Ischemic defects in the electron transport chain increase the production of reactive oxygen species from isolated rat heart mitochondria

Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed whether ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6-mo-old Fisher 344 rat hearts following 25 min global ischemia or following 40 min of perfusion alone as controls. H(2)O(2) release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of H(2)O(2) was increased by 178 +/- 14% and 179 +/- 17% in SSM and IFM (n = 9), respectively, following ischemia compared with controls (n = 8). With succinate as substrate in the presence of rotenone, H(2)O(2) increased by 272 +/- 22% and 171 +/- 21% in SSM and IFM, respectively, after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased H(2)O(2) production by 179 +/- 24% and 155 +/- 14% in SSM and IFM, respectively, following ischemia. Ischemia also increased the antimycin A-stimulated production of H(2)O(2) from complex III. Thus ischemic damage to the ETC increased both the capacity and the net production of H(2)O(2) from complex I and complex III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury.     

Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria

Mitochondria contribute to myocyte injury during ischemia. After 30 and 45 min of ischemia in the isolated perfused rabbit heart, subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, sustain a decrease in oxidative phosphorylation through cytochrome oxidase. In contrast, oxidation through cytochrome oxidase in interfibrillar mitochondria (IFM), located between the myofibrils, remains unaffected. Cytochrome oxidase activity in the intact membrane requires an inner mitochondrial membrane lipid environment enriched in cardiolipin. During ischemia, the content of cardiolipin decreased only in SSM, whereas the content of other phospholipids was preserved. Ischemia did not alter the composition of the cardiolipin that remained in SSM. Cardiolipin content was preserved in IFM during ischemia. Thus cardiolipin is a relatively early target of ischemic mitochondrial damage, leading to loss of oxidative phosphorylation through cytochrome oxidase in SSM.     

Ischemic injury to mitochondrial electron transport in the aging heart: damage to the iron-sulfur protein subunit of electron transport complex III

The aging heart sustains greater injury during ischemia and reperfusion compared to adult hearts. Aging decreases oxidative function in interfibrillar mitochondria (IFM) that reside among the myofibers, while subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, remain unaltered. Aging decreases complex III activity selectively in IFM via alteration of the cytochrome c binding site. With 25 min of global ischemia, complex III activity decreases in SSM and further decreases in IFM in the aging heart. Ischemia leads to a marked decrease in the electron paramagnetic resonance signal of the iron-sulfur protein (ISP) in both SSM and IFM, despite a preserved content of ISP peptide. Thus, ischemia results in a functional decrease in the iron-sulfur center in ISP without subunit peptide loss. In the aging heart, at the onset of reperfusion, IFM contain two tandem defects in the path of electron flow through complex III, providing a likely mechanism for enhanced oxidant production and reperfusion damage.     

Blockade of electron transport during ischemia protects cardiac mitochondria

Subsarcolemmal mitochondria sustain progressive damage during myocardial ischemia. Ischemia decreases the content of the mitochondrial phospholipid cardiolipin accompanied by a decrease in cytochrome c content and a diminished rate of oxidation through cytochrome oxidase. We propose that during ischemia mitochondria produce reactive oxygen species at sites in the electron transport chain proximal to cytochrome oxidase that contribute to the ischemic damage. Isolated, perfused rabbit hearts were treated with rotenone, an irreversible inhibitor of complex I in the proximal electron transport chain, immediately before ischemia. Rotenone pretreatment preserved the contents of cardiolipin and cytochrome c measured after 45 min of ischemia. The rate of oxidation through cytochrome oxidase also was improved in rotenone-treated hearts. Inhibition of the electron transport chain during ischemia lessens damage to mitochondria. Rotenone treatment of isolated subsarcolemmal mitochondria decreased the production of reactive oxygen species during the oxidation of complex I substrates. Thus, the limitation of electron flow during ischemia preserves cardiolipin content, cytochrome c content, and the rate of oxidation through cytochrome oxidase. The mitochondrial electron transport chain contributes to ischemic mitochondrial damage that in turn augments myocyte injury during subsequent reperfusion.     

Endoplasmic reticulum stress-mediated mitochondrial dysfunction in aged hearts

Aging impairs the mitochondrial electron transport chain (ETC), especially in interfibrillar mitochondria (IFM). Mitochondria are in close contact with the endoplasmic reticulum (ER). Induction of ER stress leads to ETC injury in adult heart mitochondria. We asked if ER stress contributes to the mitochondrial dysfunction during aging. Subsarcolemmal mitochondria (SSM) and IFM were isolated from 3, 18, and 24 mo. C57Bl/6 mouse hearts. ER stress progressively increased with age, especially in 24 mo. mice that manifest mitochondrial dysfunction. OXPHOS was decreased in 24 mo. IFM oxidizing complex I and complex IV substrates. Proteomic analysis showed that the content of multiple complex I subunits was decreased in IFM from 24 mo. hearts, but remained unchanged in in 18 mo. IFM without a decrease in OXPHOS. Feeding 24 mo. old mice with 4-phenylbutyrate (4-PBA) for two weeks attenuated the ER stress and improved mitochondrial function. These results indicate that ER stress contributes to the mitochondrial dysfunction in aged hearts. Attenuation of ER stress is a potential approach to improve mitochondrial function in aged hearts.     

