Reza A. Ghiladi

Photobactericidal Porphyrin-Cellulose Nanocrystals: Synthesis, Characterization, and Antimicrobial Properties

Adherence and survival of pathogenic bacteria on surfaces leading to concomitant transmission to new hosts significantly contributes to the proliferation of pathogens, which in turn considerably increases the threat to human health, particularly by antibiotic-resistant bacteria. Consequently, more research into effective surface disinfection and alternative materials (fabrics, plastics, or coatings) with antimicrobial and other bioactive characteristics is desirable. This report describes the synthesis and characterization of cellulose nanocrystals that were surface-modified with a cationic porphyrin. The porphyrin was appended onto the cellulose surface via the Cu(I)-catalyzed Huisgen-Meldal-Sharpless 1,3-dipolar cycloaddition having occurred between azide groups on the cellulosic surface and porphyrinic alkynes. The resulting, generally insoluble, crystalline material, CNC-Por (5), was characterized by infrared and diffusion (1)H NMR spectroscopies, gel permeation chromatography, and thermogravimetric analysis. Although only suspended, and not dissolved, in an aqueous system, CNC-Por (5) showed excellent efficacy toward the photodynamic inactivation of Mycobacterium smegmatis and Staphylococcus aureus , albeit only slight activity against Escherichia coli . The synthesis, properties, and activity of CNC-Por (5) described herein serve as a benchmark toward our overall objectives of developing novel, potent, bioactive, photobactericidal materials that are effective against a range of bacteria, with potential utilization in the health care and food preparation industries.

The Met-Tyr-Trp cross-link in Mycobacterium tuberculosis catalase-peroxidase (KatG): autocatalytic formation and effect on enzyme catalysis and spectroscopic properties.

Catalase-peroxidases (KatG) are bifunctional enzymes possessing both catalase and peroxidase activities. Three crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link that has been suggested to impart catalatic activity to the KatGs. High-performance liquid chromatographic separation of the peptide fragments resulting from tryptic digestion of recombinant Mycobacterium tuberculosis WT KatG identified a peptide with unusual UV-visible spectroscopic features attributable to the Met255-Tyr229-Trp107 cross-link, whose structure was confirmed by mass spectrometry. WT KatG lacking the Met-Tyr-Trp cross-link was prepared, making possible studies of its formation under oxidizing conditions that generate either compound I (peroxyacetic acid, PAA) or compound II (2-methyl-1-phenyl-2-propyl hydroperoxide, MPPH). Incubation of this “cross-link-free” WT KatG with PAA revealed complete formation of the Met-Tyr-Trp structure after six equivalents of peracid were added, whereas MPPH was unable to promote cross-link formation. A mechanism for Met-Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies of WT KatG and KatG(Y229F), a mutant in which the cross-link cannot be formed, were performed with MPPH and revealed an unusual compound II spectrum for WT KatG, best described as (P·)FeIII, where P· represents a protein-based radical. This contrasts with the oxoferryl compound II spectrum observed for KatG(Y229F) under identical conditions. The structure-function-spectroscopy relationship in KatG is discussed with relevance to the role that the Met-Tyr-Trp cross-link plays in the catalase-peroxidase mechanism.

Isoniazid‐resistance conferring mutations in Mycobacterium tuberculosis KatG: Catalase, peroxidase, and INH‐NADH adduct formation activities

