Evidence of the Direct Involvement of the Substrate TCP Radical in Functional Switching from Oxyferrous O₂ Carrier to Ferric Peroxidase in the Dual-Function Hemoglobin/Dehaloperoxidase from <i>Amphitrite ornata</i>

The coelomic O₂-binding hemoglobin dehaloperoxidase (DHP) from the sea worm <i>Amphitrite ornata</i> 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 copresence of substrate 2,4,6-trichlorophenol (TCP) and H₂O₂ was found to be essential for the conversion of oxy-DHP to enzymatically active ferric DHP. On the basis of 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 and 4-bromophenol (4-BP)] and nonrelevant (ferrocyanide) compounds. At <50 μM H₂O₂, all of these conversion reactions are completely inhibited by ferric heme ligands (KCN and imidazole), indicating the involvement of ferric DHP. Furthermore, the spin-trapping reagent 5,5-dimethyl-1-pyrroline-<i>N</i>-oxide (DMPO) effectively inhibits the TCP/4-BP (but not ferrocyanide)-triggered conversion of oxy-DHP to ferric DHP. These results and O₂ concentration-dependent conversion rates observed in this study demonstrate that substrate TCP triggers the conversion of oxy-DHP to a peroxidase by TCP^• oxidation of the deoxyferrous state. TCP^• is progressively generated, by increasingly produced amounts of ferric DHP, upon H₂O₂ oxidation of TCP catalyzed initially by trace amounts of ferric enzyme 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

The Use of Deuterated Camphor as a Substrate in ¹H ENDOR Studies of Hydroxylation by Cryoreduced Oxy P450cam Provides New Evidence of the Involvement of Compound I

Electron paramagnetic resonance and ¹H electron nuclear double resonance (ENDOR) spectroscopies have been used to analyze intermediate states formed during the hydroxylation of (1<i>R</i>)-camphor (H₂-camphor) and (1<i>R</i>)-5,5-dideuterocamphor (D₂-camphor) as induced by cryoreduction (77 K) and annealing of the ternary ferrous cytochrome P450cam–O₂–substrate complex. Hydroxylation of H₂-camphor produced a primary product state in which 5-<i>exo</i>-hydroxycamphor is coordinated with Fe­(III). ENDOR spectra contained signals derived from two protons [Fe­(III)-bound C5-OH_<i>exo</i> and C5-H_<i>endo</i>] from camphor. When D₂-camphor was hydroxylated under the same condition in H₂O or D₂O buffer, both ENDOR H_<i>exo</i> and H_<i>endo</i> signals are absent. For D₂-camphor in H₂O buffer, H/D exchange causes the C5-OH_<i>exo</i> signal to reappear during relaxation upon annealing to 230 K; for H₂-camphor in D₂O, the magnitude of the C5-OH_<i>exo</i> signal decreases via H/D exchange. These observations clearly show that Compound I is the reactive species in the hydroxylation of camphor in P450cam

Characterization of Heme Ligation Properties of Rv0203, a Secreted Heme Binding Protein Involved in <i>Mycobacterium tuberculosis</i> Heme Uptake

The secreted <i>Mycobacterium tuberculosis</i> (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

Complexes of Dual-Function Hemoglobin/Dehaloperoxidase with Substrate 2,4,6-Trichlorophenol Are Inhibitory and Indicate Binding of Halophenol to Compound I

The hemoglobin of sea worm <i>Amphitrite ornata</i>, which for historical reasons is abbreviated as DHP for dehaloperoxidase, has two physiological functions: it binds dioxygen in the ferrous state and dehalogenates halophenols, such as 2,4,6-trichlorophenol (TCP), using hydrogen peroxide as the oxidant in the ferric state. The crystal structures of three DHP variants (Y34N, Y34N/S91G, and L100F) with TCP bound show two mutually exclusive modes of substrate binding. One of them, the internal site, is deep inside the distal pocket with the phenolic OH moiety forming a hydrogen bond to the water molecule coordinated to the heme Fe. In this complex, the distal histidine is predominantly located in the closed position and also forms a hydrogen bond to the phenolic hydroxide. The second mode of TCP binding is external, at the heme edge, with the halophenol molecule forming a lid covering the entrance to the distal cavity. The distal histidine is in the open position and forms a hydrogen bond to the OH group of TCP, which also hydrogen bonds to the hydroxyl of Tyr38. The distance between the Cl4 atom of TCP and the heme Fe is 3.9 Å (nonbonding). In both complexes, TCP molecules prevent the approach of hydrogen peroxide to the heme, indicating that the complexes are inhibitory and implying that the substrates must bind in an ordered fashion: hydrogen peroxide first and TCP second. Kinetic studies confirmed the inhibition of DHP by high concentrations of TCP. The external binding mode may resemble the interaction of TCP with Compound I, the catalytic intermediate to which halophenols bind. The measured values of the apparent <i>K</i>_m for TCP were in the range of 0.3–0.8 mM, much lower than the concentrations required to observe TCP binding in crystals. This indicates that during catalysis TCP binds to Compound I. Mutant F21W, which likely has the internal TCP binding site blocked, has ∼7% of the activity of wild-type DHP

