Revisiting Kadenbach: Electron flux rate through cytochrome c-oxidase determines the ATP-inhibitory effect and subsequent production of ROS

Abstract

Mitochondrial respiration is the predominant source of ATP. Excessive rates of electron transport cause a higher production of harmful reactive oxygen species (ROS). There are two regulatory mechanisms known. The first, according to Mitchel, is dependent on the mitochondrial membrane potential that drives ATP synthase for ATP production, and the second, the Kadenbach mechanism, is focussed on the binding of ATP to Cytochrome c Oxidase (CytOx) at high ATP/ADP ratios, which results in an allosteric conformational change to CytOx, causing inhibition. In times of stress, ATP-dependent inhibition is switched off and the activity of CytOx is exclusively determined by the membrane potential, leading to an increase in ROS production. The second mechanism for respiratory control depends on the quantity of electron transfer to the Heme aa3 of CytOx. When ATP is bound to CytOx the enzyme is inhibited, and ROS formation is decreased, although the mitochondrial membrane potential is increased.

Keywords: allosteric inhibition; cytochrome c oxidase; enzyme kinetics; ischaemic preconditioning; phosphodiesterase inhibitors; reactive oxygen species.

Figure 1

Polarographic measurements of CytOx kinetics in bovine heart tissue homogenate (A) and mitochondria (B). Bovine heart tissue that was frozen at −80 °C was thawed, homogenized on ice in 5 volumes of standard isolation buffer (250 mM sucrose, 20 mM Hepes, 1 mM EDTA, pH 7.4) and used directly for kinetic measurements of CytOx activity or subjected to isolation of mitochondria by standard isolation procedures 14. Rats were sacrificed by decapitation and rat hearts were homogenized in the standard isolation medium (as used in the bovine heart procedure, but in addition contained 0.2% fatty acid‐free BSA) using a homogenizer after cutting the tissue into small pieces using a scissor. This rat heart tissue homogenate was either used directly for kinetic measurements of CytOx activity (C) or used for the isolation of mitochondria (D). Kinetic measurements were performed in the sucrose buffer in the presence of 5 mM ADP or 5 mM ATP, 10 mM Phosphoenolpyruvate and 160 U/mL pyruvate kinase at increasing concentrations of cytochrome c, along with 18 mM ascorbate to reduce cytochrome c. The concentration of aa₃ in the bovine heart tissue homogenate that was used for oxygen measurements was 113.57 ± 7.73 nM, and that used in the bovine heart mitochondria was 567.84 ± 38.76 nM and was determined spectrophotometrically (n =4). Although the aa3 content in the rat heart tissue homogenate was 48 ± 5.54 nM, in the rat heart mitochondria it was 251.32 ± 20.16 (n = 3). *Wilcoxon‐Mann‐Whitney‐rank sum test p < 0.05.

