Cardiac mitochondria and reactive oxygen species generation

Abstract

Mitochondrial reactive oxygen species (ROS) have emerged as an important mechanism of disease and redox signaling in the cardiovascular system. Under basal or pathological conditions, electron leakage for ROS production is primarily mediated by the electron transport chain and the proton motive force consisting of a membrane potential (ΔΨ) and a proton gradient (ΔpH). Several factors controlling ROS production in the mitochondria include flavin mononucleotide and flavin mononucleotide-binding domain of complex I, ubisemiquinone and quinone-binding domain of complex I, flavin adenine nucleotide-binding moiety and quinone-binding pocket of complex II, and unstable semiquinone mediated by the Q cycle of complex III. In mitochondrial complex I, specific cysteinyl redox domains modulate ROS production from the flavin mononucleotide moiety and iron-sulfur clusters. In the cardiovascular system, mitochondrial ROS have been linked to mediating the physiological effects of metabolic dilation and preconditioning-like mitochondrial ATP-sensitive potassium channel activation. Furthermore, oxidative post-translational modification by glutathione in complex I and complex II has been shown to affect enzymatic catalysis, protein-protein interactions, and enzyme-mediated ROS production. Conditions associated with oxidative or nitrosative stress, such as myocardial ischemia and reperfusion, increase mitochondrial ROS production via oxidative injury of complexes I and II and superoxide anion radical-induced hydroxyl radical production by aconitase. Further insight into cellular mechanisms by which specific redox post-translational modifications regulate ROS production in the mitochondria will enrich our understanding of redox signal transduction and identify new therapeutic targets for cardiovascular diseases in which oxidative stress perturbs normal redox signaling.

Keywords: electron transport chain complex proteins; mitochondria; myocardial infarction; reactive oxygen species.

Figure 1

Schematic representation illustrating the mechanism of oxygen free radical(s) generation mediated by electron transport chain, the proton motive force (PMF, Δp), and the aconitase of Krebs cycle in mitochondria. Blue arrows show the path of electron transport from NADH or FADH₂ to O₂, or reverse electron flow from FADH₂-linked succinate to complex I. Brown dashed arrows indicate the sites mediating ^•O₂⁻ generation in mitochondria. As electrons pass through the chain, protons are pumped from the mitochondrial matrix to the inter-membrane space, thereby establishing an electrochemical potential gradient or called proton motive force (Δp) across the inner membrane. The positive and negative charges on the membrane denote the membrane potential (ΔΨ). A proton gradient is denoted by ΔpH for the difference of pH across the membrane. Δp can contribute to ^•O₂⁻ generation in the respiratory conditions of state 2 and state 4. Black circles show aconitase of the Krebs Cycle that generates NADH and FADH₂ as the substrates of the ETC that are the source of hydroxyl radical production induced by ^•O₂⁻. The common inhibitors used for studying the ETC components, ΔpH, and ΔΨ are indicated in brick red italics.

Figure 2

A, Homology model of the 51 kDa subunit of bovine heart complex I using the crystal structure of respiratory complex I from T. thermophilius with a protein data bank accession code, 3I9V (pdb 3I9V), as a template (36). Arrows show the domains of peptide pGSCA206 and peptide p51, denoted by green and blue ribbons. B, Model of the 75 kDa subunit of bovine heart complex I using 3I9V as a template. Arrows show the domains of peptide pGSCB367 and peptide p75, denoted by red and blue ribbons.

Figure 3

Hydrophobic residues and polar interactions (dashed lines) in the ubiquinone-binding site (Q_p site) of mammalian complex II. The quinone-binding pocket is revealed by the X-ray structure (pdb 1ZOY), and involves the residues Trp B172, Trp B173, His B216, Arg C46, Asp D90, Tyr D91. The residues of the ubiquinone-binding site are determined by X-ray structure to be Trp B173 and Tyr D91. UQ denotes ubiquinone.

Figure 4

(A) Superoxide generation mediated by the Q-cycle mechanism in complex III. The scheme is adapted from Ref. (43) with modification. Gray areas symbolize the reactions involved in ^•O₂⁻ production. P, O, and C represent positive, outside, and cytoplasmic side respectively. N, I, and M stands for negative, inside, and matrix side, respectively. Solid bars in brick red show the opposition of electron transfer from the b_L to b_H by the membrane potential (ΔΨ) and inhibition of b_H reoxidation by antimycin A. (B) X-ray structure (pdb 1PPJ) of complex III shows Q_o site (occupied by Q_o site inhibitor, stigmatellin in green color) is located immediately next to the inter-membrane space. Structure also shows the Q_i site (occupied by Q_i site inhibitor, antimycin A in blue color) is located next to the matrix site. The subunits of cytochrome b, cytochrome c₁, and Rieske iron sulfur protein (RISP) are denoted by cyan, pale green, and yellow ribbons, respectively.

Figure 5

Schematic depiction showing the proposed physiological (highlighted with green boxes) and pathophysiological (highlighted with red boxes and bold) roles of ^•O₂⁻ generation by each respiratory electron transport complex, the proton motive force (Δp), and the aconitase of Krebs cycle. The H₂O₂ derived from ^•O₂⁻ by complex I and complex III can function as EDHF to mediate the intracellular signaling of metabolic dilation via smooth cell (SMC) relaxation. Intra-mitochondrial H₂O₂ formed at the matrix site and extra-mitochondrial H₂O₂ formed at the Q_o site can diffuse to the cytosol to trigger the physiological responses. Unlike complex I, complex II-mediated ^•O₂⁻ may modulate pre-conditioning via mK_ATP channel activation. However, it remains unclear the physiological role of ^•O₂⁻ mediated by the Qp site of complex II. In addition to the physiological role of EDHF, complex III-mediated ^•O₂⁻ also has been linked to diazoxide-induced transient opening of mK_ATP. Under the pathophysiological conditions of myocardial ischemia and reperfusion (I/R), the ^•O₂⁻ generation by complexes I, III, and flavin protein of complex II all mediate I/R injury and increase pro-oxidant activity of aconitase, thus further augmenting I/R injury. PKA-mediated phosphorylation of complex IV under ischemic conditions predisposes complex IV to generate ^•O₂⁻ and augment I/R injury. Proton motive force is proposed as a source of ^•O₂⁻ for I/R injury because reperfusion partially restores the membrane potential. Overproduction of ^•O₂⁻ from the Qp site of complex II is associated with the disease of head-to-neck paraganglioma, however, it’s pathological role in I/R injury remains unexplored.