Soluble meso and deuteroporphyrin analogs of the malaria pigment hematin anhydride.

Two soluble heme analogs of the insoluble malaria pigment hematin anhydride (HA, or ß-hematin), [Fe(III)(protoporphyrin)]2, with either mesoporphyrin (MHA) or deuteroporphyrin (DHA) are characterized by elemental analysis, SEM, IR spectroscopy, electronic spectroscopy, paramagnetic 1H NMR spectroscopy and solution magnetic susceptibility. While prior single crystal and X-ray powder diffraction results indicate all three have a common propionate linked dimer motif, there is considerable solid state variation in the conformation. This is associated with enhanced solubility of MHA and DHA. As with HA, DHA undergoes thermally promoted reversible hydration/dehydration in the solid state. Solution 1H NMR studies of DHA suggest a high spin dimeric structure with the porphyrin methyls distributed between two isomers which are also present in the solid state. These soluble iron(III)porphyrin dimers allow for the first direct solution studies by NMR and UV-Vis spectroscopies of these key species. Taken together the results illustrate the importance and utility of varying the substituents on the periphery of the porphyrin for studying heme aggregation and malaria pigment formation.

Probing the mechanism of the dedicated NO sensor [4Fe-4S] NsrR: the effect of cluster ligand environment.

NsrR from Streptomyces coelicolor is a bacterial nitric oxide (NO) sensor/nitrosative stress regulator as its primary function, and has been shown to have differential response at low, mid, and high levels of NO. These must correspond to discrete structural changes at the protein-bound [4Fe-4S] cluster in response to stepwise nitrosylation of the cluster. We have investigated the effect of the monohapto carboxylate ligand in the site differentiated [4Fe-4S] cluster cofactor of the protein NsrR on modulating its reactivity to NO with a focus on indentifying mechanistic intermediates. We have prepared a synthetic model [4Fe-4S] cluster complex with tripodal ligand and one single site differentiated site occupied by either thiolate or carboxylate ligand. We report here the mechanistic details of sequential steps of nitrosylation as observed by ESI MS and IR spectroscopy. Parallel non-denaturing mass spectrometry analyses were performed using site-differentiated variants of NsrR with the native aspartic acid, cysteine, or alanine in the position of the forth ligand to the cluster. A mono-nitrosylated synthetic [4Fe-4S] cluster was observed for the first time in a biologically-relevant thiolate-based coordination environment. Combined synthetic and protein data give unprecedented clarity in the modulation of nitrosylation of a [4Fe-4S] cluster.

Hematin anhydride (ß-hematin): An analogue to malaria pigment hemozoin possesses nonlinearity.

Hematin anhydride (ß-hematin), the synthetic analogue of the malaria pigment, "hemozoin", is a heme dimer produced by reciprocal covalent bonds among carboxylic acid groups on the protoporphyrin-IX ring and the iron atom present in the two adjacent heme molecules. Hemozoin is a disposal product formed from the digestion of hemoglobin present in the red blood cells infected with hematophagous malaria parasites. Besides, as the parasites invade red blood cells, hemozoin crystals are eventually released into the bloodstream, where they accumulate over time in tissues. Severe malaria infection leads to significant dysfunction in vital organs such as the liver, spleen, and brain in part due to the autoimmune response to the excessive accumulation of hemozoin in these tissues. Also, the amount of these crystals in the vasculature correlates with disease progression. Thus, hemozoin is a unique indicator of infection used as a malaria biomarker and hence, used as a target for the development of antimalarial drugs. Hence, exploring various properties of hemozoin is extremely useful in the direction of diagnosis and cure. The present study focuses on finding one of the unknown properties of ß-hematin in physiological conditions by using the Z-scan technique, which is simple, sensitive, and economical. It is observed that hemozoin possesses one of the unique material properties, i.e., nonlinearity with a detection limit of âˆ¼ 15 µM. The self-defocusing action causes ß-hematin to exhibit negative refractive nonlinearity. The observed data is analyzed with a thermal lensing model. We strongly believe that our simple and reliable approach to probing the nonlinearity of ß-hematin will provide fresh opportunities for malaria diagnostics & cure in the near future.