Mycobacterium tuberculosis catalase‐peroxidase (KatG) is a bifunctional hemoprotein that has been shown to activate isoniazid (INH), a pro‐drug that is integral to frontline antituberculosis treatments. The activated species, presumed to be an isonicotinoyl radical, couples to NAD+/NADH forming an isoniazid‐NADH adduct that ultimately confers anti‐tubercular activity. To better understand the mechanisms of isoniazid activation as well as the origins of KatG‐derived INH‐resistance, we have compared the catalytic properties (including the ability to form the INH‐NADH adduct) of the wild‐type enzyme to 23 KatG mutants which have been associated with isoniazid resistance in clinical M. tuberculosis isolates. Neither catalase nor peroxidase activities, the two inherent enzymatic functions of KatG, were found to correlate with isoniazid resistance. Furthermore, catalase function was lost in mutants which lacked the Met‐Tyr‐Trp crosslink, the biogenic cofactor in KatG which has been previously shown to be integral to this activity. The presence or absence of the crosslink itself, however, was also found to not correlate with INH resistance. The KatG resistance‐conferring mutants were then assayed for their ability to generate the INH‐NADH adduct in the presence of peroxide (t‐BuOOH and H2O2), superoxide, and no exogenous oxidant (air‐only background control). The results demonstrate that residue location plays a critical role in determining INH‐resistance mechanisms associated with INH activation; however, different mutations at the same location can produce vastly different reactivities that are oxidant‐specific. Furthermore, the data can be interpreted to suggest the presence of a second mechanism of INH‐resistance that is not correlated with the formation of the INH‐NADH adduct.

Porphyrin‐Cellulose Nanocrystals: A Photobactericidal Material that Exhibits Broad Spectrum Antimicrobial Activity†

Towards our overall objectives of developing potent antimicrobial materials to combat the escalating threat to human health posed by the transmission of surface‐adhering pathogenic bacteria, we have investigated the photobactericidal activity of cellulose nanocrystals that have been modified with a porphyrin‐derived photosensitizer (PS). The ability of these previously synthesized porphyrin‐cellulose‐nanocrystals (CNC‐Por (1)) to mediate bacterial photodynamic inactivation was investigated as a function of bacterial strain, incubation time and illumination time. Despite forming an insoluble suspension, CNC‐Por (1) showed excellent efficacy toward the photodynamic inactivation of Acinetobacter baumannii, multidrug‐resistant Acinetobacter baumannii (MDRAB) and methicillin‐resistant Staphylococcus aureus (MRSA), with the best results achieving 5–6 log units reduction in colony forming units (CFUs) upon illumination with visible light (400–700 nm; 118 J cm−2). CNC‐Por (1) mediated the inactivation of Pseudomonas aeruginosa, although at reduced activity (2–3 log units reduction). Confocal laser scanning microscopy of CNC‐Por (1) after incubation with A. baumannii or S. aureus suggested a lack of internalization of the PS. Research into alternative materials such as CNC‐Por (1) may lead to their application in hospitals and healthcare‐related industries wherein novel materials with the capability of reducing the rates of transmission of a wide range of bacteria, particularly antibiotic resistant strains, are desired.

Characterization of Dehaloperoxidase Compound ES and Its Reactivity with Trihalophenols

Dehaloperoxidase (DHP), the oxygen transport hemoglobin from the terebellid polychaete Amphitrite ornata, is the first globin identified to possess a biologically relevant peroxidase activity. DHP has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a substrate. Herein, we demonstrate that the first detectable intermediate following the addition of hydrogen peroxide to ferric DHP contains both a ferryl heme and a tyrosyl radical, analogous to Compound ES of cytochrome c peroxidase. Furthermore, we provide a detailed kinetic description for the reaction of preformed DHP Compound ES with the substrate 2,4,6-trichlorophenol and demonstrate the catalytic competency of this intermediate in generating the product 2,4-dichloroquinone. Using rapid-freeze-quench electron paramagnetic resonance spectroscopy, we detected a g approximately 2.0058 signal confirming the presence of a protein radical in DHP Compound ES. In the absence of substrate, DHP Compound ES evolves to a new species, Compound RH, which is functionally unique to dehaloperoxidase. We propose that this intermediate plays a protective role against heme bleaching. While unreactive toward further oxidation, Compound RH can be reduced and subsequently bind dioxygen, generating oxyferrous DHP, which may represent the catalytic link between peroxidase and oxygen transport activities in this bifunctional protein.

