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Apr 17, 2017 - Ceruloplasmin (Cp) is one of the most complex multicopper oxidase enzymes and plays an essential role in the metabolism of iron in mamm...
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Molecular Aspects of a Robust Assay for Ferroxidase Function of Ceruloplasmin Laura Cortes,†,‡ Blaine R. Roberts,‡ Anthony G. Wedd,† and Zhiguang Xiao*,†,‡ †

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia ‡ The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria 3010, Australia S Supporting Information *

ABSTRACT: Ceruloplasmin (Cp) is one of the most complex multicopper oxidase enzymes and plays an essential role in the metabolism of iron in mammals. Ferrous ion supplied by the ferroportin exporter is converted by Cp to ferric ion that is accepted by plasma metallo-chaperone transferrin. Study of the enzyme at the atomic and molecular level has been hampered by the lack of a suitable ferrous substrate. We have developed the classic chromophoric complex FeIIHx(Tar)2 (H2Tar, 4-(2-thiazolylazo)resorcinol; x = 0−2; overall charge omitted) as a robust substrate for evaluation of the ferroxidase function of Cp and related enzymes. The catalysis can be followed conveniently in real time by monitoring the solution absorbance at 720 nm, a fingerprint of FeIIHx(Tar)2. The complex is oxidized to its ferric form FeIIIHx(Tar)2 via the overall reaction sequence FeIIHx(Tar)2 → FeII-Cp → FeIII-Cp → FeIIIHx(Tar)2: i.e., Fe(II) is transferred formally from FeIIHx(Tar)2 to the substrate docking/ oxidation (SDO) site(s) in Cp, followed by oxidation to product Fe(III) that is trapped again by the ligand. Each Tar ligand in the above bis-complex coordinates the metal center in a meridional tridentate mode involving a pH-sensitive −OH group (pKa > 12), and this imposes rapid Fe(II) and Fe(III) transfer kinetics to facilitate the catalytic process. The formation constants of both the ferrous and ferric complexes at pH 7.0 were determined (log β2′ = 13.6 and 21.6, respectively), as well as an average dissociation constant of the SDO site(s) in Cp (log KD′ = −7.2).

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INTRODUCTION Uptake and distribution of iron into eukaryotic cells is dependent upon molecules that bind copper.1,2 Consequently, the metabolisms of these two essential metal nutrients are linked inextricably and their inappropriate handling is associated with many disease states.3,4 The key link in mammals is ceruloplasmin (Cp) and its analogues. These multicopper oxidase enzymes mediate transfer of the iron from the cell export pump ferroportin (Fpn) to the plasma metallo-chaperone transferrin (Tf; Figure 1). Specifically, they are responsible for conversion of Fe(II) to Fe(III) (eq 1), the form in which it is transported in the blood by Tf. 4Fe(II) + O2 + 4H+ → 4Fe(III) + 2H 2O

Figure 1. Ferroxidase activity of ceruloplasmin (Cp) during efflux of iron from cells into blood. Abbreviations: Fpn, ferroportin; Tf, transferrin.

(1)

This ferroxidase activity is compromised in motor neuron and Wilson’s diseases and other conditions associated with impaired iron and copper homeostasis.5−8 The complex multicopper oxidase Cp contains multiple T1 sites for substrate oxidation and a T2/T3 trinuclear cluster for O2 reduction (Figure 2). As a metallo-oxidase,9,10 it also features at least two substrate docking/oxidation (SDO) sites (Figure 2b, green spheres) specific for divalent metal ions such as substrate Fe(II). These sites are adjacent to electron-accepting T1 sites and to additional sites assigned to binding of the product Fe(III).11,12 However, much of the intrinsic character of Cp and its mechanism of action in mammals remains a mystery due to the © 2017 American Chemical Society

complexity of this N-linked glyco-protein (ca. 132 kDa; Figure 2b). Its basic molecular structure was defined 20 years ago by the monumental work of Lindley (Figure 2b).11,12 Pathfinding studies of its intramolecular electron transfer pathways were reported 15 years ago by Solomon.13,14 Little further substantive chemistry has emerged in the interim. A major obstacle is the unsatisfactory and/or indirect nature of existing assay procedures.15−20 Received: February 10, 2017 Published: April 17, 2017 5275

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(H2Tar, ligand 4-(2-thiazolylazo)resorcinol; x = 0−2, see below; Figure 3). It is able to both deliver reactant Fe(II) to Cp and trap product Fe(III) via eq 3.

Figure 3. Metal ligands with coordinating atoms shown in red. +O2 /Cp

[Fe IIHx(Tar)2 ]x − 2 ⎯⎯⎯⎯⎯⎯⎯⎯→ [Fe IIIHx(Tar)2 ]x − 1

This substrate overcomes each of the five fundamental problems discussed above, and both reactant and product can be followed directly and sensitively in real time at physiological pH in air by monitoring the change in solution absorbance characteristic of the complexes [FeIIHx(Tar)2]x−2 and [FeIIIHx(Tar)2]x−1 (overall charge omitted hereafter). This new assay resembles closely the ferroxidase function of Cp (Figure 1). It is 1 order of magnitude more sensitive than the existing assays. An extra bonus of this new assay is the flexibility to tune the apparent ferroxidase activity of the enzymes by variation of the molar ratio of H2Tar to Fe(II), the pH, or the temperature and also by inclusion of other divalent metal ions. This study explored the fundamental chemistry and molecular aspects of this new assay, which will expedite the study of the key ferroxidase function of this important enzyme at the molecular level in biological fluids.