Dioxygen controls the nitrosylation reactions of a protein-bound [4Fe4S] cluster.

Iron-sulfur clusters are exceptionally tuneable protein cofactors, and as one of their many roles they are involved in biological responses to nitrosative stress. Both iron-sulfur proteins and synthetic model clusters are extremely sensitive to nitrosylation, tending towards rapid multi-step reaction and cluster degradation. Reaction of protein-bound iron-sulfur clusters with nitric oxide can be stopped at partial nitrosylation in vivo, and repair of protein-bound nitrosylated clusters is possible in the cellular environment. We have used a combination of infrared, EPR, and UV-visible spectroscopies to show that a model [4Fe4S] cluster-containing protein, A. ferroxidans high potential iron-sulfur protein (HiPIP), reacts with NO to give a product mixture dominated by Roussin's Black Salt (RBS) and Roussin's Red Ester (RRE) species. We have shown that O2 plays a critical role in controlling the major product of nitrosylation, with RBS-like products favoured under strictly anaerobic conditions and RRE favoured in the presence of trace O2. Moreover, addition of trace O2 to anaerobically nitrosylated samples induces conversion of RBS-like products to RRE. These findings may have implications for mechanisms of iron-sulfur cluster repair following nitrosative stress, suggest a crucial role for trace O2, and provide an important link between nitrosylation chemistry of iron-sulfur proteins and the well-established reactivity of synthetic iron-sulfur clusters.

Hydrating the Bispropionate Notch in Malaria Pigment: A New Structural Motif in the Iron(III)(deuteroporphyrin) Dimer.

Treating deuterohemin, chloro(deuteroporphyrinato)iron(III), with a non-coordinating base in DMSO/methanol allows for the isolation of [(deuteroporphyrinato)iron(III)]2 , deuterohematin anhydride (DHA), an analogue of malaria pigment, the natural product of heme detoxification by malaria. The structure of DHA obtained from this solvent system has been solved by X-ray powder diffraction analysis and displays many similarities, yet important structural differences, to malaria pigment. Most notably, a water molecule of solvation occupies a notch created by the propionate side chains and stabilizes a markedly bent propionate ligand coordinated with a long Fe-O bond, and a carboxylate cluster associated with water molecules is generated. Together, these features account for its increased solubility and more open structure, with an increased porphyrin-porphyrin separation. The IR spectroscopic signature associated with this structure also accounts for the strong IR band at 1587 cm-1 seen for many amorphous preparations of synthetic malaria pigment, and it is proposed that stabilizing these structures may be a new objective for antimalarial drugs. The important role of the vinyl substituents in this biochemistry is further demonstrated by the structure of deuterohemin obtained by single-crystal X-ray diffraction analysis.

Solution and Solid State Correlations of Antimalarial Drug Actions: NMR and Crystallographic Studies of Drug Interactions with a Heme Model.

Solution NMR has been used in tandem with a diamagnetic non-iron heme model compound as a simple and effective tool to rapidly probe the structures of the bound complexes formed between the metalloporphyrin and antimalarial drugs from the 4-aminoquinoline, 4-methylenehydroxylquinoline, and 8-aminoquinoline subfamilies. The ability of gallium(III) protoporphyrin IX to mimic heme chemistry is exploited. The 4-aminoquinolines quinacrine and amodiaquine and two novel 3-halo chloroquine analogues are found to bind to the metalloporphyrin through hydrogen-bonding and stacking interactions, while halofantrine and the 4-methylenehydroxylquinolines, quinine and mefloquine bind through the alcohol group of the drug. In each case, detailed structural information is available from the NMR assessment. The mefloquine model is confirmed crystallographically. The 8-aminoquinoline primaquine does not interact strongly. These tools show promise for future applications in assessing antimalarials in preclinical development for heme-binding drug targets.

