Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH

Abstract

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe³⁺cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H₂O₂) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe³⁺cyt c in the presence of H₂O₂. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD(•), which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe³⁺cyt c may play a key role in the mitochondrial "ROS-induced ROS-release" signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.

Figure 1

Room temperature EPR spectra of the superoxide radical adduct of DMPO, DMPO-OOH. All the reactions were performed in 50 mM phosphate buffer (pH = 7.4) containing 0.1 mM DTPA. Spectrum A: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), NADH (1 mM), and H₂O₂ (0.5 mM). Spectrum B: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), NADH (1 mM), and H₂O₂ (0.5 mM). Spectrum C: DMPO (50 mM), Fe³⁺cyt c (0.1 mM), NADH (1 mM), and H₂O₂ (0.5 mM). Spectrum D: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), SOD1 (500 U/mL), NADH (1 mM), and H₂O₂ (0.5 mM). Spectrum E: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), and H₂O₂ (0.5 mM). Spectrum F: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), and NADH (1 mM). The observed isotropic hyperfine values of the DMPO-OOH adducts are a_N = 13.49 G, a_H1 = 10.78 G, and a_H2 = 1.39 G. EPR instrument parameters used were as described in the materials and methods section.

Figure 2

Room temperature EPR spectra of the superoxide radical adduct of DMPO, DMPO-OOH. All the reactions were performed in 50 mM phosphate buffer (pH = 7.4) containing 0.1 mM DTPA. Spectrum A: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), NADH (0.1 mM), and H₂O₂ (0.5 mM). Spectrum B: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), NADH (0.5 mM), and H₂O₂ (0.5 mM). Spectrum C: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), NADH (1 mM), and H₂O₂ (0.5 mM). Spectrum D: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), NADH (2 mM), and H₂O₂ (0.5 mM). EPR instrument parameters used were as described in the materials and methods section.

Figure 3

(A) Plot of the concentration of superoxide radical adduct of DMPO (DMPO-OOH) vs time for various concentrations of NADH. Experiments were performed with DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), H₂O₂ (0.5 mM) and various concentrations of NADH in phosphate buffer containing DTPA (0.1 mM). (B) Plot of initial rate of formation of superoxide radical adducts of DMPO (DMPO-OOH) vs NADH concentrations. Rates in panel B were obtained from the initial slope of the data from panel A. EPR spectra were quantified by computer simulation of the spectra and comparison of the double integral of the observed signal with that of a TEMPO standard (1 µM) measured under the identical conditions. Data represent means ± S.E (n = 3).

Figure 4

Room temperature EPR spectra of the superoxide radical adduct of DMPO, DMPO-OOH. All the reactions were performed in 50 mM phosphate buffer (pH = 7.4) containing 0.1 mM DTPA. Spectrum A: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), NADH (1 mM), and H₂O₂ (0.05 mM). Spectrum B: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), NADH (1 mM), and H₂O₂ (0.1 mM). Spectrum C: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), NADH (1 mM), and H₂O₂ (0.25 mM). Spectrum D: DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), NADH (1 mM), and H₂O₂ (0.5 mM). EPR instrument parameters used were as described in the materials and methods section.

Figure 5

(A) Plot of the concentration of superoxide radical adduct of DMPO (DMPO-OOH) vs time for various concentrations of H₂O₂. Experiments were performed with DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), NADH (1 mM), and various concentrations of H₂O₂ in phosphate buffer containing DTPA (0.1 mM). (B) Plot of initial rate of formation of superoxide radical adducts of DMPO (DMPO-OOH) vs H₂O₂ concentrations. Rates in panel B were obtained from the initial slope of the data from panel A. EPR spectra were quantified by computer simulation of the spectra and comparison of the double integral of the observed signal with that of a TEMPO standard (1 µM) measured under the identical conditions. Data represent means ± S.E (n = 3).

Figure 6

Room temperature EPR spectra of the superoxide radical adduct of DMPO, DMPO-OOH. All the reactions were performed in 50 mM phosphate buffer (pH = 7.4) containing 0.1 mM DTPA. The reaction mixture contains DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c (0.1 mM), NADH (1 mM), and H₂O₂ (0.5 mM). Spectrum A: The reaction mixture was prepared inside the nitrogen filled glove box. Spectrum B: The reactants were taken out from the glove box and the reaction mixture was prepared under room air. EPR instrument parameters used were as described in the materials and methods section.

Figure 7

Plot of the oxygen consumption in phosphate buffer containing Fe³⁺cyt c (0.1 mM), NADH (1 mM), and H₂O₂ (0.5 mM) using the EPR oximetry probe LiPc. Line width from the EPR spectra were converted into partial pressure of oxygen using the calibration formula of pO₂ = (Line width G − 0.1748)/0.005684. EPR instrument parameters used were microwave frequency 9.775, modulation frequency 100 kHz, modulation amplitude 0.1 G, microwave power 1 mW, number of scans 1, scan time 10.5 s, and time constant 82 ms. The partial pressure of oxygen was converted into dissolved concentration based on the solubility of oxygen in water at room air of 0.265 mM [38].

Figure 8

Oxidation of Fe³⁺cyt c to M-Fe³⁺cyt c by HOCl increases the initial rate of formation of superoxide radical adducts of DMPO (DMPO-OOH). The preparation of M-Fe³⁺cyt c is described in the materials and methods section. EPR spin trapping experiments were performed with DMPO (50 mM), methyl-β-cyclodextrin (0.1 M), Fe³⁺cyt c/M-Fe³⁺cyt c (0.1 mM), NADH (1 mM), and H₂O₂ (0.5 mM) in phosphate buffer containing DTPA (0.1 mM). A: Fe³⁺cyt c. B: M-Fe³⁺cyt c. EPR spectra were quantified by computer simulation of the spectra and comparison of the double integral of the observed signal with that of a TEMPO standard (1 µM) measured under the identical conditions. Initial rates were calculated and mean ± S.E shown in graph (n = 3).

Figure 9

UV-visible absorption spectra of cyt c in phosphate buffer, pH 7.4. Experiments were performed with Fe³⁺cyt c, NADH, and H₂O₂. Reactions were initiated by the addition of H₂O₂. Spectra were recorded five minutes after mixing all the reactants. A: Fe³⁺cyt c (0.1 mM), B: Fe³⁺cyt c (0.1 mM) and H₂O₂ (0.5 mM), C: Fe³⁺cyt c (0.1 mM) and NADH (2 mM), D: Fe³⁺cyt c (0.1 mM), NADH (2 mM), and H₂O₂ (0.5 mM).

Figure 10

The effect of NADH and H₂O₂ on the reduction of Fe³⁺cyt c in phosphate buffer, pH 7.4. Experiments were performed with Fe³⁺cyt c, NADH, and H₂O₂. Reactions were initiated by the addition of H₂O₂. UV-visible absorption spectra were recorded five minutes after mixing all the reactants. A: Fe³⁺cyt c (0.1 mM), NADH (0.1 – 2 mM), and H₂O₂ (0.5 mM). B: Fe³⁺cyt c (0.1 mM), NADH (1 mM), and H₂O₂ (0.05 – 0.5 mM). Data represent means ± S.E (n = 3).

Figure 11

Proposed model of cytochrome c mediated removal of H₂O₂, oxidation of NADH, generation of superoxide radical, and alternative electron transfer pathway in mitochondria.

Scheme 1

Scheme 2