Figure 2. (a) Active site of a simple multicopper oxidase with arrows showing substrate reactions and electron flow (T1 site, blue; T2 site, teal; T3 (binuclear) site, purple). (b) Ribbon representation of the Cp molecule (PDB: 1KCW) highlighting the six structural domains (D1, red; D2, green; D3, bronze; D4, gray; D5, teal; D6, yellow), six defined copper ions (T1, blue; T2, teal; T3, purple), and two Fe(II)binding sites (green).

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Activity assays for clinical use have relied on monitoring oxidation of o-dianisidine or 1,4-hydroquinone, used originally as substrates for simpler amine oxidase enzymes (cf. Figure 2a).21−23 These indirect assays do not measure the native ferroxidase activity. The membrane pump Fpn (Figure 1) is not yet available as a ferrous donor for in vitro study. Consequently, existing ferroxidase assays are based on eq 2:15−20

RESULTS A search for a suitable ferrous substrate included the classic chromophores [FeII(Fz)3]4− and FeIIHxL2 (L = Tar, Par (=4(2-pyridylazo)resorcinol); cf. Figure 3). The ligand Ferrozine (Fz) has been used frequently to trap and quantify the ion Feaq2+ (cf. eq 2).18,20 However, [FeII(Fz)3]4− reacts slowly with dioxygen in the presence of Cp, presumably due to the slow rates of its ligand exchange reactions (see the Supporting Information, including Figures S1 and S2). In contrast, FeIIHxL2 species are fairly air-stable and are oxidized by Cp at an acceptable rate. Both were characterized as substrates for comparison purposes. Spectroscopic Characterization. Titration of Feaq2+ into a solution of H2Tar (44 μM) in the sodium salt of 3-(Nmorpholino)propanesulfonic acid (Mops) buffer (50 mM, pH 7.0, 100 mM NaCl) induced stepwise changes in the absorbance spectra with a tight isosbestic point at 490 nm and an end point at Fe:H2Tar = 1:2, consistent with formation of the well-defined complex FeIIHx(Tar)2 in solution (Figure 4a,b). The spectrum is characterized by two absorbance bands with λmax 490 and 720 nm, respectively (Figure 4a). The former overlaps the ligand absorbance at λmax 470 nm while the latter does not, providing an excellent quantitative tool. The documented behavior of related ligand H2Par (Figure 3)29 suggests that H2Tar acts as a meridional tridentate anionic ligand whose stereochemistry is enforced by the NN double bond of the azo backbone. Hence, FeIIHx(Tar)2 is likely to exhibit a distorted-octahedral geometry, as shown for one of two geometric isomers in the inset of Figure 4a. Free ligand H2Tar is characterized by two pKa values assigned to the p- and o-hydroxyl groups of the benzenediol

+O2 /Cp

Feaq 2 + ⎯⎯⎯⎯⎯⎯⎯⎯→ Feaq 3 +

(3)

(2)

These assays are problematic for the following reasons: (i) Neither “free” Feaq2+ nor Feaq3+ at high concentrations is physiologically relevant (Figure 1). (ii) Feaq2+ at high concentration is unstable in solution at physiological pH and is subject to direct oxidation by O2. (iii) Feaq3+ is unstable and prone to precipitation as the “hydroxide” (rust) at physiological pH. (iv) Both cations at high concentrations are sensitive to precipitation or chelation by many anions typically present in serum or assay media, including bicarbonate, chloride, citrate, phosphate, and sulfate (see Figure 10). (v) Methods assessing Feaq2+ loss or Feaq3+ gain are complex, noncontinuous and time-consuming, and have low sensitivity (cf. Table S1 in the Supporting Information). The current ferroxidase assays were examined recently, and a protocol based on a combination of three of them was proposed.20 This led to an integrated, but time-consuming, approach that enables careful assessment of apparent differences in assay results. However, the combined protocol is still based upon eq 1 and does not solve the fundamental problems therein. A concerted search for a robust model ferrous substrate in this work led to the chromophoric complex [FeIIHx(Tar)2]x−2 5276

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Consequently, the ionization equilibrium of eq 4 is likely to exist in solution in the pH range 4−8, as confirmed for ZnIIHx(Par)2.26 The forms of the free ligands in solution will be Hx′Tar or Hx′Par (x′ = 1, 2) but the notation is simplified to Tar or Par hereafter. pK a1≈ 5

MII(HTar)2 ←⎯⎯⎯⎯→ [MII(HTar)(Tar)]− pK a2 ≈ 7 − 8

←⎯⎯⎯⎯⎯⎯⎯→ [MII(Tar)2 ]2 −

(4)

The solution spectrum of FeIIHx(Tar)2 is also pH-dependent due to access to the different ionization states of eq 4. For example, titrations of Feaq2+ into the same Tar solution diluted into buffers of different pH generated similar turning points at Fe2+:Tar = 1:2, but with different final absorbance intensities at 720 nm (Figure 4b). Influences due to perturbed binding equilibria are unlikely, since addition of excess Feaq2+ did not lead to further increases in the solution absorbance (Figure 4b). It is likely that the molar absorptivity of the Fe(II)−Tar system is pH-dependent and the empirical formula ε = 4.14pH − 10 mM−1 cm−1 holds in the range pH 6.0−8.0 (Figure 4c and Table 1). Equivalent experiments with Par provided comparable data and similar conclusions for FeIIHx(Par)2 (Figure S3 in the Supporting Information and Table 1). Titration of divalent metal ions M2+ (M = Ni, Zn) into either a Tar or Par solution also induced spectral changes in the visible region consistent with formation of the respective 1:2 complex MIIHxL2 (L = Tar, Par; Table 1). Each complex MIIHxL2 is characterized by an intense absorption band in the visible spectrum which overlaps with that of the free ligand. Complexes FeIIHxL2 exhibit an additional unique absorbance band at λ >700 nm (Figure 4a and Figure S3a in the Supporting Information). Notably, both ligands react with Fe(III) to yield 1:2 complexes that lack absorbance at λ >700 nm but are characterized by absorbances at ∼540 nm which overlap partially those of the free ligands and/or the corresponding Fe(II) complexes (Table 1; see also Figure 5b and Figure S8b in the Supporting Information). Consequently, the characteristic absorbances at λ >700 nm for FeIIHxL2 provide an advantage for quantitative characterization.

