Characterization of the second conserved domain in the heme uptake protein HtaA from Corynebacterium diphtheriae.

HtaA is a heme-binding protein that is part of the heme uptake system in Corynebacterium diphtheriae. HtaA contains two conserved regions (CR1 and CR2). It has been previously reported that both domains can bind heme; the CR2 domain binds hemoglobin more strongly than the CR1 domain. In this study, we report the biophysical characteristics of HtaA-CR2. UV-visible spectroscopy and resonance Raman experiments are consistent with this domain containing a single heme that is bound to the protein through an axial tyrosine ligand. Mutants of conserved tyrosine and histidine residues (Y361, H412, and Y490) have been studied. These mutants are isolated with very little heme (≤5%) in comparison to the wild-type protein (~20%). Reconstitution after removal of the heme with butanone gave an alternative form of the protein. The HtaA-CR2 fold is very stable; it was necessary to perform thermal denaturation experiments in the presence of guanidinium hydrochloride. HtaA-CR2 unfolds extremely slowly; even in 6.8M GdnHCl at 37°C, the half-life was 5h. In contrast, the apo forms of WT HtaA-CR2 and the aforementioned mutants unfolded at much lower concentrations of GdnHCl, indicating the role of heme in stabilizing the structure and implying that heme transfer is effected only to a partner protein in vivo.

Corynebacterium diphtheriae HmuT: dissecting the roles of conserved residues in heme pocket stabilization.

The heme-binding protein HmuT is part of the Corynebacterium diphtheriae heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of potential importance for heme binding: R237, Y272 and M292. In this study, site-directed mutations at these three positions provided insight into the nature of axial heme binding to the protein and its effect on the thermal stability of the heme-loaded protein fold. UV-visible absorbance, resonance Raman (rR) and thermal unfolding experiments, along with collision-induced dissociation electrospray ionization mass spectrometry, were used to probe the contributions of each mutated residue to the stability of ϖ HmuT. Thermal unfolding and rR experiments revealed that R237 and M292 are important residues for heme binding. Arginine 237 is a hydrogen-bond donor to the phenol side chain of Y235, which serves as an axial heme ligand. Methionine 292 serves a supporting structural role, favoring the R237 hydrogen-bond donation, which elicits a, heretofore, unobserved modulating influence on π donation by the axial tyrosine ligand in the heme carbonyl complex, HmuT-CO.

Heme-bound SiaA from Streptococcus pyogenes: Effects of mutations and oxidation state on protein stability.

The protein SiaA (HtsA) is part of a heme uptake pathway in Streptococcus pyogenes. In this report, we present the heme binding of the alanine mutants of the axial histidine (H229A) and methionine (M79A) ligands, as well as a lysine (K61A) and cysteine (C58A) located near the heme propionates (based on homology modeling) and a control mutant (C47A). pH titrations gave pKa values ranging from 9.0 to 9.5, close to the value of 9.7 for WT SiaA. Resonance Raman spectra of the mutants suggested that the ferric heme environment may be distinct from the wild-type; spectra of the ferrous states were similar. The midpoint reduction potential of the K61A mutant was determined by spectroelectrochemical titration to be 61±3mV vs. SHE, similar to the wild-type protein (68±3mV). The addition of guanidine hydrochloride showed two processes for protein denaturation, consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket (the half-life for the slower process ranged from less than half a day to two days). The ease of protein unfolding was related to the strength of interaction of the residues with the heme. We hypothesize that kinetically facile but only partial unfolding, followed by a very slow approach to the completely unfolded state, may be a fundamental attribute of heme trafficking proteins. Small motions to release/transfer the heme accompanied by resistance to extensive unfolding may preserve the three dimensional form of the protein for further uptake and release.

Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment.

The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type (WT) HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicates that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A is reduced readily in the presence of dithionite. The FeCO Raman shifts in WT, H136A, and Y235A HmuT-CO complexes provide further evidence of the axial ligand assignments. Additionally, these frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A-CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.

Molecular dynamics of anthraquinone DNA intercalators with polyethylene glycol side chains.

The interactions of a homologous series of four anthraquinone (AQ) intercalators with increasing lengths of polyethylene glycol (PEG) side chains with DNA have been studied via molecular dynamics (MD) simulations. The geometry, conformation, interactions, and hydration of the complexes were examined. The geometries of the four ligands were similar with parallel stacking to the long axis of the adjacent DNA base pairs. Hydrogen bonding between the AQ amide and DNA led to a preference for the trans-syn conformer. As the side chain lengthened, binding to DNA reduced the conformational space, resulting in an increase in unfavorable entropy. Increased localization of the PEG side chain in the DNA groove, indicating some interaction of the side chain with DNA, also contributed unfavorably to the entropy. The changes in free energy of binding due to entropic considerations (-3.9 to -6.3 kcal/mol) of AQ I-IV were significant. The hydration of the PEG side chain decreased upon binding to DNA. Understanding of side chain conformations, interactions, and hydration changes that accompany the formation of a ligand-DNA complex may be important in the development of new applications of pegylated small molecules that target biological macromolecules.