Figure 2

Schematic representation of the modified molecular structure of CytOx subunits, originally from Herrmann et al. 123. The mitochondrial‐encoded SU I, II and III have the central stage, whereas the nuclear‐ encoded SU surround the central column. The blue arrow represents the binding of oxygen to the transmembrane helices of SU I and II 124. Cytochrome c (molecule on the left, Hoffmeister K, Wikimedia commons) transfers electrons to CytOx (grey arrow ‘e^‐’). It is proposed that during enzyme turnover the enzyme cycles between two conformers, one with a substrate binding site on subunit II, and the other along the interface of subunits II, IV and VIb. Structural analyses suggests that Glu112, Glu113, Glu114 and Asp125 of subunit IV, and Glu40, Glu54, Glu78, Asp35, Asp49, Asp73 and Asp74 of subunit VIb are residues that could possibly be involved 125. Cytochrome c binding affects the conformation of cytochrome a within CytOx 126. A: Proposed model representing the influence of ADP or ATP binding to SU IV and SU VIII on the enzymatic activity of CytOx. Ten binding sites for adenine nucleotides are known. At seven sites, ADP and ATP are exchanged 127. One binding site for ATP or ADP, located at the matrix‐oriented domain of the heart‐type subunit VIaH, increases the H+/e‐ stoichiometry of the enzyme in heart or skeletal muscle from 0.5 to 1.0 when bound ATP is exchanged by ADP. Two further binding sites for ATP or ADP are located at the cytosolic and the matrix domain of subunit IV. Although the additional binding site on SU VIa has been confirmed by Taanman et al. 128 (not shown), most binding sites were found on SU IV and VIII using radioactive ATP analogues, suggesting that these two nuclear‐coded polypeptides may play a regulatory role 129, 130. Especially, SU IV is essential for the assembly and respiratory function of the complete enzyme complex 131. Because of the negative charges associated with ATP (fourfold), and the dipole moment of cytochrome c 132, 133,the holoenzyme creates an electrostatic field (negative sign on the cycle) that finally regulates the internal electron‐transfer reactions by its electric field strength 134. This explains how CytOx acts like an ‘electro‐catalyst for oxygen reduction’ 135. Furthermore, Craig et al. 136, 137 and Lin et al. 138 found that ATP binding to cytochrome c diminishes electron flow in the mitochondrial respiratory pathway and respiration is shut down. B: In the case of the exchange of ADP to ATP on the seven nucleotide binding sites the electrostatic field becomes weaker because of less negative charge with ADP. Subsequently, electron transfer from cytochrome c to SU II becomes accelerated. C: Modified model for subunit order inside the CytOx molecule according Tsukihara et al. 139, 140 and shows again the proposed mechanism of ATP binding to SU IV and VIII. The subunits of CytOx in the molecule centre are shown with blue (SU I), pink (SU II) and dark grey cycles (SU III). Roman numbers represents the helices. Blue dotted lines mark the entry of Helix I/II/III to Oxygen pathway 1 and the entry of Helix IV/V to Oxygen pathway 2. The binding of ATP (small grey cycles with white minus signs) at seven positions to SU IV and VIII results in a higher negative charge for the molecular dipole. The more negative ‘cloud’ induces tilting and bending of the molecule, and the binding of cytochrome c (black dotted line) is influenced, resulting in alterations of the subunit positioning (here helices XI, XII, I and II) together with a reduction in the distance between haeme a and haeme a₃. The influence of an electric potential field and the effect of ionic strength on the reaction rate of cytochrome c have been described by Koppenol et al. 141. D: The same molecular model features the situation after binding of ADP to all the binding sites of SU IV and VIII. A less negatively charged ‘cloud’ (left side) widens the distances between Helices XI, XII, I and II and finally induces a ‘more open’ angle between haeme a and haeme a₃ for acceleration of electron transfer and increased Dioxygen turn over. However, the question of a pH‐dependent polarity change at the binuclear centre 142 remains unanswered, although the proton K‐pathway is known to become sufficiently flexible for internal water molecules to alternately occupy upper and lower parts of the oxygen pathways, which are associated with conserved Thr‐359 and Lys‐362 residues. Subsequent intramolecular ‘constrictions’ 143 could support the already known effect of dielectric relaxation of CytOx 144.

Figure 3

Freshly isolated rat heart mitochondria were used for the kinetic measurements of CytOx activity without (A) and with 1 μg/mL Milrinone (B), and without (C) and with 1 μg/mL Euphylong (D). Each phosphodiesterase inhibitor was added directly to the sucrose‐containing buffer. Oxygen consumption as a function of CytOx activity was measured in the presence of either 5 mM ADP or 5 mM ATP + 10 mM Phosphoenolpyruvate and 160 U/mL pyruvate kinase at increasing concentrations of cytochrome c (0–40 μM) using 18 mM ascorbate as a substrate. Mitochondrial stock protein concentrations were determined by the BCA method and were 26.6 ± 1.30 mg/ml (n = 3). Concentrations of mitochondria between Figure A and C were 5 to 1. *Wilcoxon‐Mann‐Whitney‐rank sum test p < 0.05.