Figure 4. Spectroscopic characterization of FeIIHx(Tar)2: (a) change in solution spectrum of H2Tar (44 μM; blue) in Na-Mops (50 mM, pH 7.0, 100 mM NaCl) upon titration with Fe2+ (inset: proposed molecular structure of a geometric isomer of FeIIHx(Tar)2); (b) variation of A720 at three pH values; (c) variation of ε720 nm with pH (ε = 4.14pH − 10 mM−1 cm−1).

ring (Figure 3 and Table 1).24 The o-OH (pKa2 = 12.8) is involved in metal chelation, while the p-OH (pKa1 = 7.4) is not. The high energy cost required for deprotonation of the o-OH ligand is compensated partially by the formation of two adjacent five-membered chelate rings (Figure 4a, inset). The ionization states of the uncoordinated p-OH functions are determined by solution conditions. For several metal complexes MIIHx(Tar)2 in 50% (v/v) dioxane/water, pKa1 values have been estimated to be ∼7, similar to that of the free ligand, while pKa2 estimates are 1−2 pH units higher.24,30 However, a recent estimate for H2Par revealed that the pKa value for this p-OH is about 1.5 pH units lower in 1% DMSO water solution (Table 1).26

Table 1. Spectroscopic Data and Formation Constants for Ligands and Metal Complexes UV−vis data at pH 7.0a

formation constant

complex

λmax (nm)

ε (mM−1 cm−1)

log K1c

log K2c

H2Tar FeIIHx(Tar)2 FeIIIHx(Tar)2 NiIIHx(Tar)2 ZnIIHx(Tar)2 H2Par

470 720 540 500 490 412

24.8 19.0b 46.5

12.8

7.4

FeIIHx(Par)2 FeIIIHx(Par)2 NiIIHx(Par)2 ZnIIHx(Par)2 HFz [FeII(Fz)3]4−

705 495 495 490

18.6b 135.2

562

27.9

37.8

12.9 11.1 12.4 12.10f

13.2 12.4 ∼ 3.2

11.8 10.1 6.9 5.44f

12.8 11.1

log β2c

log β2′d

24.8 21.2

13.6 21.6 13.2 9.6

26.0 23.5

15.2 12.7 15.6g

ref 24 this this 24 24 25 26 this this 25 25 27 28

worke worke

worke worke

This work; the absorption maxima of most metal complexes MIIHxL2 (M ≠ Fe) partially overlap that of the ligand. bThese values are pH-dependent: ε = 4.14pH − 10 for FeIIHx(Tar)2, and ε = 3.6pH − 6.6 for FeIIHx(Par)2. cAbsolute formation constants as determined by potentiometric titration in 50% (v/v) dioxane/water as reported. dConditional formation constant at pH 7.0 estimated via eq S3 in the Supporting Information using the reported pKa for the coordinating o-OH of the ligand (pKa = 12.8 and 12.4 for H2Tar and H2Par, respectively). epH 7.0. f Values determined in 1% DMSO/water solution. gReported conditional log β3′ determined via direct metal ion titration. a

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Inorganic Chemistry Table 2. Reduction Potential Dataa potential (mV vs SHE) complex or enzyme II

Fe Hx(Tar)2 FeIIHx(Par)2 Fet3 Cp

pH

E1/2

Ec

Ea

ΔE

6.0 7.0 6.6 7.0 7.0 7.0

+387(3) +314(2) +437(4) +408(4) +427b +448b

+351 +280 +398 +375

+436 +349 +475 +440

85 69 76 65

a

Electrochemical conditions: pyrolytic graphite electrode; scan rate 10−150 mV s−1; Na-Mops buffer (50 mM, pH 7.0; 0.1 M NaClO4); [Fe]tot = 0.25 mM; [H2L]tot = 1.0 mM; bDetermined by poised potential redox titration in refs 13 and 14.

Figure 5. Ferroxidase activity assay with substrate FeIIHx(Tar)2 (40 μM, Fe(II):Tar = 1:2) in air-saturated Na-Mops buffer (50 mM, pH 7.0; 100 mM NaCl): (a) A720 time course in the absence (i) and presence (ii) of Cp (0.15 μM); (b) visible spectra of (i) FeIIHx(Tar)2, (ii) FeIIIHx(Tar)2 (via FeIIHx(Tar)2 catalyzed by Cp), and (iii) Tar.

