Cytoglobin has potent superoxide dismutase function

Abstract

Cytoglobin (Cygb) was discovered as a novel type of globin that is expressed in mammals; however, its functions remain uncertain. While Cygb protects against oxidant stress, the basis for this is unclear, and the effect of Cygb on superoxide metabolism is unknown. From dose-dependent studies of the effect of Cygb on superoxide catabolism, we identify that Cygb has potent superoxide dismutase (SOD) function. Initial assays using cytochrome c showed that Cygb exhibits a high rate of superoxide dismutation on the order of 10⁸ M^-1 ⋅ s^-1 Spin-trapping studies also demonstrated that the rate of Cygb-mediated superoxide dismutation (1.6 × 10⁸ M^-1 ⋅ s^-1) was only ∼10-fold less than Cu,Zn-SOD. Stopped-flow experiments confirmed that Cygb rapidly dismutates superoxide with rates within an order of magnitude of Cu,Zn-SOD or Mn-SOD. The SOD function of Cygb was inhibited by cyanide and CO that coordinate to Fe³⁺-Cygb and Fe²⁺-Cygb, respectively, suggesting that dismutation involves iron redox cycling, and this was confirmed by spectrophotometric titrations. In control smooth-muscle cells and cells with siRNA-mediated Cygb knockdown subjected to extracellular superoxide stress from xanthine/xanthine oxidase or intracellular superoxide stress triggered by the uncoupler, menadione, Cygb had a prominent role in superoxide metabolism and protected against superoxide-mediated death. Similar experiments in vessels showed higher levels of superoxide in Cygb^-/- mice than wild type. Thus, Cygb has potent SOD function and can rapidly dismutate superoxide in cells, conferring protection against oxidant injury. In view of its ubiquitous cellular expression at micromolar concentrations in smooth-muscle and other cells, Cygb can play an important role in cellular superoxide metabolism.

Keywords: EPR; ROS; SOD; free radical; superoxide.

Fig. 1.

Measurement of the purity of Cygb by SDS PAGE and MALDI-TOF MS. (A) The gel lanes are the following: Lane 1: Molecular weight markers, Lane 2: 0.5 μg Cygb, Lane 3: 1.0 μg Cygb, and Lane 4: 5.0 μg Cygb. Densitometry of the 5 μg lane of the gel is shown on the right. Cygb accounts for >99% of the total density of that gel lane. (B) MALDI-TOF MS data showing the Cygb peak at ∼21.4 kDa with very high purity. Peaks are labeled by mass with the peak areas in parenthesis.

Fig. 2.

Measurement of the SOD activity of Cygb and Cu,Zn-SOD assayed by ferricytochrome c reduction. The reaction mixture contained 50 μM ferricytochrome c, 50 μM X, and 0.1 mM EDTA in 50 mM phosphate buffer (pH 7.8) and was initiated by adding XO (70 nM final concentration). Increase in optical absorbance at 550 nm was followed in the absence or presence of various concentrations of Cygb (A) or Cu,Zn-SOD (SOD1) (C) with initial rates of cyt c reduction measured in the absence (V₀) and presence (V_i) of Cygb or Cu,Zn-SOD. Graphs of the concentration-dependent inhibition of the rate of cyt c reduction by Cygb (B) or Cu,Zn-SOD (D). Graphs show the mean ± SEM of three independent measurements fitted with nonlinear regression.

Fig. 3.

EPR measurements of the decrease in the rate of O₂^•− trapping with increasing concentrations of Cygb, Cu,Zn-SOD, or Mn-SOD. Stack plots of the EPR signal observed as a function of protein concentration are shown for Cygb (A), Cu,Zn-SOD (SOD1) (C), and Mn-SOD (SOD2) (E) ∼5 min after initiating the X/XO reaction. Initial rates of formation of the DIPPMPO-OOH signal were determined in the absence (V₀) and presence (V_i) of various concentrations of Cygb (B), Cu,Zn-SOD (D), and Mn-SOD (F). Each data point is the mean ± SEM of three measurements and fitted with a hyperbolic function.

Fig. 4.

Rates of O₂^•− decay measured by stopped-flow spectroscopy for Cu,Zn-SOD, Mn-SOD, and Cygb. The O₂^•− absorbance was monitored at 245 nm for Cu,Zn-SOD (SOD1), Mn-SOD (SOD2), and Cygb in 100 mM Hepes and 0.1 mM EDTA, pH 7.8, at RT. The slope of the plot of k_obs versus [protein] yielded the bimolecular dismutation rate for each protein: Cu,Zn-SOD ∼1.0 × 10⁹ M⁻¹ ⋅ s⁻¹, Mn-SOD ∼5.3 × 10⁸ M⁻¹ ⋅ s⁻¹, and Cygb ∼1.0 × 10⁸ M⁻¹ ⋅ s⁻¹. The data shown are the mean ± SEM of at least five independent measurements.

Fig. 5.

Binding of CN or CO to the Fe³⁺ or Fe²⁺ heme inhibits Cygb-mediated O₂^•− dismutation. EPR spin-trapping was performed using DIPPMPO in the presence of X and XO as described in Fig. 3. (A) Spectra shown are 40-s acquisitions acquired after ∼2 min of the reaction. (B) Relative intensities from the EPR spectra in A. While 50 nM Cygb quenches the observed O₂^•− radical adduct generation, this is largely abolished with CN-Fe³⁺-Cygb (CN-Cygb) or CO-Fe²⁺-Cygb (CO-Cygb). Thus, either complexation of CN to the ferric heme or CO to the ferrous heme blocks the SOD activity of Cygb. Data are the mean ± SEM, n = 10 to 14. **P < 0.01 versus 50 nM Cygb.