Substituent effect on the preferred DNA binding mode and affinity of a homologous series of naphthalene diimides.

A combination of isothermal titration calorimetry (ITC), topoisomerase I DNA unwinding assays, and ethidium bromide displacement studies were employed to investigate the binding of a homologous series of naphthalene diimides (NDI) to DNA. Our results suggest that the nature of the substituent plays a significant role in both the preferred binding mode and relative binding affinity of the compounds of this study. Only intercalative-type binding (K=15±3×10(6)M(-1)) was observed for the NDI with the smallest substituent (trimethyl-ethylamino), while larger members of the series (diethylmethyl-, dipropylmethyl- and dibutylmethyl-ethylamino substituents) adopted an additional binding mode of higher affinity (K(1)=31-78×10(6)M(-1)).

Synthesis and electrochemical evaluation of conjugates between 2'-deoxyadenosine and modified anthraquinone: probes for hole-transfer studies in DNA.

Photoexcitation of anthraquinones (AQ) in association with DNA results in DNA damage mainly at guanine residues, with products from thymine oxidation also observed. Studies of adenine oxidation will be aided by systems with an increased driving force for charge transfer, achieved by adding electron-withdrawing groups to the AQ ring. Attaching AQ derivatives to adenine via a bridge with two carbon atoms should enable the intended regiocontrol within the DNA duplex structure. Herein we report the synthesis of conjugates between AQ and adenine in which the AQ moieties have been modified with a formyl, a trifluoroacetyl, and two methyl ester groups. These have been synthesized by palladium coupling of tert-butyldiphenylsilyl 5'-protected 8-ethynyl-2'-deoxyadenosine with the corresponding bromoanthraquinone intermediates. Bromo intermediates bearing formyl or trifluoroacetyl were prepared by monolithiation of 2,6-dibromoanthraquinone, a step that required protection of the anthraquinone carbonyls. A bromo intermediate bearing two methyl ester groups was obtained from 1,2,4-trimethylbenzene by Friedel-Crafts acylation with 4-bromobenzoyl chloride followed by oxidation to the tricarboxylic acid, cyclization to form the anthraquinone ring, and finally esterification. Hydrogenation of the ethynyl linker gave the ethanyl linker. Cyclic voltammetry showed that the conjugate with the two ester groups and ethynyl linker was the most easily reduced of the derivatives synthesized. These derivatives, with reduction potentials favorable for electron transfer, can be used in studies of adenine oxidation in DNA.

Photon activation therapy and brachytherapy.

PURPOSE: In photon activation therapy (PAT), energy deposition at critical sites within a tumor can be increased by complexing the DNA with higher Z atoms, and provoking the emission of Auger electrons after inducing a photoelectric effect. This in vivo study evaluates the hypothesis using X-rays from palladium-103 seeds to excite the L-edge of platinum (Pt) atoms bound to the DNA of cancerous cells. METHODS AND MATERIALS: Pt (II) tetrakis(N-methyl-4-pyridyl) porphyrin chloride was used to locate Pt atoms adjacent to the DNA of the KHJJ murine mammary carcinoma; a 2.3-mCi palladium-103 seed was implanted in the tumor. RESULTS: The tumor periphery received subtherapeutic doses. The rate of tumor growth in mice treated with PAT was slower than in mice treated with brachytherapy only. CONCLUSIONS: The tumor growth delay for PAT-treated mice is attributed to Auger emission from Pt atoms that produced substantial local damage. However, other co-existing mechanisms cannot be ruled out.

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.

Parameters of inhibition of HIV-1 infection by small anionic microbicides.

Sulfonated porphyrins and phthalocyanines have been shown to have anti-HIV activity and are under consideration as microbicides. Both categories of compounds are small negatively charged molecules and both were previously shown to inhibit cell fusion induced by the HIV Env protein and to block binding of gp120 to the CD4 receptor. In the present study we show that these compounds inhibit transmission of cell-associated HIV, inactivate a broad range of HIV-1 primary isolates and are active against DS polyanion-resistant virus. The compounds tested are active over a range of pH values, and possess no detectable activity against normal bacterial flora. These results support the conclusion that anionic tetrapyrroles are promising candidates as microbicides for HIV prevention.

Identification of amino acid residues involved in heme binding and hemoprotein utilization in the Porphyromonas gingivalis heme receptor HmuR.

