the Neisseria meningitidis hemoglobin binding receptor

Background

For many pathogenic microorganisms, iron acquisition represents a significant stress during the colonization of a mammalian host. Heme is the single most abundant source of soluble iron in this environment. While the importance of iron assimilation for nearly all organisms is clear, the mechanisms by which heme is acquired and utilized by many bacterial pathogens, even those most commonly found at sites of infection, remain poorly understood.

Methods

An alternative protocol for the production and purification of the outer membrane hemoglobin receptor (HmbR) from the pathogen Neisseria meningitidis has facilitated a biophysical characterization of this outer membrane transporter by electronic absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman techniques.

Results

HmbR co-purifies with 5-coordinate high spin ferric heme bound. The heme binding site accommodates exogenous imidazole as a sixth ligand, which results in a 6-coordinate, low-spin ferric species. Both the 5- and 6-coordinate complexes are reduced by sodium hydrosulfite. Four HmbR variants with a modest decrease in binding efficiency for heme have been identified (H87C, H280A, Y282A, and Y456C). These findings are consistent with an emerging paradigm wherein the ferric iron center of bound heme is coordinated by a tyrosine ligand.

Conclusion

In summary, this study provides the first spectroscopic characterization for any heme or iron transporter in Neisseria meningitidis, and suggests a coordination environment heretofore unobserved in a TonB-dependent hemin transporter.

General Significance

A detailed understanding of the nutrient acquisition pathways in common pathogens such as N. meningitidis provides a foundation for new antimicrobial strategies.     

Mutation of the five conserved histidines in the endothelial nitric-oxide synthase hemoprotein domain. No evidence for a non-heme metal requirement for catalysis.

Five conserved histidine residues are found in the human endothelial nitric-oxide synthase (NOS) heme domain: His-420, His-421, and His-461 are close to the heme, whereas His-146 and His-214 are some distance away. To investigate whether the histidines form a non-heme iron-binding site, we have expressed the H146A, H214A, H420A, H421A, and H461A mutants. The H420A mutant could not be isolated, and the H146A and H421A mutants were inactive. The H214A mutant resembled the wild-type enzyme in all respects. The H461A mutant had a low-spin heme, but high concentrations of L-Arg and tetrahydrobiopterin led to partial recovery of activity. Laser atomic emission showed that the only significant metal in NOS other than calcium and iron is zinc. The activities of the NOS isoforms were not increased by incubation with Fe(2+), but were inhibited by high Fe(2+) or Zn(2+) concentrations. The histidine mutations altered the ability of the protein to dimerize and to bind heme. However, the protein metal content, the inability of exogenous Fe(2+) to increase catalytic activity, and the absence of evidence that the conserved histidines form a metal site provide no support for a catalytic role for a non-heme redox-active metal.     

Genetic variability of the heme uptake system among different strains of the fish pathogen Vibrio anguillarum: identification of a new heme receptor

The ability to utilize heme compounds as iron sources was investigated in Vibrio anguillarum strains belonging to serotypes O1 to O10. All strains, regardless of their serotype or isolation origin could utilize hemin and hemoglobin as sole iron sources. Similarly, all of the isolates could bind hemin and Congo red, and this binding was mediated by cell envelope proteins. PCR and Southern hybridization were used to assay the occurrence of heme transport genes huvABCD, which have been previously described in serotype O1. Of 23 strains studied, two serotype O3 isolates proved negative for all huvABCD genes, whereas nine strains included in serotypes O2, O3, O4, O6, O7, and O10 tested negative for the outer membrane heme receptor gene huvA. A gene coding for a novel outer membrane heme receptor was cloned and characterized in a V. anguillarum serotype O3 strain lacking huvA. The new heme receptor, named HuvS, showed significant similarity to other outer membrane heme receptors described in Vibrionaceae, but little homology (39%) to HuvA. This heme receptor was present in 9 out of 11 of the V. anguillarum strains that tested negative for HuvA. Furthermore, complementation experiments demonstrated that HuvS could substitute for the HuvA function in Escherichia coli and V. anguillarum mutants. The huvS and huvA sequences alignment, as well as the analysis of their respective upstream and downstream DNA sequences, suggest that horizontal transfer and recombination might be responsible for generating this genetic diversity.     

Conformational mobility in the active site of a heme peroxidase

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds "on" to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing "off" the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes.