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.

Dehaloperoxidase (DHP) from Amphitrite ornata is a heme protein that can function both as a hemoglobin and 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 NεH 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 discloses the multiconformational nature of the ferrous DHP precursor. The observation and characterization of Cpds I, II, and ES in the absence and 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.

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 identified 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 heme binding to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggest 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 heme binding to Rv0203-His and Rv0203-notag are similar, 115 (μM s)-1 and 133 (μM 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 s-1 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.