Thin-layer spectroelectrochemistry of the Fe(III)/Fe(II) redox reaction of dehaloperoxidase-hemoglobin
Highlights
► Dehaloperoxidase (DHP) is a hemoglobin with high intrinsic peroxidase activity. ► We measured Fe(III/II) reduction potential via thin-layer spectroelectrochemistry. ► Reduction potential value is more positive than other known globins. ► Distal histidine position and conformation lead to positive potential shift. ► A thermodynamic square scheme couples conformational change to electron transfer.
Introduction
Hemoglobin (Hb) and myoglobin (Mb) are heme proteins (“globins”) known universally for their respiratory functions of dioxygen transport and storage in vertebrate and some invertebrate organisms [1]. Understanding the electrochemical properties of globins has been a topic of longstanding interest as evidenced by the publication in 1923 of Fe(III)/Fe(II) reduction potentials (E°′) for mammalian Hb and Mb [2], [3]. Reduction potentials of globins have moderately positive values (100 ± 50 mV vs SHE) commensurate with preferential stabilization of the ferrous heme state that is required for dioxygen binding. Interfacial electron transfer (ET) properties of hemoglobin and myoglobin have also been investigated over a sustained period of time with reports first appearing in the 1970s [4], [5], [6]. Although interfacial ET rates for globin Fe(III)/Fe(II) heme conversion were found to be sluggish in comparison to electron transport proteins such as cytochromes [7], [8], suitably rapid and reproducible kinetics were achieved in due course that furthered significant advances [9], [10], [11], [12]. In recent years, the literature on globin electrochemistry has burgeoned with the growing recognition that hemoglobin and myoglobin can serve as effective transduction elements in amperometric biosensors for the detection of H2O2, trichloroacetic acid, nitric oxide, and other substrates [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]. To develop a biosensor of this type requires an effective electronic coupling of the enzymatic reactions of an immobilized globin to the substrate electrode. The work of Rusling’s group in the 1990s on the confinement of electroactive heme proteins in organized lipid and layer-by-layer films and the electrochemical enabling of their high valent Fe(IV) states provided much of the groundwork for these recent advances in sensor development [25], [26], [27], [28]. Although the ability of myoglobin and hemoglobin to utilize their Fe(IV) redox states to perform peroxidase-like chemistry is well established [29], [30], [31], [32], [33], their catalytic efficiencies are known to be considerably inferior to true peroxidases (vide infra).
In this paper, we report on the electrochemistry of a unique dual-function globin, Amphitrite ornata dehaloperoxidase-hemoglobin (DHP), a globin of classic 3-over-3 α-helical structure that, in addition to having a conventional dioxygen-binding capability [34], [35], features a much higher level of intrinsic peroxidase activity than do common hemoglobins and myoglobins [33], [36], [37], [38], [39]. In light of the mandatory role played by Fe(IV)-based catalysis in globin-based biosensors (vide supra), DHP may represent an attractive target protein for the development of more efficient devices of this kind. In A. ornata, two genes (dhp A and dhp B) [40] code for slightly different DHP isoforms (DHP A and DHP B), both of which exhibit enhanced peroxidase activity [41]. All data reported herein pertain to the DHP A isoform.
The organism that produces DHP, A. ornata, is a marine annelid that thrives in estuarine mud flats cohabited by other annelids such as Saccoglossus kowalewskii and Notomastus lobatus, which secrete 2,4,6-tribromophenol (2,4,6-TBP) and other haloaromatic toxicants for territorial defense [42], [43], [44]. In this environment, the survival of A. ornata relies on DHP’s ability to enzymatically dehalogenate brominated phenols to less toxic species [45]. In the presence of H2O2, DHP catalyzes the 2-electron oxidation of 2,4,6-TBP to 2,6-dibromoquinone (2,6-DBQ) [33], [45], a capability that extends to 2,4,6-trichloro- and 2,4,6-trifluorophenols [41], [46], [47]. Stopped-flow kinetics experiments have shown that DHP oxidizes 2,4,6-TBP to 2,6-DBQ at rates approximately tenfold slower than horseradish peroxidase but approximately tenfold faster than HHMb [33]. The net reaction can be represented as follows:
The peroxidase activity of DHP can also be judged by considering rates of O–O bond cleavage, a key step in the mechanism of peroxidase catalysis. The initial reaction step, in which H2O2 binds to the ferric heme iron, gives rise to an oxo-ferryl species (Compound ES or Compound I) that functions as the substrate oxidant (see Scheme 1). For DHP A at pH 7.0, the rate constant for O–O bond cleavage has been reported as k2 = 3.56 × 104 M−1 s−1 [47]. In comparison, for globins and peroxidases, the respective values are ∼102 M−1 s−1 and upwards of 107 M−1 s−1 [48]. Although less potent than a true peroxidase, the level of catalytic activity exhibited by DHP A is unprecedented among globins.