Crystal structures of the NO sensor NsrR reveal how its iron-sulfur cluster modulates DNA binding.

NsrR from Streptomyces coelicolor (Sc) regulates the expression of three genes through the progressive degradation of its [4Fe-4S] cluster on nitric oxide (NO) exposure. We report the 1.95 Å resolution crystal structure of dimeric holo-ScNsrR and show that the cluster is coordinated by the three invariant Cys residues from one monomer and, unexpectedly, Asp8 from the other. A cavity map suggests that NO displaces Asp8 as a cluster ligand and, while D8A and D8C variants remain NO sensitive, DNA binding is affected. A structural comparison of holo-ScNsrR with an apo-IscR-DNA complex shows that the [4Fe-4S] cluster stabilizes a turn between ScNsrR Cys93 and Cys99 properly oriented to interact with the DNA backbone. In addition, an apo ScNsrR structure suggests that Asn97 from this turn, along with Arg12, which forms a salt-bridge with Asp8, are instrumental in modulating the position of the DNA recognition helix region relative to its major groove.

NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function.

The Rrf2 family transcription factor NsrR controls expression of genes in a wide range of bacteria in response to nitric oxide (NO). The precise form of the NO-sensing module of NsrR is the subject of controversy because NsrR proteins containing either [2Fe-2S] or [4Fe-4S] clusters have been observed previously. Optical, Mössbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that Streptomyces coelicolor NsrR (ScNsrR), previously reported to contain a [2Fe-2S] cluster, can be isolated containing a [4Fe-4S] cluster. ChIP-seq experiments indicated that the ScNsrR regulon is small, consisting of only hmpA1, hmpA2, and nsrR itself. The hmpA genes encode NO-detoxifying flavohemoglobins, indicating that ScNsrR has a specialized regulatory function focused on NO detoxification and is not a global regulator like some NsrR orthologues. EMSAs and DNase I footprinting showed that the [4Fe-4S] form of ScNsrR binds specifically and tightly to an 11-bp inverted repeat sequence in the promoter regions of the identified target genes and that DNA binding is abolished following reaction with NO. Resonance Raman data were consistent with cluster coordination by three Cys residues and one oxygen-containing residue, and analysis of ScNsrR variants suggested that highly conserved Glu-85 may be the fourth ligand. Finally, we demonstrate that some low molecular weight thiols, but importantly not physiologically relevant thiols, such as cysteine and an analogue of mycothiol, bind weakly to the [4Fe-4S] cluster, and exposure of this bound form to O2 results in cluster conversion to the [2Fe-2S] form, which does not bind to DNA. These data help to account for the observation of [2Fe-2S] forms of NsrR.

Orienting the heterocyclic periphery: a structural model for chloroquine's antimalarial activity.

The antimalarial drug chloroquine binds to gallium proto-porphyrin-IX in methanol and in the solid state and represents a unique drug-heme model.

Soluble diamagnetic model for malaria pigment: coordination chemistry of gallium(III)protoporphyrin-IX.

The facile axial ligand exchange properties of gallium(III) protoporphyrin IX in methanol solution were utilized to explore self-association interactions by NMR techniques. Structural changes were observed, as well as competitive behavior with the ligands acetate and fluoride, which differed from that seen with the synthetic analogue gallium(III) octaethylporphyrin which lacks acid groups in its side-chains and has less solution heterogeneity as indicated by absorption and MCD spectroscopies. The propionic acid side chains of protoporphyrin IX are implicated in all such interactions of PPIX, and both dynamic metal-propionic interactions and the formation of propionate-bridged dimers are observed. Fluoride coordination provides an unusual example of slow ligand exchange, and this allows for the identification of a fluoride bridged dimer in solution. An improved synthesis of the chloride and hydroxide complexes of gallium(III) protoporphyrin IX is reported. An insoluble gallium analogue of hematin anhydride is described. In general, the interactions between solvent and the metal are found to confer very high solubility, making [Ga(PPIX)](+) a useful model for ferric heme species.