Reactivity of Deoxy- and Oxyferrous Dehaloperoxidase B from Amphitrite ornata: Identification of Compound II and Its Ferrous—Hydroperoxide Precursor

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. The bifunctional nature of DHP as a globin peroxidase appears to be at odds with the traditional starting oxidation state for each individual activity. Namely, reversible oxygen binding is only mediated via a ferrous heme in globins, and peroxidase activity is initiated from ferric centers and to the exclusion of the oxyferrous oxidation state from the peroxidase cycle. Thus, to address what appears to be a paradox, herein we report the details of our investigations into the DHP catalytic cycle when initiated from the deoxy- and oxyferrous states using biochemical assays, stopped-flow UV-visible, and rapid-freeze-quench electron paramagnetic resonance spectroscopies, and anaerobic methods. We demonstrate the formation of Compound II directly from deoxyferrous DHP B upon its reaction with hydrogen peroxide and show that this occurs both in the presence and in the absence of trihalophenol. Prior to the formation of Compound II, we have identified a new species that we have preliminarily attributed to a ferrous-hydroperoxide precursor that undergoes heterolysis to generate the aforementioned ferryl intermediate. Taken together, the results demonstrate that the oxyferrous state in DHP is a peroxidase competent starting species, and an updated catalytic cycle for DHP is proposed in which the ferric oxidation state is not an obligatory starting point for the peroxidase catalytic cycle of dehaloperoxidase. The data presented herein provide a link between the peroxidase and oxygen transport activities, which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system.

Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B from Amphitrite ornata

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. Of the two DHP isoenzymes identified to date, much of the recent focus has been on DHP A, whereas very little is known pertaining to the activity, substrate specificity, mechanism of function, or spectroscopic properties of DHP B. Herein, we report the recombinant expression and purification of DHP B, as well as the details of our investigations into its catalytic cycle using biochemical assays, stopped-flow UV-visible, resonance Raman, and rapid freeze-quench electron paramagnetic resonance spectroscopies, and spectroelectrochemistry. Our experimental design reveals mechanistic insights and kinetic descriptions of the dehaloperoxidase mechanism which have not been previously reported for isoenzyme A. Namely, we demonstrate a novel reaction pathway in which the products of the oxidative dehalogenation of trihalophenols (dihaloquinones) are themselves capable of inducing formation of oxyferrous DHP B, and an updated catalytic cycle for DHP is proposed. We further demonstrate that, unlike the traditional monofunctional peroxidases, the oxyferrous state in DHP is a peroxidase-competent starting species, which suggests that the ferric oxidation state may not be an obligatory starting point for the enzyme. The data presented herein provide a link between the peroxidase and oxygen transport activities which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system.

Compound ES of Dehaloperoxidase Decays via Two Alternative Pathways Depending on the Conformation of the Distal Histidine

Dehaloperoxidase (DHP) is a respiratory hemoglobin (Hb) that has been shown to catalyze the conversion of trihalophenols to dihaloquinones in the presence of hydrogen peroxide. Ferric heme states of the resting DHP and the free radical intermediates formed under H2O2 treatment were studied by low-temperature electron paramagnetic resonance spectroscopy in the range of reaction times from 50 ms to 2 min at three different pH values. Two high-spin ferric heme forms were identified in the resting enzyme and assigned to the open and closed conformations of the distal histidine, His55. Two free radicals were found in DHP activated by H2O2: the radical associated with Compound ES (the enzyme with the heme in the oxoferryl state and a radical on the polypeptide chain) has been assigned to Tyr34, and the other radical has been assigned to Tyr38. The Tyr34 radical is formed with a very high relative yield (almost 100% of heme), atypical of other globins. High-performance liquid chromatography analysis of the reaction products showed a pH-dependent formation of covalent heme-to-protein cross-links. The stable DHP Compound RH, formed under H2O2 in the absence of the trihalophenol substrates, is proposed to be a state with the ferric heme covalently cross-linked to Tyr34. A kinetic model of the experimental data suggests that formation of Compound RH and formation of the Tyr38 radical are two alternative routes of Compound ES decay. Which route is taken depends on the conformation of His55: in the less populated closed conformation, the Tyr38 radical is formed, but in the major open conformation, Compound ES decays, yielding Compound RH, a product of safe termination of the two oxidizing equivalents of H2O2 when no substrate is available.