Equivalent experiments on the Par system demonstrated a similar process at E1/2 = 414(4) mV, which is 115 mV more positive than that of the Tar system (Figure S7a,c in the Supporting Information and Table 2). The electrochemistry is pH-sensitive, and the reduction potentials for both systems shift positively with decreasing pH (Table 2 and Figure S7d): i.e., oxidation of the Fe(II) forms becomes more difficult as the pH decreases. FeIIHx(Tar)2 Is an Effective Formal Substrate for Cp. The more reactive FeIIHx(Tar)2 system was studied systematically with appropriate comparison with less reactive FeIIHx(Par)2 (Figure S8 in the Supporting Information). The Tar complex is stable under anaerobic conditions within the pH range 6.0−8.0. It oxidizes slowly in air-saturated buffers, but the oxidation rate is accelerated by the Cp enzyme (Figure 5a). The spectrum of the soluble single product was indistinguishable quantitatively from that of FeIIIHx(Tar)2 (Figure 5b(ii)), confirming catalysis of ferrous oxidase (reaction 5):

Formation Constants. The formation constants of several divalent metal complexes MIIHxL2 (L = Tar, Par) have been determined by potentiometric titration in 50% (v/v) dioxane/ water and are expected to be pH-dependent.24,25,29 The conditional formation constants log β2′ at a specific pH may be calculated via eq S3 in the Supporting Information using the reported pKa for the coordinating ortho−OH function; values at pH 7.0 are listed in Table 1. However, the critical data for FeIIHx(Tar)2 and FeIIIHx(Tar)2 are not available, while those for FeIIHx(Par)2 and FeIIIHx(Par)2 have been reported31−33 but are not well-defined, presumably due to their respective redox and hydrolytic instabilities. This work has estimated the β2′ values for FeIIHx(Tar)2 around physiological pH by competition with appropriate ligands Y: Fe IIHx(Tar)2 + Y ⇌ Fe IIY + 2Hx ′Tar

That for FeIIIHx(Tar)2 could then be extracted with electrochemistry via the Nernst relationship of eq S5 in the Supporting Information. The results are summarized in Table 1 and Table S2 in the Supporting Information. Further details are given in the Supporting Information, including Figures S4 and S5. The stability of FeIIHx(Tar)2 toward other divalent metal ions 2+ M (M = Zn, Ni) was also estimated via metal competition (eq S4 in the Supporting Information) and is consistent with the reported respective formation constants (Figure S6 in the Supporting Information and Table 1). The rates of equivalent reactions for Par are too slow for meaningful estimation of equivalent β2′ values (Figure S4c,d in the Supporting Information). However, this work demonstrated clearly that Par has a higher affinity for Fe2+ than does Tar (Figure S4b), consistent with general observations that Par has a higher affinity for divalent metal ions by 1−2 orders of magnitude (cf. Table 1). Redox Chemistry. Cyclic voltammograms in Na-Mops buffer (50 mM; pH 7.0; NaClO4, 100 mM) containing Fe(II):Tar = 1:4 exhibited a redox process centered at E1/2 = 314(2) mV vs SHE (Figure S7a,b in the Supporting Information). The derived parameters are typical of a reversible one-electron-redox process (Figure S7b and Table 2), which can be assigned to the redox couple FeIIIHx(Tar)2/ FeIIHx(Tar)2. Variation of the total molar ratio of Tar to Fe had little impact on the electrode response, demonstrating the chemical stability of both complexes under the conditions and their direct response on the electrode as intact complexes.

Cp

4Fe IIHx(Tar)2 + O2 + 4H+ → 4Fe IIIHx(Tar)2 + 2H 2O (5)

As demonstrated below, ligand Tar transfers ferrous ion formally from the complex FeIIHx(Tar)2 to the Fe(II)-binding SDO site(s) in Cp and the metal ion must be transferred prior to oxidation. Product Fe(III) is trapped again by the same Tar ligand as the soluble complex FeIIIHx(Tar)2. In this sense, Tar mimics the functions of the physiological partners Fpn and Tf of the Cp enzyme (Figure 1) and offers the convenience of monitoring reaction 5 in real time via the fingerprint absorbance at 720 nm for the formal reactant FeIIHx(Tar)2 under all conditions (Figure 5). This constitutes a robust assay for the ferroxidase functions of Cp and related enzymes. The molecular aspect of the catalytic reaction of eq 5 is explored and documented below under a variety of conditions. In the Presence of Excess Ligand Tar, Free Feaq2+ Is Not the Major Direct Substrate. A stock solution of FeIIHx(Tar)2 with the molar ratio Tar:Fe(II) = 2.2:1.0 was prepared in Na-Mops buffer (50 mM, pH 7.0). This imposes [Tar]free = 0.2[FeIIHx(Tar)2] for the initial solution in airsaturated Na-Mops buffer in a half-cell of a mixing cuvette. The other half-cell contained Cp in the same volume of air-saturated buffer. The reaction was started by reagent mixing and followed by the absorbance at 720 nm. Note that the reactants and catalyst are diluted by a factor of 2 upon mixing. Ferroxidase activity increased with the absolute concentration of FeIIHx(Tar)2 at the fixed initial molar ratio Tar:Fe(II) = 2.2:1.0 (black trace, Figure 6a). Equation 6 is a form of the 5278

