Coordinate and redox interactions of epinephrine with ferric and ferrous iron at physiological pH

Abstract

Coordinate and redox interactions of epinephrine (Epi) with iron at physiological pH are essential for understanding two very different phenomena - the detrimental effects of chronic stress on the cardiovascular system and the cross-linking of catecholamine-rich biopolymers and frameworks. Here we show that Epi and Fe³⁺ form stable high-spin complexes in the 1:1 or 3:1 stoichiometry, depending on the Epi/Fe³⁺ concentration ratio (low or high). Oxygen atoms on the catechol ring represent the sites of coordinate bond formation within physiologically relevant bidentate 1:1 complex. Redox properties of Epi are slightly impacted by Fe³⁺. On the other hand, Epi and Fe²⁺ form a complex that acts as a strong reducing agent, which leads to the production of hydrogen peroxide via O₂ reduction, and to a facilitated formation of the Epi-Fe³⁺ complexes. Epi is not oxidized in this process, i.e. Fe²⁺ is not an electron shuttle, but the electron donor. Epi-catalyzed oxidation of Fe²⁺ represents a plausible chemical basis of stress-related damage to heart cells. In addition, our results support the previous findings on the interactions of catecholamine moieties in polymers with iron and provide a novel strategy for improving the efficiency of cross-linking.

Figure 1

UV/Vis spectra of Epi and ferric iron in 10 mM Tris buffer, pH 7.4. (a) 0.2 mM Epi and 0.2 mM Fe³⁺. (b) 0.2 mM Epi in the presence of 0.05, 0.1, 0.2, or 0.4 mM Fe³⁺ (30 min incubation). Dashed lines represent sums of experimental spectra for different [Epi]/[Fe³⁺] = 4 and [Epi]/[Fe³⁺] = 1 (divided by 2; dark); and for [Epi]/[Fe³⁺] = 1 and free [Fe³⁺] = 0.2 mM (pale). (c) 0.4 mM Epi in the presence of 0.1, 0.4 or 0.6 mM Fe³⁺ (30 min incubation). The dashed lines represent the sum of experimental spectra for [Epi]/[Fe³⁺] = 1 and free [Fe³⁺] = 0.2 mM. (d) Stability of Epi in the presence of Fe³⁺, measured by HPLC. (e) Changes in UV/Vis spectra for different [Epi]/[Fe³⁺] ratios, during a 30 min incubation period. In all systems [Epi] = 0.2 mM. For clarity, the ranges 400–700 nm and 260–350 nm (gray line represents absorption from Epi only) are shown separately.

Figure 2

Low-T EPR spectra of Fe³⁺ in 10 mM Tris buffer, pH 7.4. (a) 100 K EPR spectra of Fe³⁺ in the absence or presence of Epi. (b) 100 K EPR spectra (left) and the intensity of the g = 4.26 Fe³⁺ signal (right) for different [Epi]/[Fe³⁺]. [Fe³⁺] = 0.1 mM in all samples. (c) 20 K EPR spectra of 0.067 mM and 0.2 mM Fe³⁺ in the presence of 0.2 mM Epi. Line-widths are given in mT. All samples were frozen after 15 min incubation at 293 K.

Figure 3

Raman spectra of 0.2 mM Epi with or without 0.2 mM Fe³⁺ in 10 mM phosphate buffer, pH 7.4. The spectrum of 0.2 mM Fe³⁺ is shown for comparison. Spectra were obtained after 15 min incubation period, using the λ = 532 nm laser excitation line. Inset: Two bands contributing to the signal at ∼535 cm^-1.

Figure 4

Cyclic voltammograms of 0.2 mM Epi in 10 mM Tris buffer, pH 7.4, containing different Fe³⁺ concentrations, at the boron doped diamond electrode. (a) From top to bottom: Epi (dark lines) and Fe³⁺ (0.2 mM; pale line), and [Epi]/[Fe³⁺] = 4, 2, and 1. The positions of oxidation/anodic (E_pa) and reduction/cathodic (E_pc) potentials are marked with dotted lines (dark – iron-free system; pale – all other settings). E_pa and E_pc are presented as mean values ± SE (mV). (b) Mean values (±SE) of anodic (I_pa; open circles) and cathodic (I_pc; closed circles) peak currents in CV of Epi with different [Fe³⁺]. Scan rate was 0.1 V/s. E_pa and E_pc, and I_pa and I_pc not sharing a common letter were significantly different (P < 0.05).

Figure 5

Redox interactions of 0.2 mM Epi with Fe²⁺ at pH 7.4. (a) UV/Vis spectra showing the oxidation of 0.2 mM Fe²⁺ to Fe³⁺ in 10 mM Tris. Inset: The accumulation of Fe³⁺ during spontaneous oxidation of 0.1 and 0.2 mM Fe²⁺; [Fe³⁺] was calculated using the absorbance at 300 nm and the FeCl₃ calibration curve. Exponential fits are presented (R² > 0.990). (b) UV/Vis spectra of Epi/Fe²⁺ systems after 1 min incubation in 10 mM Tris. (c) UV/Vis spectra of Epi/Fe²⁺ systems after 1 min incubation in 100 mM Tris. Dashed line represents the sum of experimental spectra. (d) 20 K EPR spectrum of the [Epi]/[Fe²⁺]_i = 2 system in 10 mM Tris after 1 min of incubation. The high field part of the spectrum was multiplied 10× for clarity (right). (e) Time-dependent changes of CV and peak currents (I_pa and I_pc) of [Fe²⁺]_i = 0.2 mM in 10 mM Tris at boron doped diamond electrode. Black line – CV of Fe³⁺ (0.2 mM). (f) Changes (marked with arrows) of anodic and cathodic E and I of Fe²⁺ and Epi in the [Epi]/[Fe²⁺]_i = 2 system.

Figure 6

Changes in O₂ concentration and redox potential (E_h) in Epi/Fe²⁺ systems in 10 mM Tris, pH 7.4. (a) Changes of [O₂] and rate of O₂ consumption induced by different concentrations of Fe²⁺ in the absence or presence of 0.2 mM Epi. Top-down peaks in the right panel represent the initial rate of O₂ consumption following the addition of Fe²⁺. (b) Quantification of O₂ consumption and H₂O₂ accumulation, 30 s after the addition of Fe²⁺ or CAT, respectively. H₂O₂ accumulation was quantified by CAT-induced O₂ release (2H₂O₂ → 2H₂O + O₂; [H₂O₂] = 2 × Δ[O₂]). Data are presented as means ± SE. Closed circles represent [O₂] and open circles represent [H₂O₂]. (c) Changes in the redox potential of 10 mM Tris buffer with or without Epi, following the addition of Fe²⁺. (d) Changes in the redox potential of O₂-free 10 mM Tris buffer (under N_2(g)) with or without Epi, following the addition of Fe²⁺. Dashed lines denote the redox potentials of referent systems (stable over time).