Electron Paramagnetic Resonance and Electron-Nuclear Double Resonance Studies of the Reactions of Cryogenerated Hydroperoxoferric–Hemoprotein Intermediates

The fleeting ferric peroxo and hydroperoxo intermediates of dioxygen activation by hemoproteins can be readily trapped and characterized during cryoradiolytic reduction of ferrous hemoprotein–O₂ complexes at 77 K. Previous cryoannealing studies suggested that the relaxation of cryogenerated hydroperoxoferric intermediates of myoglobin (Mb), hemoglobin, and horseradish peroxidase (HRP), either trapped directly at 77 K or generated by cryoannealing of a trapped peroxo-ferric state, proceeds through dissociation of bound H₂O₂ and formation of the ferric heme without formation of the ferryl porphyrin π-cation radical intermediate, compound I (Cpd I). Herein we have reinvestigated the mechanism of decays of the cryogenerated hydroperoxyferric intermediates of α- and β-chains of human hemoglobin, HRP, and chloroperoxidase (CPO). The latter two proteins are well-known to form spectroscopically detectable quasistable Cpds I. Peroxoferric intermediates are trapped during 77 K cryoreduction of oxy Mb, α-chains, and β-chains of human hemoglobin and CPO. They convert into hydroperoxoferric intermediates during annealing at temperatures above 160 K. The hydroperoxoferric intermediate of HRP is trapped directly at 77 K. All studied hydroperoxoferric intermediates decay with measurable rates at temperatures above 170 K with appreciable solvent kinetic isotope effects. The hydroperoxoferric intermediate of β-chains converts to the <i>S</i> = 3/2 Cpd I, which in turn decays to an electron paramagnetic resonance (EPR)-silent product at temperature above 220 K. For all the other hemoproteins studied, cryoannealing of the hydroperoxo intermediate directly yields an EPR-silent majority product. In each case, a second follow-up 77 K γ-irradiation of the annealed samples yields low-spin EPR signals characteristic of cryoreduced ferrylheme (compound II, Cpd II). This indicates that in general the hydroperoxoferric intermediates relax to Cpd I during cryoanealing at low temperatures, but when this state is not captured by reaction with a bound substrate, it is reduced to Cpd II by redox-active products of radiolysis

Heme Binding Properties of Glyceraldehyde-3-phosphate Dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for the insertion of cellular heme into inducible nitric oxide synthase [Chakravarti, R., et al. (2010) <i>Proc. Natl. Acad. Sci. U.S.A. 107</i>, 18004–18009], we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin complex with UV–visible maxima at 362, 418, and 537 nm and when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were as follows: <i>k</i>_on = 17800 M^–1 s^–1, <i>k</i>_off1 = 7.0 × 10^–3 s^–1, and <i>k</i>_off2 = 3.3 × 10^–4 s^–1 (giving approximate affinities of 19–390 nM). Ferrous heme bound more poorly to GAPDH and dissociated with a <i>k</i>_off of 4.2 × 10^–3 s^–1. Magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data on the ferric, ferrous, and ferrous–CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in the ferric complex was not displaced by CN^– or N₃^– but in the ferrous complex could be displaced by CO at a rate of 1.75 s^–1 (for >0.2 mM CO). Studies with heme analogues revealed selectivity toward the coordinating metal and porphyrin ring structure. The GAPDH–heme complex was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-aminolevulinic acid. Our finding of heme binding to GAPDH expands the protein’s potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH are consistent with it performing heme sensing or heme chaperone-like functions in cells

Heme Binding by <i>Corynebacterium diphtheriae</i> HmuT: Function and Heme Environment

The heme uptake pathway (hmu) of <i>Corynebacterium diphtheriae</i> 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