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Depletion of these reactive species would lead to their rapid replacement via dissociation of the dominant FeIIHx(Tar)2 complex due to simultaneous consumption of the Tar ligand by product Fe(III). Consequently, steady-state concentrations of these reactive species appeared to maintain a steady-state reaction rate (Figure 6b(i)). This will be discussed in more detail below. Estimation of Fe(II) Affinity of the Cp SDO Site(s). The rate of oxidation of FeIIHx(Tar)2 (40 μM) decreased by about 50% upon change of the molar ratio Tar:Fe(II) from 1.8 to 2.1 (Figure 6b). Consequently, the free Feaq2+ concentration in the solution is a measure of KD′ of the Cp Fe(II)-SDO site(s) under these conditions. The conditional log β2′ = 13.6 determined in this work at pH 7.0 for the Tar system (Table 1) allows estimation of log KD′ ≈ −7.2. This is consistent with a previous estimation of log KD′ < −5.0.22 M(II) Ions Influence Transfer of Fe(II) from FeIIHx(Tar)2 to Cp. The affinities of Tar for Fe(II) and Ni(II) are similar (Table 1 and Figure S6 in the Supporting Information), and NiIIHx(Tar)2 is inactive in the assay. Titration of NiIISO4 into a substrate solution with Tar:Fe(II) = 2.4:1.0 initially enhanced ferroxidase activity (Figure 7a), apparently by reducing the

Figure 6. Ferroxidase activity (black traces) of Cp (0.1−0.3 μM) on FeIIHx(Tar)2: (a) increasing substrate concentration at the fixed molar ratio Fe(II):Tar = 1.0:2.2, i.e., with decreasing initial free [Fe2+] (red curve); (b) increasing molar ratio Tar:Fe(II) at a fixed initial concentrations of (i) [Tar]tot = 80 μM at [Tar]tot:[Fe]tot < 2.0 and (ii) [Fe(II)]tot = 40 μM at [Tar]tot:[Fe]tot > 2.0. Note that the initial [FeIIHx(Tar)2] concentrations in (i) and (ii) are identical at 40 μM (red trace). Reactions were carried out in Na-Mops buffer (50 mM, NaCl 0.15 M) and monitored at 720 nm using a mixing cuvette.

equation defining β2′, and the first term on the right-hand side is a constant under the conditions: ⎛ 1 [Fe IIH (Tar) ] ⎞ x 2 ⎟⎟ − log[Tar]free log[Fe 2 +]free = log⎜⎜ [Tar]free ⎝ β2′ ⎠ (6) II

Consequently, an increase in [Fe Hx(Tar)2] imposes an increase in [Tar]free and a decrease in [Fe2+]free (red trace in Figure 6a). If “free Feaq2+” was the major direct substrate, the Cp activity should decrease with an increase in [FeIIHx(Tar)2] under the constraint Tar:Fe(II) = 2.2:1.0. However, this was clearly not the case under the conditions and, instead, the activity was observed to increase (black trace, Figure 6a). In the Presence of Excess Ligand Tar, Fe(II) Must Be Transferred from Fe II H x (Tar) 2 to Cp Directly for Oxidation. Under conditions of excess ligand (Tar:Fe(II) > 2.0) but at a constant initial concentration of FeIIHx(Tar)2 (40 μM, red trace in Figure 6b), the apparent activity is inhibited sensitively by increasing concentrations of free ligand Tar (black trace in Figure 6b(ii)). It is apparent that the intact complex FeIIHx(Tar)2 cannot be an outer-sphere substrate for direct nonspecific reduction on the Cp surface and that the Fe(II) center in FeIIHx(Tar)2 must be transferred to the enzyme reaction SDO site(s) for oxidation. Excess free ligand Tar (but not the intact FeIIHx(Tar)2 complex, cf. Figure 6a, black trace) competes for Fe(II) with the Cp site(s), limits the Fe(II) transfer process, and decreases the overall catalytic rate. Support for this interpretation was provided by the contrasting behavior of the electrochemistry experiments: neither the reduction potentials nor the electrode currents for the electrochemistry of FeIIHx(Tar)2 and FeIIIHx(Tar)2 were sensitive to the concentration of free ligand, confirming that it is the intact complexes that are the redox-active species at the electrode surface under the conditions (vide supra). In the Presence of Limiting Ligand Tar, Dissociated Feaq2+ May Become a Major Direct Substrate in Equilibrium with Intact FeIIHx(Tar)2. Under conditions of slightly excess ferrous iron (Tar:Fe(II) < 1.8) but a fixed total ligand concentration, the ferroxidase activity remains constant at a maximum rate comparable to that using FeII(NH4)2(SO4)2 as a substrate (see Figures 6b(i) and 9b). This is intriguing and may be related to the fact that, under the conditions of excess Fe(II), Feaq2+ and/or related labile Fe(II)-Tar species at increased relative concentrations may compete strongly as direct substrates.

Figure 7. Ferroxidase activity of Cp on FeIIHx(Tar)2 in the presence of Ni(II) and Zn(II): (a) titration of NiIISO4 into Cp (0.5 μM) and FeIIHx(Tar)2 ([Fe(II)]tot = 40 μM; [Tar]tot = 96 μM) (initial [FeIIHx(Tar)2] is shown in red); (b) titration of ZnIISO4 into Cp (0.1 μM) and FeIIHx(Tar)2 ([Fe]tot = 47 μM; [Tar]tot = 90 μM) (decrease in initial [FeIIHx(Tar)2] is shown in red).