The dehaloperoxidase paradox

The dual functions of the dehaloperoxidase-hemoglobin of Amphitrite ornata leads to a paradox. Peroxidase and hemoglobin functions require ferric and ferrous resting states, respectively. Assuming that hemoglobin function is the dominant function, the starting point for peroxidase activation would be the oxyferrous state. Activation of that state leads to the ferryl intermediate, followed by one-electron oxidation of the substrate, which results in the ferric state. Since no exogenous reductant is known, there is no return to the ferrous form or hemoglobin function. The observation that an internal binding site for 4-bromophenol leads to inhibition leads to a further paradox that the enzyme would be inhibited immediately upon activation under ambient conditions in benthic ecosystems where the inhibitor, 4-bromophenol is present in greater concentration than the substrate, 2,4,6-tribromophenol. In this review, we explore the unresolved aspects of the reaction scheme that leads to the apparent paradox. Recent data showing activation of the oxyferrous state, an extremely high reduction potential and exogenous reduction by the 2,6-dibromoquinone product present a potential resolution of the paradox. These aspects are discussed in the context of control of reactivity radical pathways and reactivity by the motion of the distal histidine, H55, which in turn is coupled to the binding of substrate and inhibitor.

Molecular oxygen and sulfur reactivity of a cyclotriveratrylene derived trinuclear copper(I) complex.

Our continuing efforts into developing copper coordination chemistry relevant to dioxygen-processing copper proteins has led us to design and synthesize a cyclotriveratrylene (CTV)-based trinucleating ligand, CTV-TMPA, which employs tetradentate tris(2-pyridylmethyl)-amine chelates (TMPA) for their copper ion binding sites. Binding of three copper ions per CTV-TMPA unit was established by various chemical and spectroscopic methods such as UV-vis and resonance Raman (rR) spectroscopies. The following complexes were observed: A tricopper(I) complex [(CTV-TMPA)Cu(I)(3)](3+) (1), a CO adduct [(CTV-TMPA)Cu(I)(3)(CO)(3)](3+) (1-CO; nu(C=O) = 2094 cm(-1)), a triphenylphosphine adduct [(CTV-TMPA)Cu(I)(3)(PPh(3))(3)](3+) (1-PPh(3)), a tricopper(II) complex [(CTV-TMPA)Cu(II)(3)](3+) (1-Ox), and its tris-monochloride or tris-monobromide adducts. Also, introduction of dioxygen to the -80 degrees C solutions of 1 leads to O(2)-adducts, the first example of a synthetic copper complex which can stabilize a mononuclear Cu(II)-superoxo and dinuclear peroxo species simultaneously within one complex {[Cu] = 1.53 mM in THF: (mu-1,2-peroxo complex, lambda(max) = 543 (epsilon 9650) nm): nu(O-O) = 825 ((Delta(18)O(2)) = -47) cm(-1); nu(Cu-O) = 506 ((Delta(18)O(2)) = -26) cm(-1): (superoxo complex, lambda(max) = 427 (epsilon 3150) nm): nu(O-O) = 1129 ((Delta(18)O(2)) = -60) cm(-1); nu(Cu-O) = 463 ((Delta(18)O(2)) = -27) cm(-1)}. Elemental sulfur reacts reversibly with 1 leading to a (proposed) hexanuclear species [{(CTV-TMPA)Cu(II)(3)}(2)(mu-1,2-S(2)(2-))(3)](6+) (1-S) {lambda(max) = 544 (epsilon 7270) nm}, possessing one dicopper(II)-disulfide structural type: {THF solvent) nu(S-S) = 489 ((Delta(34)S) = -10) cm(-1); nu(Cu-S) = 307 ((Delta(34)S) = -5) cm(-1)}. Derivation of spectroscopic, structural, and chemical conclusions were aided by the study of a close mononuclear analogue with one pyridyl group of the TMPA parent possessing a 6-CH(2)OCH(3) substituent, this being part of the CTV-TMPA architecture.