Structure of malaria pigment and related propanoate-linked metalloporphyrin dimers.

Malaria pigment, the heme detoxification product of malaria's invasion, digestion, and growth inside mammalian red blood cells, is an insoluble phase of iron(III)protoporphyrin-IX. Even though its structure was determined in 2000 by powder X-ray diffraction, significant questions remain about its formation and possible interaction with quinoline antimalarial drugs. A recent structural study, also with X-ray powder diffraction, has reconfirmed that the material isolated from the parasite is isostructural with its synthetic equivalent. It was recently suggested that other isomers may also be formed and may be present in synthetic samples. In particular, a series of stereoisomers are possible for the arrangement of vinyl groups on the periphery of the dimerized porphyrin rings. In principle, any given dimer can have vinyl groups at the α or ß sites, and at γ or δ sites. In this paper, several models are evaluated, both biphasic and homogeneous methyl/vinyl disorder, against several sets of diffraction data, both published and new. We conclude that methyl/vinyl disorder is intrinsic to the system, and that the evidence at hand does not support the existence of any other crystalline isomers in carefully prepared samples of either natural or synthetic samples. Finally, the porphyrin-porphyrin interactions are evaluated using Scheidt's indicies for porphyrin π-stacking, and we find modest to weak π-interactions in these condensed phases.

Spectroscopic and theoretical studies of Ga(III)protoporphyrin-IX and its reactions with myoglobin.

Ga(III)protoporphyrin-IX (Ga-PP) has been proposed as a model for the key interporphyrin interactions in malaria pigment. Unlike the paramagnetic parent iron heme derivatives, Ga-PP is readily soluble in methanol (MeOH). We report optical, mass spectroscopic, and theoretical results for Ga-PP as well as its reactions with myoglobin. UV-visible absorption and MCD spectroscopy show that Ga-PP exhibits a typical spectrum for a main group metal: a Q-band at 539 nm and a B band at 406 nm when dissolved in MeOH. We also report optical data for Zn(II)protoporphyrin IX (Zn-PP) dissolved in MeOH, which exhibits a Q-band at 545 nm and a B band at 415 nm. ESI mass spectral data for Ga-PP dissolved in MeOH show the presence of predominantly monomers, with smaller fractions of dimers [(Ga-PP)(2)] and trimers. UV-visible and MCD absorption spectroscopy and ESI mass spectral data demonstrate the successful insertion of monomeric Ga-PP into apo-Mb. Ga-PP-Mb exhibits a B band at 417 nm and Q bands at 545 and 584 nm, which are all red-shifted from the free Ga-PP values. The calculated electronic structures and frontier molecular orbitals of Ga-PP, (Ga-PP)(2) and Zn-PP fit the previously reported trends in band energies and oscillator strengths as a function of molecular orbital energies. These new data can be applied to explain the experimentally observed optical spectroscopy. The observed Q-band energies are accounted for by calculated (HOMO-LUMO) gap of the frontier MOs, while the split in the two top occupied MOs accounts for the magnitude of the Q-band oscillator strength as well as the experimentally observed Q to B band energy separation. Although Ga-PP shares more spectroscopic properties with Zn-PP than it does with Fe(III)PPIX, the trivalent oxidation state allows this molecule to be used as a model for ferric hemes in heme proteins.

[Gallium(III) protoporphyrin IX]2: a soluble diamagnetic model for malaria pigment.

Gallium(III) protoporphyrin IX forms a dimeric propionate-bridged dimer, 2, that is a soluble diamagnetic analogue of hematin anhydride. The single-crystal structure of 2 corresponds to a nondisordered inversion-symmetric dimer similar to malaria pigment but, unlike it, has a six-coordinate metal and an intraporphyrin rather than an interporphyrin hydrogen bond. NMR NOE correlations demonstrate the presence of the propionate linkage in solutions with pyridine. Taken together, this is the first single-crystal X-ray diffraction study of a propionate-linked dimer as found in malaria pigment and the first evidence for its presence in solution.