We have previously identified and characterized a heme/hemoglobin receptor, HmuR, in Porphyromonas gingivalis. To analyze the conserved amino acid residues of HmuR that may be involved in hemin/hemoprotein binding and utilization, we constructed a series of P. gingivalis A7436 hmuR mutants with amino acid replacements and characterized the ability of these mutants to utilize hemin and hemoproteins. Site-directed mutagenesis was employed to introduce mutations H95A, H434A, H95A-H434A, YRAP420-423YAAA, and NPDL442-445NAAA into HmuR in both P. gingivalis and Escherichia coli. Point mutations at H95 and H434 and in the NPDL motif of HmuR resulted in decreased binding to hemin, hemoglobin, and human serum albumin-hemin complex. Notably, mutations of these conserved sites and motifs led to reduced growth of P. gingivalis when human serum was used as the heme source. Analysis using a three-dimensional homology model of HmuR indicated that H95, H434, and the NPDL motif are present on apical or extracellular loops of HmuR, while the YRAP motif is present on the barrel wall. Taken together, these results support a role for H95, H434, and the NPDL motif of the P. gingivalis HmuR protein in heme binding and utilization of serum hemoproteins and the HmuR YRAP motif in serum hemoprotein utilization.

Sulfonated naphthyl porphyrins as agents against HIV-1.

Sulfonated 5,10,15,20-tetra(1-naphthyl)porphyrin (T1NapS) and 5,10,15,20-tetra(2-naphthyl)porphyrin (T2NapS) and their copper and iron chelates show activity against the human immunodeficiency virus (HIV-1). The porphyrins were prepared by sulfonation of the parent structures with sulfuric acid. More highly sulfonated structures were prepared by sulfonation for longer times. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry showed species with as many as eight sulfonates. Some of the mass spectral peaks for the copper chelates were consistent with loss of water, apparently from intramolecular sulfone formation between two adjacent naphthalene rings that took place during copper insertion. The compounds could be separated using capillary electrophoresis; addition of beta- or gamma-cyclodextrin gave substantially better separation of the components. Activity against HIV was evaluated using an epithelial HeLa-CD4-CCR5 cell line; EC50 values for HIV-1 IIIB and HIV-1 JR-FL ranged from 1 to 15 microg/ml. The compounds exhibit low toxicity for human epithelial cells and have potential as microbicides which might be used to provide protection against sexual transmission of HIV.

Binding of a homologous series of anthraquinones to DNA.

The DNA binding characteristics of a series of homologous 2,6-disubstituted anthraquinone threading intercalators bearing one to four ethylene glycol units in their side chains have been studied. Binding constants were measured via surface plasmon resonance (SPR). These compounds bind to an AT-rich hairpin with slightly higher affinity than to a GC-rich hairpin. The binding constants decrease as the length of the side chain increases.

Prevention of HIV-1 infection by phthalocyanines.

The ability of selected phthalocyanines and metallophthalocyanines to block HIV infection has been evaluated in an epithelial HeLa-CD4 cell line with an integrated LTR-beta-galactosidase gene. Sulfonated phthalocyanine itself (PcS), as well as its copper, nickel, and vanadyl chelates, were the most effective in blocking viral infection. These compounds were also very effective in blocking the fusion activity of the viral Env proteins. All of these compounds are expected to bind axial ligands weakly or not at all. In contrast, sulfonated phthalocyanines bearing metals expected to bind axial ligands more tightly (aluminum, cobalt, chromium, iron, silicon, and zinc) were less effective in blocking HIV infection and also less effective at inhibiting fusion. A number of active compounds were found to block binding of gp120 to CD4. Selected cationic and carboxy phthalocyanines, as well as porphyrazines, were also evaluated. Our results indicate that at least some of the compounds render the virus noninfectious, i.e. that they are virucidal. These compounds have potential as microbicides that might be used to provide protection against sexually transmitted HIV.

Prevention of poxvirus infection by tetrapyrroles.

BACKGROUND: Prevention of poxvirus infection is a topic of great current interest. We report inhibition of vaccinia virus in cell culture by porphyrins and phthalocyanines. Most previous work on the inhibition of viruses with tetrapyrroles has involved photodynamic mechanisms. The current study, however, investigates light-independent inhibition activity. METHODS: The Western Reserve (WR) and International Health Department-J (IHD-J) strains of vaccinia virus were used. Virucidal and antiviral activities as well as the cytotoxicity of test compounds were determined. RESULTS: Examples of active compounds include zinc protoporphyrin, copper hematoporphyrin, meso(2,6-dihydroxyphenyl)porphyrin, the sulfonated tetra-1-naphthyl and tetra-1-anthracenylporphyrins, selected sulfonated derivatives of halogenated tetraphenyl porphyrins and the copper chelate of tetrasulfonated phthalocyanine. EC50 values for the most active compounds are as low as 0.05 microg/mL (40 nM). One of the most active compounds was the neutral meso(2,6-dihydroxyphenyl)porphyrin, indicating that the compounds do not have to be negatively charged to be active. CONCLUSIONS: Porphyrins and phthalocyanines have been found to be potent inhibitors of infection by vaccinia virus in cell culture. These tetrapyrroles were found to be active against two different virus strains, and against both enveloped and non-enveloped forms of the virus, indicating that these compounds may be broadly effective in their ability to inhibit poxvirus infection.

