Jing Du

University of South Carolina, Columbia, South Carolina, United States

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Publications (12)53.83 Total impact

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    ABSTRACT: The coelomic O2-binding hemoglobin dehaloperoxidase (DHP) from the sea worm Amphitrite ornata is a dual function heme protein that also possesses a peroxidase activity. Two different starting oxidation states are required for reversible O2 binding (ferrous) and peroxidase (ferric) activity bringing into question how DHP manages the two functions. In our previous study, the co-presence of substrate 2,4,6-trichorophenol (TCP) and H2O2 was found to be essential for the conversion of oxy-DHP to enzymatically active ferric DHP. Based on that study, a functional switching mechanism involving substrate radicals (TCP•) was proposed. To further support this mechanism, herein we report details of our investigations into the H2O2-mediated conversion of oxy-DHP to the ferric or ferryl ([TCP] < [H2O2]) state triggered by both biologically relevant [TCP, 4-bromophenol (4-BP)] and non-relevant (ferrocyanide) compounds. When [H2O2] < 50 μM, all of these conversion reactions are completely inhibited by ferric heme ligands (KCN, imidazole), indicating an involvement of ferric DHP. Furthermore, the spin-trapping reagent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) effectively inhibits the TCP/4-BP (but not ferrocyanide)-triggered oxy- to ferric DHP conversion. These results and [O2]-dependent conversion rates observed in this study demonstrate that substrate TCP triggers the conversion of oxy-DHP into a peroxidase by TCP• oxidation of the deoxyferrous state. TCP• is cumulatively generated, with increasingly produced ferric DHP, upon H2O2-oxidation of TCP catalyzed initially by trace amounts of ferric DHP present in the oxy-DHP sample. The data presented herein further address the mechanism of how the halophenolic substrate triggers the conversion of hemoglobin DHP into a peroxidase.
    Biochemistry 06/2014; · 3.38 Impact Factor
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    ABSTRACT: Sea worm, Amphitrite ornata, has evolved its globin (an O2 carrier) also to serves as a dehaloperoxidase (DHP) to detoxify haloaromatic pollutants generated by competing species. A previous mutagenesis study by our groups on both DHP and sperm whale myoglobin (SW Mb) revealed some structural factors that influence the dehaloperoxidase activities (significantly lower for Mb) of both proteins. Using an isocyanide/O2 partition constant measurement method in this study, we have examined the effects of these structural factors on the O2 equilibrium constants (KO2) of DHP, SW Mb, and their mutants. A clear trend of decreasing O2 affinity and increasing catalytic activity along with the increase in the distal His N(ε2)-heme iron distance is observed. An H93K/T95H Mb double mutant mimicking the DHP proximal His positioning exhibited markedly enhanced O2 affinity, confirming the essential effect of proximal His rotation on the globin function of DHP. For DHP, the L100F, T56G and M86E variants showed the effects of distal volume, distal His flexibility and proximal electronic push, respectively, on the O2 affinity. This study provides insights into how DHP has evolved its heme environment to gain significantly enhanced peroxidase capability without compromising its primary function as an O2 carrier.
    Archives of Biochemistry and Biophysics 01/2014; · 3.37 Impact Factor
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    ABSTRACT: It is generally accepted that the inactive P420 form of cytochrome P450 (CYP) involves the protonation of the native cysteine thiolate to form a neutral thiol heme ligand. On the other hand, it has also been suggested that recruitment of a histidine to replace the native cysteine thiolate ligand might underlie the P450→P420 transition. Here we discuss resonance Raman investigations of the H93G myoglobin (Mb) mutant in the presence of tetrahydrothiophene (THT) or cyclopentathiol (CPSH), and on pressure-induced cytochrome P420cam (CYP101), that show a histidine becomes the heme ligand upon CO binding. The Raman mode near 220 cm-1, normally associated with the Fe-histidine vibration in heme proteins, is not observed in either reduced P420cam or the reduced H93G Mb samples, indicating that histidine is not the ligand in the reduced state. The absence of a mode near 220 cm-1 is also inconsistent with a generalization of the suggestion that the 221 cm-1 Raman mode, observed in the P420-CO photoproduct of inducible nitric oxide synthase (iNOS), arises from a thiol-bound ferrous heme. This leads us to assign the 218 cm-1 mode observed in the 10 ns P420cam-CO photoproduct Raman spectrum to a Fe-histidine vibration, in analogy to many other histidine bound heme systems. Additionally, the inverse correlation plots of the νFe-His and νCO