Fig. 6.

Spectrophotometric measurements of the reaction of Cygb with O₂^•−. (A) The spectrum of CygbFe³⁺ (10 µM) with Soret peak at 416 nm and α/β peaks at 533 and 563 nm (solid line). After addition of dithionite, CygbFe²⁺ is formed with shift of Soret peak to 428 nm and α/β peaks to 532 and 560 nm (dash dot line), and following subsequent introduction of O₂, CygbFe²⁺-O₂ is formed with Soret peak at 417 nm and α/β peaks at 540 and 578 m (dashed line). (B) Stiochiometric addition (1:1) of KO₂ to CygbFe²⁺-O₂ (solid line), formed as in A, triggers a shift back to CygbFe³⁺ (dashed line). (C) Addition (1:1) of KO₂ to CygbFe³⁺ (solid line) results in only trace change in the spectra (dotted line), and with further addition of KO₂, no further spectral changes were seen. From the minimal spectral change, it is estimated that less than 10% of CygbFe³⁺ is converted to CygbFe²⁺-O₂. (D) Upon repeat of the experiment in C in the presence of CO-saturated buffer, the Fe³⁺Cygb spectrum is converted to that of CygbFe²⁺-CO (dashed line), with a Soret band shift to 420 nm, and shifts in the α/β region to 569 nm/541 nm, respectively, with increased absorbance. With further addition of KO₂, no further spectral changes were seen. Thus, O₂^•− reduces Cygb from the ferric to the ferrous state, which is stabilized by CO binding. As shown in C, in the absence of CO, little CygbFe²⁺-O₂ accumulates, likely due to further rapid reaction with O₂^•−. Spectra of 0.5 s were recorded with mixing and start of spectral acquisition within ∼4 s. For each experiment, the reactions were completed in the initial spectrum as depicted with no further changes in repeat scans.

Fig. 7.

Spectra of CygbFe³⁺ and CygbFe²⁺-O₂ and measurement of CygbFe²⁺-O₂ formation with constant flux of O₂^•−. Spectra (A) and difference spectra (B) of the α/β region of CygbFe³⁺ and CygbFe²⁺-O₂. (C) Measurement of CygbFe²⁺-O₂ formation from CygbFe³⁺ (24 µM) in the presence of constant O₂^•− generation from XO (1 µM) and X (400 µM). A maximum of ∼4% of the Cygb was converted to CygbFe²⁺-O₂.

Fig. 8.

Addition of Cygb quenches extracellular radical generation and prevents cell death. SMCs, 2 × 10^6/mL, were incubated with the O₂^•− generating system X, 300 μM, and XO, 0.1 μM, and (X/XO) with or without 0.5 µM Cygb. (A) EPR spin-trapping was performed in the presence of 10 mM DIPPMPO, with spectra shown recorded ∼5 min postaddition of X/XO. Prominent signals of DIPPMPO-OOH peaks labeled (+) and DIPPMPO-OH peaks labeled (⋆) were seen that were quenched in the presence of Cygb. (B) Bar graph of the data from triplicate experiments as in A. (C) Data from parallel experiments measuring cell death by trypan blue exclusion with measurements at 5 and 15 min postaddition of X/XO. Increase in cell death was seen with exposure to the radical-generating system that was decreased by Cygb. Thus, Cygb addition quenched the radical generation and decreased radical-induced cell death. Data are the mean ± SEM, n = 3. **P < 0.01 versus No Cygb.

Fig. 9.

Effect of Cygb expression on menadione-induced intracellular O₂^•− generation and cell death. Cygb expression was knocked down in SMCs using Cygb siRNA with matched Scr siRNA control. (A and B) With siRNA treatment, Cygb expression was decreased by ∼85% compared to the Scr siRNA control SMCs. (C) EPR spin-trapping of menadione-treated SMCs showed that menadione induced O₂^•−-mediated radical generation that was increased in the Cygb siRNA–treated SMCs in which Cygb expression was knocked down. (D) From triplicate experiments, Cygb KD increased the observed radical generation by over threefold. This radical generation was quenched by an SOD_m that enters cells, M40403 (50 μM). When these cells were preincubated with Cygb either 0.5 or 2.5 μM, radical generation was also decreased. (E) From parallel experiments measuring cell death by trypan blue uptake, with measurements at 30 min postmenadione addition, cell death increased in Cygb siRNA–treated cells but not in control Scr siRNA–treated cells. Thus, KD of Cygb increased the levels of O₂^•− detected, and this correlated with increased cell death. Data are the mean ± SEM, n = 3. **P < 0.01 versus Scr siRNA.

Fig. 10.

Effect of Cygb-KO on menadione-induced O₂^•− generation in aorta of WT and Cygb^−/− mice. Aortic sections from C57BL/6 (WT) and Cygb^−/− (KO) mice were incubated with 10 µM DHE alone, 10 µM menadione plus DHE, or with 100 µM SOD_m (GC4419) together with DHE and menadione. Sections were visualized with confocal fluorescence microscopy. As seen in the images, red fluorescence arises from O₂^•−-mediated oxidation of DHE. The bar graph shows quantitation of the fluorescence from a series of repeat measurements. Incubation with SOD_m quenched the observed fluorescence, confirming that it is O₂^•− derived. The menadione-induced O₂^•− generation in aortic sections was much higher in Cygb-KO aorta compared to WT. Data shown are the mean ± SEM, n = 6. *P < 0.01 versus WT.