excess of inhibiting free ligand Tar. The effect was tempered once excess ligand was consumed and the concentration of FeIIHx(Tar)2 started to decrease (Figure 7a). This latter aspect appears to arise from the competition between Ni2+ and Fe2+ for the SDO site(s) in Cp. It is consistent with inhibition by free Ni2+ of the ferroxidase activity of Cp on the substrate FeII(NH4)2(SO4)2 (Figure S10a in the Supporting Information). Interestingly, an equivalent experiment with Zn2+ inhibited the activity (Figure 7b). The affinity of Tar for Zn(II) is 4 orders of magnitude weaker than that for Fe(II) (Table 1), and so, in contrast to Ni2+, Zn2+ ion competes but weakly with Fe2+ for free ligand Tar (Figure 7b, red trace). However, Zn2+ has been demonstrated to bind competitively to the SDO site(s) in Cp and to inhibit the ferroxidase activity of Cp on the substrate FeII(NH4)2(SO4)2 at pH 6−7.22,34 This latter effect was confirmed here (Figure S10b in the Supporting Information). Zn2+ also inhibits the ferroxidase activity of FeIIHx(Par)2, but much less sensitively (cf. Figure S11 in the Supporting Information versus Figure 7b). This is consistent with the increased affinity of Zn2+ for ligand Par relative to that for Tar (Table 1). The differing effects of Ni2+ and Zn2+ on oxidation of FeIIHx(Tar)2 by Cp can be attributed to their contrasting affinities for the competing ligands Tar and Cp: Ni2+ binds Tar much more strongly than does Zn2+ but appears to bind the Cp 5279

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Inorganic Chemistry SDO site(s) more weakly than does Zn2+. This conclusion is supported by previous observations.22,34 Ligand Tar Is Adapted for Rapid Rates of Substitution of Fe(II) in FeIIHx(Tar)2. The submicromolar affinity of the SDO site(s) in Cp for Fe(II) estimated above provides a thermodynamic basis for extraction of Fe(II) from FeIIHx(Tar)2. On the other hand, the ligand structure of Tar (Figure 8a) supports rapid Fe(II) transfer kinetics. The meridional coordination mode

of free Bca that inhibits the activity significantly along the time course.35−37 In contrast, the reaction rate in the present situation decreased slowly along the reaction time course (Figure 9a), consistent

Figure 9. Cp ferroxidase activities: (a) FeIIHx(Par)2 at [Cp] (μM) (i) 0, (ii) 0.027, (iii) 0.054, (iv) 0.098, (v) 0.13, (vi) 0.27, and (vii) 0.54; (b) plots of oxidation rate for substrates (i) FeII(NH4)2(SO4)2 (cf. Figure S13b in the Supporting Information), (ii) FeIIHx(Tar)2, (iii) FeIIHx(Par)2 (cf. Figure S13a). Initial solutions: FeIIHxL2 (L = Tar, Par); [Fe(II)], 40 μM; [L], 72 μM in Na-Mops buffer (50 mM, pH 7.0, 150 mM NaCl); [FeII(NH4)2(SO4)2], 40 μM in Na-Hepes buffer (50 mM, pH 7.0, 200 mM Na2SO4).

with the effective free ligand concentration [Tar] remaining constant. This is consistent with the fact that release of the Tar ligand from the reactant FeIIHx(Tar)2 is compensated by its consumption in the formation of the product FeIIIHx(Tar)2 complex. Consequently, the reaction kinetics is simplified by this constant steady-state free Tar concentration during the catalysis and the Cp ferroxidase activity at the initial substrate concentration may be determined reliably from the initial oxidation rates as a function of the enzyme concentration. Plots of the initial oxidation rate versus [Cp] were linear, allowing derivation of a consistent enzyme turnover number under specific conditions (Figure 9b(ii)). The comparative behavior of substrates FeIIHx(Par)2 and [FeII(Fz)3]4− support this analysis. The former behaves similarly but is less reactive (Figure S13a in the Supporting Information and Figure 9b(iii)), consistent with the higher affinity of Par for Fe(II). The slow exchange properties of [FeII(Fz)3]4− are the source of its low reactivity (Figure S2b,c in the Supporting Information). Further details are provided in the Supporting Information. In contrast, the ferroxidase activity of Cp on substrate FeII(NH4)2(SO4)2 (40 μM) in the sodium salt of Hepes (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (50 mM; pH 7.0; Na2SO4, 0.2 M) was ∼20% higher than that for equimolar FeIIHx(Tar)2 (Tar:Fe(II) ≤ 1.8) (Figure S13b in the Supporting Information and Figure 9b(i)). The weak ligand SO42− at high relative concentration is able to stabilize “free Fe2+” and inhibit its auto-oxidation. Overall, the Cp ferroxidase activity (min−1) for different substrates under conditions specified in Figure 9 and Figure S1 in the Supporting Information increases in the order

Figure 8. (a) Kinetic lability in ligand substitution for FeIIHx(Tar)2 and related metal complexes. (b, c) pH dependence of ferroxidase activity of Cp on substrate FeIIHx(Tar)2: (b) change in substrate concentration with time at pH = 6.5, 6.7, 6.9 and 7.1; (c) initial oxidation rate between 0.5 and 1.5 min in (b). Conditions: Cp (0.5 μM), [Fe(II)]tot = 23 μM, Fe:Tar = 1.0:2.4 in air-saturated Na-Mops buffer (50 mM, NaCl 0.1 M).