frequencies for the CO adducts of P420cam and the H93G analogs provide supporting evidence that histidine is the heme ligand in the P420-CO bound state. We conclude that, when CO binds to the ferrous P420 state, a histidine ligand is recruited as the heme ligand. The common existence of a HXC-Fe motif in many CYP systems allows the C→H ligand switch to occur with only minor conformational changes. One suggested conformation of P420-CO involves the addition of another turn in the proximal L helix so that, when the protonated Cys ligand is dissociated from the heme, it can become part of the helix and the heme is ligated by the His residue from the adjoining loop region. In other systems, such as iNOS and CYP3A4 (where the HXC-Fe motif is not found) a somewhat larger conformational change would be necessary to recuit a nearby histidine.
    Biochemistry 08/2013; · 3.38 Impact Factor
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    ABSTRACT: The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work, we use spectroscopic (absorption, electron paramagnetic resonance, and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low-spin in both ferric and ferrous oxidation states. Rv0203-notag is high-spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen-ligated heme proteins. Mutagenesis experiments determined that residues Tyr59, His63, and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63, and His89 are required for the binding of heme to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggests the heme ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for binding of heme to Rv0203-His and Rv0203-notag are similar, 115 and 133 μM(-1) s(-1), respectively. However, the heme off rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 and 0.0002 s(-1), respectively, and Rv0203-notag has a single off rate of 0.082 s(-1). The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His tag interferes with heme binding. Furthermore, these results imply that the His tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme transport proteins, implying that Rv0203 may act as an extracellular heme transporter.
    Biochemistry 02/2012; 51(7):1518-31. · 3.38 Impact Factor
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    ABSTRACT: Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O(2) transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity ∼10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of "peroxidase-like" Mb mutants and "Mb-like" DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned ∼0.3 and ∼0.8 Å, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the "DHP-like" position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the molecular evolution of the dual-function enzyme DHP.
    Biochemistry 07/2011; 50(38):8172-80. · 3.38 Impact Factor
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    ABSTRACT: Preparation of heme model complexes is a challenging subject of long-standing interest for inorganic chemists. His93Gly sperm whale myoglobin (H93G Mb) has the proximal His replaced with the much smaller non-coordinating Gly. This leaves a cavity on the proximal side of the heme into which a wide variety of exogenous ligands can be delivered. The end result is a remarkably versatile scaffold for the preparation of model heme adducts to mimic the heme iron coordination structure of native heme proteins. In this review, we first summarize the quantitative evidence for differential ligand binding affinities of the proximal and distal pockets of the H93G Mb cavity mutant that facilitates the preparation of mixed-ligand derivatives. Then we review our use of magnetic circular dichroism and electronic absorption spectroscopy to characterize nitrogen-, oxygen-, and sulfur-donor-ligated H93G Mb adducts with an emphasis on species not easily prepared by other heme model system approaches and those that serve as spectroscopic models for native heme proteins.
    Coordination Chemistry Reviews 04/2011; 255(7-8):700-716. · 11.02 Impact Factor
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    ABSTRACT: Cytochrome P450-mediated monooxygenation generally proceeds via a reactive ferryl intermediate coupled to a ligand radical [Fe(IV)═O]+• termed Compound I (Cpd I). The proximal cysteine thiolate ligand is a critical determinant of the spectral and catalytic properties of P450 enzymes. To explore the effect of an increased level of donation of electrons by the proximal ligand in the P450 catalytic cycle, we recently reported successful incorporation of SeCys into the active site of CYP119, a thermophilic cytochrome P450. Here we report relevant physical properties of SeCYP119 and a detailed analysis of the reaction of SeCYP119 with m-chloroperbenzoic acid. Our results indicate that the selenolate anion reduces rather than stabilizes Cpd I and also protects the heme from oxidative destruction, leading to the generation of a new stable species with an absorbance maximum at 406 nm. This stable intermediate can be returned to the normal ferric state by reducing agents and thiols, in agreement with oxidative modification of the selenolate ligand itself. Thus, in the seleno protein, the oxidative damage shifts from the heme to the proximal ligand, presumably because (a) an increased level of donation of electrons more efficiently quenches reactive species such as Cpd I and (b) the protection of the thiolate ligand provided by the protein active site structure is insufficient to shield the more oxidizable selenolate ligand.