Inactivation of human immunodeficiency virus type 1 by porphyrins.

We have evaluated the anti-human immunodeficiency virus (HIV) activity of a series of natural and synthetic porphyrins to identify compounds that could potentially be used as microbicides to provide a defense against infection by sexually transmitted virus. For assays we used an epithelial HeLa-CD4 cell line with an integrated long terminal repeat-beta-galactosidase gene. For structure-activity analysis, we divided the porphyrins tested into three classes: (i) natural porphyrins, (ii) metallo-tetraphenylporphyrin tetrasulfonate (metallo-TPPS4) derivatives, and (iii) sulfonated tetra-arylporphyrin derivatives. None of the natural porphyrins studied reduced infection by more than 80% at a concentration of 5 micro g/ml in these assays. Some metal chelates of TPPS4 were more active, and a number of sulfonated tetra-aryl derivatives showed significantly higher activity. Some of the most active compounds were the sulfonated tetranaphthyl porphyrin (TNapPS), sulfonated tetra-anthracenyl porphyrin (TAnthPS), and sulfonated 2,6-difluoro-meso-tetraphenylporphine [TPP(2,6-F2)S] and its copper chelate [TPP(2,6-F2)S,Cu], which reduced infection by 99, 96, 94, and 96%, respectively. Our observations indicate that at least some of these compounds are virucidal, i.e., that they render the virus noninfectious. The active compounds were found to inhibit binding of the HIV type 1 gp120 to CD4 and also to completely inhibit the ability of Env proteins expressed from recombinant vectors to induce cell fusion with receptor-bearing target cells. These results support the conclusion that modified porphyrins exhibit substantial activity against HIV and that their target is the HIV Env protein.

Effect of DNA Scaffolding on Intramolecular Electron Transfer Quenching of a Photoexcited Ruthenium(II) Polypyridine Naphthalene Diimide.

Intramolecular emission quenching of a photoexcited ruthenium(II) polypyridine by a covalently linked naphthalene diimide (NDI) has been measured in aqueous buffer both without and with calf thymus DNA. The complex consists of a Ru(2,2'-bipyridine)(2)(2,2'-bipyridine-5-carboxamide)(2+) electron donor covalently attached by way of a -CH(2)CH(2)CH(2)- linker to a 1,4,5,8-naphthalene diimide acceptor (Ru-NDI, 1). The NDI portion of the complex intercalates in calf thymus DNA, as indicated by the hypochromism of its optical absorbance bands and observation of an induced circular dichroism spectrum in the same region. Emission quenching in Ru-NDI has been measured relative to a Ru tris-bpy model lacking the NDI moiety by both lifetime and emission quantum yield techniques. Using lifetime averages, the relative emission quenching is, respectively, 99.1% and 97.9% in aqueous buffer solutions without and with DNA. The emission quenching is ascribed to intramolecular electron transfer within the Ru-NDI complex with an estimated driving force (-DeltaG degrees ) of 0.33 eV. In buffer, the emission decays of Ru-NDI alone are fit well with a triexponential model with lifetimes of 0.34 (0.88), 1.99 (0.11), and 12.6 (0.008) ns (relative amplitude). The emission decays of the DNA-intercalated Ru-NDI complex are also fit well with a triexponential model with lifetimes of 0.31 (0.79), 2.00 (0.13), and 11.8 (0.08) ns. Thus, the fractional amplitudes of the lifetimes change upon DNA intercalation of the complex, while the lifetimes themselves remain essentially the same. The average rates of electron transfer in aqueous buffer without and with DNA are, respectively, 1.6 x 10(9) and 6.8 x 10(8) s(-)(1). The striking result of this study is that the overall character of electron transfer quenching in Ru-NDI is very similar whether or not it is bound to DNA. Intercalation of the NDI in DNA apparently has negligible consequences for electron transfer, implying either that the activation energy and electronic coupling in Ru-NDI are largely unaffected by this, at first glance, seemingly significant environmental change or that changes in these parameters on DNA binding cancel fortuitously.