of the Tar ligand imposes a cis arrangement on the two phenoxo functions for both structural isomers of FeIIHx(Tar)2 (Figure 8a). These ligand sites are highly basic (pKa = 12.8; Table 1) and may be dissociated readily to create empty binding sites for the incoming attacking ligands, and the process is predicted to be promoted by an increase in proton concentration. Indeed, the ferroxidase activity of Cp on FeIIHx(Tar)2 decreased considerably with increasing pH (Figure 8b,c), despite a more favorable reduction potential of FeIIHx(Tar)2 at high pH (i.e., it is more easily oxidized at higher pH; cf. Table 2 and Figure S7d in the Supporting Information). Substrate FeIIHx(Par)2 behaves equivalently (Table 2 and Figure S12 in the Supporting Information). In comparison, the oxidase activity of Cp on [FeII(Fz)3]4− is insensitive to pH, as a proton-sensitive ligand is not present above pH ∼4 for the bidentate Fz ligand (see Figure 3 and Figure S1b(iii) in the Supporting Information).27 In contrast, the oxidase activity of Cp on substrate FeII(NH4)2(SO4)2 was observed to increase (instead of decrease) with increased pH.20 These last two control experiments suggested that the pH effect observed in Figure 8b,c must arise primarily from the pH lability of the two Fe(II)−phenoxo bonds in substrate FeIIHx(Tar)2, not from the Cp enzyme. The Concentration of Free Ligand Tar Remains Constant during Catalysis. Complex [CuI(Bca)2]3− acts as a substrate for cuprous oxidase enzymes such as E. coli CueO and PcoA (multicopper oxidases).35−37 Ligand Bca binds Cu(I) with high affinity but Cu(II) with lower affinity. Consequently, oxidation of [CuI(Bca)2]3− leads to accumulation

[Fe II(Fz)3 ]4 − (0.1) < Fe IIHx(Par)2 (9.7) < Fe IIHx(Tar)2 (49.5) < Fe II(NH4)2 (SO4 )2 (58.6)

(7)

Competing Ligands Affect the Absolute Activity but Do Not Induce Auto-Oxidation. Additives that alter the affinity of ligands L = Tar, Par for Fe(II) or Fe(III) or compete for those ions may alter the oxidase activity and reaction mechanism. For example, addition of Na2SO4 (0.2 M) to the 5280

DOI: 10.1021/acs.inorgchem.7b00372 Inorg. Chem. 2017, 56, 5275−5284

Article

Inorganic Chemistry

Figure 11. (a) Change in concentration of FeIIHx(Tar)2 (30 μM; Tar:Fe = 2.2:1.0) in air-saturated Mops buffer (100 mM, pH 7.0; 150 mM NaCl) upon catalytic oxidation by O2 with Cp (0.15 μM) as catalyst in the presence of FeIII-chelate Ferron (i−iv; 0, 15, 30, 45 μM). (b) Plot of the initial ferroxidase activity versus molar ratio Ferron:Fe for experiments i−iv in (a). (c, d) Change in solution spectra for solutions containing (c) 0 or (d) 30 μM Ferron.

of Cp and related enzymes under physiological conditions. The overall reaction of eq 5 is demonstrated to proceed via a catalytic cycle mediated by the Cp enzyme that may be summarized as Scheme 1. The catalysis starts with delivery of Fe(II), formally by the ligand Tar, to the Cp SDO site(s) (step ia−ib), followed by rapid oxidation of the enzyme-bound Fe(II) to Fe(III) (step ii) and removal of the product Fe(III) by ligand Tar to regenerate the resting enzyme (step iii). Depending on the Tar/Fe(II) molar ratio and/or the reaction medium pH, the Fe(II) from the dominant complex FeIIHx(Tar)2 in solution may be delivered to the Cp reaction site(s) either directly as the Fe(II)−Tar complex FeIIHx(Tar)n (n = 1, 2) or indirectly as dissociated Feaq2+ species in equilibrium with FeIIHx(Tar)2 (step i). The free ligand Tar released from step i is formally consumed by trapping Fe(III) product in step iii. Consequently, the overall concentration of free Tar ligand remains steady during catalysis. This assay is robust and can be followed spectroscopically in real time reliably and reproducibly under a variety of reaction conditions via the change of the fingerprint absorbance at 720 nm for the starting FeIIHx(Tar)2 complex. Step i in Scheme 1 is sensitive to the free Tar concentration. In the presence of excess free Tar ligand (i.e., Tar:Fe(II) > 2), the bound Fe(II) in FeIIHx(Tar)2 is delivered directly by the Tar ligand to the substrate reaction site(s) in Cp and the trace free Feaq2+ ion, due to its low concentration under the conditions, appears to be unable to compete for the reaction site(s) (cf, Figure 6a and Scheme 1, step i, where y → 2). Both free Tar ligand and the SDO site(s) of Cp compete for Fe(II) and then step ib becomes rate-determining (see Figure 6b(ii)). This is consistent with the conclusion that the intact FeIIHx(Tar)2 complex cannot be a direct outer-sphere substrate and the bound Fe(II) cannot be oxidized without transfer to the SDO site(s) of the Cp enzyme. A ternary complex of Tar−Fe(II)−Cp may form transiently for step ib to facilitate a rapid Fe(II) transfer, and this is possible due to the lability of one or two phenoxo coordination groups for the FeIIHx(Tar)2 complex (see Figure 8a).