    Biochemistry 03/2011; 50(14):3014-24. · 3.38 Impact Factor
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    ABSTRACT: His93Gly sperm whale myoglobin (H93G Mb) has the proximal histidine ligand removed to create a cavity for exogenous ligand binding, providing a remarkably versatile template for the preparation of model heme complexes. The investigation of model heme adducts is an important way to probe the relationship between coordination structure and catalytic function in heme enzymes. In this study, we have successfully generated and spectroscopically characterized the H93G Mb cavity mutant ligated with less common alkylamine ligands (models for Lys or the amine group of N-terminal amino acids) in numerous heme iron states. All complexes have been characterized by electronic absorption and magnetic circular dichroism spectroscopy in comparison with data for parallel imidazole-ligated H93G heme iron moieties. This is the first systematic spectral study of models for alkylamine- or terminal amine-ligated heme centers in proteins. High-spin mono- and low-spin bis-amine-ligated ferrous and ferric H93G Mb adducts have been prepared together with mixed-ligand ferric heme complexes with alkylamine trans to nitrite or imidazole as heme coordination models for cytochrome c nitrite reductase or cytochrome f, respectively. Six-coordinate ferrous H93G Mb derivatives with CO, NO, and O(2) trans to the alkylamine have also been successfully formed, the latter for the first time. Finally, a novel high-valent ferryl species has been generated. The data in this study represent the first thorough investigation of the spectroscopic properties of alkylamine-ligated heme iron systems as models for naturally occurring heme proteins ligated by Lys or terminal amines.
    Inorganic Chemistry 02/2011; 50(4):1242-9. · 4.59 Impact Factor
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    ABSTRACT: Dehaloperoxidase (DHP) from Amphitrite ornata is a heme protein that can function both as a hemoglobin and as a peroxidase. This report describes the use of 77 K cryoreduction EPR/ENDOR techniques to study both functions of DHP. Cryoreduced oxyferrous [Fe(II)-O(2)] DHP exhibits two EPR signals characteristic of a peroxoferric [Fe(III)-O(2)(2-)] heme species, reflecting the presence of conformational substates in the oxyferrous precursor. (1)H ENDOR spectroscopy of the cryogenerated substates shows that H-bonding interactions between His N(ε)H and heme-bound O(2) in these conformers are similar to those in the β-chain of oxyferrous hemoglobin A (HbA) and oxyferrous myoglobin, respectively. Decay of cryogenerated peroxoferric heme DHP intermediates upon annealing at temperatures above 180 K is accompanied by the appearance of a new paramagnetic species with an axial EPR signal with g(⊥) = 3.75 and g(∥) = 1.96, characteristic of an S = 3/2 spin state. This species is assigned to Compound I (Cpd I), in which a porphyrin π-cation radical is ferromagnetically coupled with an S = 1 ferryl [Fe(IV)═O] ion. This species was also trapped by rapid freeze-quench of the ambient-temperature reaction mixture of ferric [Fe(III)] DHP and H(2)O(2). However, in the latter case Cpd I is reduced very rapidly by a nearby tyrosine to form Cpd ES [(Fe(IV)═O)(porphyrin)/Tyr(•)]. Addition of the substrate analogue 2,4,6-trifluorophenol (F(3)PhOH) suppresses formation of the Cpd I intermediate during annealing of cryoreduced oxyferrous DHP at 190 K but has no effect on the spectroscopic properties of the remaining cryoreduced oxyferrous DHP intermediates and kinetics of their decay. These observations indicate that substrate (i) binds to oxyferrous DHP outside of the distal pocket and (ii) can reduce Cpd I to Cpd II [Fe(IV)═O]. These assumptions are also supported by the observation that F(3)PhOH has only a small effect on the EPR properties of radiolytically cryooxidized and cryoreduced ferrous [Fe(II)] DHP. EPR spectra of cryoreduced ferrous DHP disclose the multiconformational nature of the ferrous DHP precursor. The observation and characterization of Cpds I, II, and ES in the absence and in the presence of F(3)PhOH provides definitive evidence of a mechanism involving consecutive one-electron steps and clarifies the role of all intermediates formed during turnover.
    Journal of the American Chemical Society 10/2010; 132(42):14995-5004. · 10.68 Impact Factor