Herein we report measurements of the anaerobic Fe(III)/Fe(II) reduction potential of DHP A. The results, when considered in the context of known globin and peroxidase reduction potentials, proved to be surprising. Globins have moderately positive reduction potentials (∼50 mV for Mb; ∼150 mV for Hb) [3], which are compatible with stabilization of the Fe(II) resting state required for binding and release of dioxygen. Peroxidases, on the other hand, feature more negative reduction potentials that serve to stabilize the Fe(III) resting state of the catalytic cycle. Cytochrome c peroxidase and horseradish peroxidase, for example, have values of −182 mV [49] and −266 mV [50], respectively. Given its decidedly peroxidase-like character, it would have been unsurprising to find the reduction potential of DHP A shifted from typical globin values towards the more negative peroxidase potential region. However, not only is such a negative shift absent but, rather, the DHP A reduction potential [E°′ =+ 202 ± 6 mV vs SHE (pH 7.0)] is actually more positive than any value previously reported for an intracellular globin. To develop insight into this unexpected finding, a comparative Gibbs free energy analysis of the crystal structures of DHP A, myoglobin, and hemoglobin, was undertaken. Our analysis implicates the distal histidine, H55, as the key to understanding the DHP A reduction potential. Because H55 is positioned further from the heme iron than in a typical globin, its ability to stabilize a ligated water molecule by hydrogen bonding is diminished, resulting in destabilization of the Fe(III) state. In addition, DHP A undergoes an unusual conformational change with H55 alternating between an internal position (“closed conformation”) and an external position (“open conformation”). This conformational change is redox state dependent with the open conformation favored by the Fe(II) state. A redox square scheme of the type introduced by Laviron and Roullier [51] is proposed as a basis for explaining how ET-coupled conformational change affects the reduction potential.
Section snippets
Chemicals
N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD, 95%), dichlorophenol–indophenol (DCIP), potassium ferricyanide ([Fe(CN)6]3+, 99+%), phenazine methosulfate (PhMS, 90+%), hexaammineruthenium(III) chloride ([Ru(NH3)6]3+, 98%), ruthenium(III) chloride hydrate, ethylenediamine (99+%), zinc dust (<10 μm, 98+%)), and all buffer salts were purchased from Sigma. Tris(ethylenediamine)ruthenium(II) tetrachlorozincate ([Ru(en)3]2+) was synthesized and re-crystallized as previously reported [52], [53]. All
Spectroelectrochemistry
Fig. 1A shows a representative set of thin layer UV–VIS spectra obtained for DHP A following attainment of equilibrium at various applied potentials. Under an oxidizing potential, such as EAPP = 0.497 V, a Soret peak is observed at 407 nm corresponding to the ferric state. At a limiting reducing potential, such as EAPP = −0.103 V, a Soret peak is observed at 432 nm, which corresponds to deoxyferrous DHP A. Fig. 1B displays the Nernst plot obtained by conventional analysis [56] of absorbance data for
Discussion
Given its characteristic globin structure and ability to reversibly bind O2, the fact that the Fe(III)/(Fe(II) reduction potential of DHP A was found to be considerably more positive than any known peroxidase was not unexpected. On the other hand, it was surprising, in light of its enhanced peroxidase activity, to find a reduction potential that was the most positive among all known intracellular globins (i.e., ∼50 mV above typical Hbs and ∼150 mV above typical Mbs [3]). Among the globins, only
Conclusions
DHP A has the highest known Fe(III)/Fe(II) reduction potential for an intracellular globin, an unusual observation given that it has enhanced peroxidase activity. To elucidate the means by which DHP A accomplishes this feat, we have undertaken a comparative Gibbs free energy analysis using available crystal structures of DHP A, myoglobin, and hemoglobin. Our analysis has identified two key factors originating on the distal side of the protein as a basis for explaining the observed potential.
Acknowledgements
This project was supported by Army Research Office Grants 51432-CH-SR (E.F.B.) and 52278-LS (S.F.). We thank Professor Reza A. Ghiladi and Dr. Matthew K. Thompson for helpful discussions.
References (79)
J. Biol. Chem.
(1923)- et al.
Biochim. Biophys. Acta
(1975) - et al.
Bioelectrochem. Bioenerg.
(1976) - et al.
Bioelectrochem. Bioenerg.
(1977) - et al.
J. Electroanal. Chem.
(1992) - et al.
J. Electroanal. Chem.
(1987) - et al.
Adv. Colloid Interface Sci.
(2005) - et al.
J. Electroanal. Chem.
(2001) - et al.
Anal. Biochem.
(2004) - et al.
Talanta
(2005)
J. Biol. Chem.
Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol.
Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol.
J. Biol. Chem.
Biochem. Biophys. Res. Commun.
Biochem. Syst. Ecol.
J. Biol. Chem.
J. Electroanal. Chem.
J. Inorg. Biochem.
J. Inorg. Biochem.
Inorg. Chim. Acta
Biochim. Biophys. Acta
Biophys. Chem.
J. Molec. Biol.
Hemoglobin and Myoglobin in their Reactions with Ligands
Oxidation–Reduction Potentials of Organic Systems
Comprehensive Treatise of Electrochemistry, Volume 10: Bioelectrochemistry
J. Am. Chem. Soc.
Chem. Lett.
J. Am. Chem. Soc.
Anal. Sci.
Microchim. Acta
Chembiochem
Biomacromolecules
Anal. Chem.
J. Am. Chem. Soc.
Langmuir
J. Phys. Chem. B
J. Am. Chem. Soc.
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Present address: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323, USA.