Figure 10. Normalized oxidation rates in the absence (black bars) and presence of Cp (0.1−0.5 μM; gray bars) and anions in Na-Mops buffer (50 mM, pH 7.0): (a) FeIIHx(Tar)2 (50 μM; Tar: Fe(II) = 2.05); (b) FeIIHx(Par)2 (50 μM; Par: Fe(II) = 2.05); (c) FeII(NH4)2(SO4)2 (50 μM).

assay solution in Na-Mops buffer (50 mM, pH 7.0) decreased the oxidase activity by 30−40% (Figure 10a,b), likely due to limiting Fe(II) transfer from the formal substrate FeIIHx(Tar)2 to the Fe(II) SDO reaction site(s) in Cp. In fact, many common anions, buffers, and ligands found in biological media affect the reactivity of FeIIHxL2. They include citrate, bicarbonate, chloride, phosphate, Tris, BisTris, TEA, and Tf (Figure 10a,b and Table S3 in the Supporting Information). However, the stabilities of FeIIHxL2 under the aerobic assay conditions ensure minimal background auto-oxidation in the presence of these common species (Figure 10a,b and Table S3 in the Supporting Information). In contrast, FeII(NH4)2(SO4)2 is more sensitive to background auto-oxidation under these conditions, particularly in the presence of bicarbonate and the common buffers phosphate and BisTris (Figure 10c and Table S3). Ferron (7-iodo-8-hydroxyquinoline-5-sulfonic acid) is a highaffinity Fe(III) ligand.38 Its addition to the assay solutions inhibited the Cp oxidase activity on the substrate FeIIHx(Tar)2 (Figure 11a,b). The changed pattern in solution spectra indicated that the Ferron ligand competes for the product Fe(III) with the Tar ligand (Figure 11c,d). This will promote accumulation of free Tar ligand along the reaction course and inhibit Fe(II) transfer to the Cp SDO site(s) for oxidation. This interpretation is supported by equivalent experiments with the metallo-chaperone Tf instead of Ferron (data not shown).

■

DISCUSSION This study explores the chemistry of the classic complex FeIIHx(Tar)2 as a ferrous substrate for the ferroxidase function 5281

DOI: 10.1021/acs.inorgchem.7b00372 Inorg. Chem. 2017, 56, 5275−5284

Article

Inorganic Chemistry Scheme 1. Proposed Mechanism for Cp as a Ferrous Oxidase Acting upon Formal Substrate FeIIHx(Tar)2 under Various Conditions (y = 0−2; See Text for Details)a

Figure 12. Proposed functional metal sites in Cp (PDB code: 1KCW): three T1 sites (blue) in domains D2, D4, and D6 for substrate oxidation; trinuclear cluster of T2 (cyan) and T3 (purple) for O2 reduction; cation-binding sites (green) in D4 and D6 for Fe(II) binding and oxidation; “holding” site (red circle) for sequestration of product Fe(III) prior to transfer to chaperone Tf. Amino acid residue numbers of potential metal ligands are given.

a

Note that the protonation state of the Tar ligand was omitted for all Tar species in the scheme for clarity.

On the other hand, in the presence of a slight excess of Fe(II) (i.e., Tar:Fe(II) < 2), the overall reaction rate reached a constant maximum that is comparable to the oxidation rate with FeII(NH4)2(SO4)2 as ferrous substrate (Figure 9b). This suggests that the nature of the direct ferrous substrate may have shifted from FeIIHx(Tar)2 to the elevated levels of free Feaq2+ and/or other labile Fe(II)−Tar complexes under the conditions. These labile Fe(II) species are buffered by the dominant FeIIHx(Tar)2 complex in solution to a constant value (cf, steps ia and ib where y = 0, 1), since there is simultaneous consumption of the Tar ligand by product Fe(III) at step iii. Consequently, under the conditions of excess free Feaq2+, the catalytic rate reaches a steady-state maximum with the ratedetermining step being shifted perhaps from step ib to step ii or iii. The free Feaq2+ concentration is maintained at a low but steadystate concentration that suppresses nonenzymatic auto-oxidation. This allows the overall catalytic process to be monitored reliably and sensitively on the basis of the the fingerprint probe of absorbance at 720 nm for the formal substrate FeIIHx(Tar)2. In the next two steps (ii and iii), the Cp-bound Fe(II) is oxidized rapidly to Fe(III) product that is trapped by the Tar ligand to regenerate the resting enzyme and to complete the catalytic cycle. A transient complex of Tar−Fe(III)−Cp may also form similarly for the last step to facilitate a rapid removal of the Fe(III) product from the enzyme. The catalytic model of Scheme 1 may be rationalized from the available structural information on the Cp enzyme. The proposed functional metal sites are shown in Figure 2b and are highlighted in Figure 12.11,12 There are three separate T1 sites in domains D2, D4, and D6 (blue spheres). Two separate M(II) binding sites (green spheres) were identified adjacent to the T1 sites in D4 and D6. They are likely to be the specific Fe(II) SDO sites and feature a mixture of His and carboxylato side chains as potential ligands (Figure 12): these are known to support the proton transfer reactions needed for the ligand attack model outlined in Figure 8a. Another potential metal binding site is adjacent to the M(II) site in D6 (Figure 12, red circle). It is displaced toward the protein surface and features four hard carboxylato residues.

It was assigned as a “holding site” for Fe(III) product for subsequent donation to metallo-chaperone Tf.12 An equivalent “holding site” may also exist for T1 in D4 but is less well-defined. This restricted structural information provides a basis for interpretation of the experimental mechanism summarized in Scheme 1. The conditional log β2′ = 13.6 at pH 7.0 for FeIIHx(Tar)2 allowed estimation of the average affinity of the SDO sites for Fe(II) as log KD ≈ −7.2 (Figure 6b). Their reduction potential cannot be more positive than that of the T1 site (448 mV13). According to the Nernst equation (eq S5 in the Supporting Information), this sets a minimum average affinity of log KD ≈ −13 for Fe(III) binding to the SDO site(s). The affinity of the “holding sites” for Fe(III) must be higher, and those of the receiving ligands must be higher still. The log β2′ = 21.6 at pH 7.0 estimated here for FeIIIHx(Tar)2 means that the Tar ligand is able to buffer free Feaq3+ concentration at