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    ABSTRACT: Amphitrite ornata dehaloperoxidase (DHP) is the first heme-containing globin possessing a native peroxidase enzymatic activity. DHP catalyzes the H(2)O(2)-dependent dehalogenation of halophenols. By possessing this detoxifying enzymatic activity, these organisms are able to thrive in an environment contaminated with toxic haloaromatics. It has been proposed that DHP evolved from a dioxygen carrier globin protein and therefore possesses dual physiological roles of O(2) carrier and dehaloperoxidase. Although DHP is isolated in the catalytically inactive oxyferrous state (oxy-DHP), we find that the combination of H(2)O(2) and the substrate 2,4,6-trichlorophenol (TCP) brings about facile switching of oxy-DHP to the enzymatically active ferric state via a process likely involving substrate radicals (TCP*). In contrast, in the absence of TCP, H(2)O(2) alone converts oxy-DHP to an inactive state (compound RH) instead of oxidizing the enzyme to the ferric state. Further, although the rate of autoxidation of oxy-DHP is somewhat enhanced by the presence of TCP, the effect is too small to be the functional switch. Instead, both substrate and H(2)O(2) are needed to convert oxy-DHP to the catalytically active ferric state. These observations provide a physiological link between the O(2) carrier role of the ferrous protein and the peroxidase activity of the ferric enzyme in this bifunctional protein.
    Biochemistry 07/2010; 49(29):6064-9. · 3.38 Impact Factor
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    ABSTRACT: The mammalian circadian clock synchronizes physical and metabolic activity with the diurnal cycle through a transcriptional-posttranslational feedback loop. An additional feedback mechanism regulating clock timing has been proposed to involve oscillation in heme availability. Period 2 (PER2), an integral component in the negative feedback loop that establishes circadian rhythms in mammals, has been identified as a heme-binding protein. However, the majority of evidence for heme binding is based upon in vitro heme-binding assays. We sought to ascertain if these largely spectral assays could distinguish between specific and nonspecific heme interactions. Heme-binding properties by a number of other well-characterized proteins, all with no known biological role involving heme interaction, corresponded to those displayed by PER2. Site-directed mutants of putative heme-binding residues identified by MCD were unable to locate a specific heme-binding site on PER2. Protein film electrochemistry also indicates that heme binds PER2 nonspecifically on the protein surface. Our results establish the inability of qualitative in vitro assays to easily distinguish between specific and nonspecific heme binding. We conclude that heme binding to PER2 is likely to be nonspecific and does not involve the hydrophobic pocket within the PER2 PAS domains that in other PAS proteins commonly recognizes cofactors. These findings also question the significance of in vivo studies that implicate heme interactions with the clock proteins PER2 and nPAS2 in biological function.
    Biochemistry 05/2010; 49(20):4327-38. · 3.38 Impact Factor
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    ABSTRACT: His93Gly sperm whale myoglobin (H93G Mb) has the proximal histidine ligand removed to create a cavity for exogenous ligand binding, making it a versatile template for the preparation of model heme complexes. In this study, we have measured the first and second ligand binding affinities of imidazole and pyridine to form mono- and bis-ligated ferric and ferrous H93G Mb complexes. Electronic absorption spectroscopy has been utilized to determine the binding affinities for the proximal (Kd1, first ligand) and distal (Kd2, second ligand) pockets of H93G Mb. Magnetic circular dichroism spectroscopy has been used to confirm the identity of the complexes. The binding affinities for the first ligand are one hundred- to one thousand-fold higher than those for the second ligand (Kd1 « Kd2) for the same exogenous ligand. This is entirely opposite to what is seen with free heme in organic solvents where Kd1 » Kd2. Thus, the proximal pocket is the high affinity binding site. The lower affinity for the distal pocket can be attributed to steric hindrance from the distal histidine. This report provides quantitative evidence for differential ligand binding affinities of the proximal and distal pockets of H93G Mb, a unique property that facilitates generation of heme iron derivatives not easily prepared with other heme model systems.
    Spectroscopy 01/2008; 22. · 0.53 Impact Factor

Publication Stats

56 Citations
53.83 Total Impact Points

Institutions

  • 2008–2014
    • University of South Carolina
      • Department of Chemistry and Biochemistry
      Columbia, South Carolina, United States