Aging selectively decreases oxidative capacity in rat heart interfibrillar mitochondria

Mitochondrial-derived oxidative injury contributes to cellular aging as well as to reperfusion-induced tissue damage. While the aging-heart suffers greater tissue damage following ischemia and reperfusion than the adult heart, the occurrence of aging-related alterations in mitochondrial oxidative metabolism in the elderly heart has remained uncertain. We determined if aging altered oxidative metabolism in either of the two populations of cardiac mitochondria, subsarcolemmal mitochondria (SSM) that reside beneath the plasma membrane or interfibrillar mitochondria (IFM) located between the myofibrils. SSM and IFM were isolated from 6-month adult and 24- and 28-month elderly Fischer 344 rat hearts. Aging-related alterations were limited to IFM, while SSM remained unaffected. Aging decreased the rate of oxidative phosphorylation in IFM, including when stimulated by electron donors specific for cytochrome oxidase. Cytochrome oxidase enzyme activity was decreased in IFM from aging hearts, while activity in SSM remained similar to adult controls. These findings allow future studies of aging-related decrements in oxidative function to focus upon IFM, while SSM provide an inherent control group of mitochondria that are free of aging-related alterations in oxidative function. The selective alteration of IFM during aging raises the possibility that the consequences of aging-induced mitochondrial dysfunction will be enhanced in specific subcellular regions of the senescent myocyte.     

Mitochondria-specific transgenic overexpression of phospholipid hydroperoxide glutathione peroxidase (GPx4) attenuates ischemia/reperfusion-associated cardiac dysfunction

Ischemia/reperfusion (I/R) injury elicits damage to mitochondria. Antioxidants provide protection from I/R-induced mitochondrial damage. The goal of this study was to determine the impact of mitochondria-specific overexpression of GPx4 (PHGPx) on cardiac function following I/R. Transgenic mice were created in which PHGPx was overexpressed solely in the mitochondrion (mPHGPx). MPHGPx and littermate control hearts were subjected to global no-flow ischemia (20 min) followed by reflow reperfusion (30, 60, and 90 min). Following I/R, mPHGPx hearts possessed significantly better rates of contraction, developed pressures, and peak-systolic pressures as compared to controls (P<0.05). No differences were observed in rates of relaxation or end-diastolic pressures. Lipid peroxidation was significantly lower in mitochondria from mPHGPx hearts as compared to controls, following I/R (P<0.05). Electron transport chain (ETC) complex I, III, and IV activities were significantly higher in mPHGPx hearts as compared to controls, following I/R (P<0.05). MPHGPx overexpression enhanced ETC complex I, III, and IV activities in subsarcolemmal mitochondria (SSM; P<0.05), and ETC complex I and III activities in interfibrillar mitochondria (IFM; P<0.05) following I/R. These results indicate that mitochondria-specific GPx4 overexpression protects cardiac contractile function and preserves ETC complex activities following I/R. These results provide further rationale for the use of mPHGPx as a therapeutic protectant.     

Ischemic preconditioning does not protect via blockade of electron transport.

Ischemic preconditioning (IPC) before sustained ischemia decreases myocardial infarct size mediated in part via protection of cardiac mitochondria. Reversible blockade of electron transport at complex I immediately before sustained ischemia also preserves mitochondrial respiration and decreases infarct size. We proposed that IPC would attenuate electron transport from complex I as a potential effector mechanism of cardioprotection. Isolated, Langendorff-perfused rat hearts underwent IPC (3 cycles of 5-min 37 degrees C global ischemia and 5-min reperfusion) or were perfused for 40 min without ischemia as controls. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated. IPC did not decrease ADP-stimulated respiration measured in intact mitochondria using substrates that donate reducing equivalents to complex I. Maximally expressed complex I activity measured as rotenone-sensitive NADH:ubiquinone oxidoreductase in detergent-solubilized mitochondria was also unaffected by IPC. Thus the protection of IPC does not occur as a consequence of a partial decrease in complex I activity leading to a decrease in integrated respiration through complex I. IPC and blockade of electron transport both converge on mitochondria as effectors of cardioprotection; however, each modulates mitochondrial metabolism during ischemia by different mechanisms to achieve cardioprotection.     

Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations

Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (∼10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (∼70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C₂₀-, C₂₂-, and C₂₄-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other.     

Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion

Mitochondria are increasingly recognized as lynchpins in the evolution of cardiac injury during ischemia and reperfusion. This review addresses the emerging concept that modulation of mitochondrial respiration during and immediately following an episode of ischemia can attenuate the extent of myocardial injury. The blockade of electron transport and the partial uncoupling of respiration are two mechanisms whereby manipulation of mitochondrial metabolism during ischemia decreases cardiac injury. Although protection by inhibition of electron transport or uncoupling of respiration initially appears to be counterintuitive, the continuation of mitochondrial oxidative phosphorylation in the pathological milieu of ischemia generates reactive oxygen species, mitochondrial calcium overload, and the release of cytochrome c. The initial target of these deleterious mitochondrial-driven processes is the mitochondria themselves. Consequences to the cardiomyocyte, in turn, include oxidative damage, the onset of mitochondrial permeability transition, and activation of apoptotic cascades, all favoring cardiomyocyte death. Ischemia-induced mitochondrial damage carried forward into reperfusion further amplifies these mechanisms of mitochondrial-driven myocyte injury. Interruption of mitochondrial respiration during early reperfusion by pharmacologic blockade of electron transport or even recurrent hypoxia or brief ischemia paradoxically decreases cardiac injury. It increasingly appears that the cardioprotective paradigms of ischemic preconditioning and postconditioning utilize modulation of mitochondrial oxidative metabolism as a key effector mechanism. The initially counterintuitive approach to inhibit mitochondrial respiration provides a new cardioprotective paradigm to decrease cellular injury during both ischemia and reperfusion. cardiolipin; cytochrome c; complex I; cytochrome oxidase     

Depletion of cardiolipin and cytochrome c during ischemia increases hydrogen peroxide production from the electron transport chain

Mitochondrial electron transport is a major source of reactive oxygen species (ROS) during cardiac ischemia and reperfusion. In the isolated rabbit heart, 30 and 45 min of ischemia decrease the contents of cardiolipin and cytochrome c in subsarcolemmal mitochondria (SSM) located beneath the plasma membrane. In contrast, interfibrillar mitochondria (IFM) in the interior of the myocyte do not sustain a decrease in cardiolipin. We proposed that the depletion of cardiolipin and the accompanying cytochrome c loss during ischemia were critical events that amplified ROS production by mitochondria. The total production of H2O2 was measured in submitochondrial particles (SMP) prepared from rabbit heart SSM and IFM after 0, 15, 30, and 45 min of ischemia. With NADH as substrate, total H2O2 production was increased only in SMP from SSM after 30 and 45 min ischemia, when ischemia decreased the content of cardiolipin and cytochrome c. In contrast, ischemia did not augment H2O2 generation in SMP from IFM with preserved cardiolipin and cytochrome c content. Thus, during the evolution of ischemic injury, H2O2 production from the electron transport chain increased after depletion of cardiolipin and the loss of cytochrome c.     

High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system

Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25 kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in ‘broken mitochondria’ (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.     

Elucidation of the effects of lipoperoxidation on the mitochondrial electron transport chain using yeast mitochondria with manipulated fatty acid content

Lipoperoxidative damage to the respiratory chain proteins may account for disruption in mitochondrial electron transport chain (ETC) function and could lead to an augment in the production of reactive oxygen species (ROS). To test this hypothesis, we investigated the effects of lipoperoxidation on ETC function and cytochromes spectra of Saccharomyces cerevisiae mitochondria. We compared the effects of Fe²⁺ treatment on mitochondria isolated from yeast with native (lipoperoxidation-resistant) and modified (lipoperoxidation-sensitive) fatty acid composition. Augmented sensitivity to oxidative stress was observed in the complex III-complex IV segment of the ETC. Lipoperoxidation did not alter the cytochromes content. Under lipoperoxidative conditions, cytochrome c reduction by succinate was almost totally eliminated by superoxide dismutase and stigmatellin. Our results suggest that lipoperoxidation impairs electron transfer mainly at cytochrome b in complex III, which leads to increased resistance to antimycin A and ROS generation due to an electron leak at the level of the Q_O site of complex III.     

Impairment of mitochondrial respiratory chain activity in aortic endothelial cells induced by glycated low-density lipoprotein

Coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated LDL (glyLDL) were detected in patients with diabetes. Our previous studies demonstrated that glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). This study examined the effects of glyLDL on oxygen consumption in mitochondria and the activities of key enzymes in the mitochondrial electron transport chain (ETC) in cultured porcine aortic EC. The results demonstrated that glyLDL treatment significantly impaired oxygen consumption in Complexes I, II/III, and IV of the mitochondrial ETC in EC compared to LDL or vehicle control detected using oxygraphy. Incubation with glyLDL significantly reduced the mitochondrial membrane potential, the NAD⁺/NADH ratio, and the activities of mitochondrial ETC enzymes (NADH-ubiquinone dehydrogenase, succinate cytochrome c reductase, ubiquinone cytochrome c reductase, and cytochrome c oxidase) in EC compared to LDL or control. The abundance of mitochondria-associated ROS and the release of ROS from EC were significantly increased after glyLDL treatment. The findings suggest that glyLDL attenuates the activities of key enzymes in the mitochondrial ETC, decreases mitochondrial oxygen consumption, reduces mitochondrial membrane potential, and increases ROS generation in EC, which potentially contribute to mitochondrial dysfunction in diabetic patients.     

Studies on the orientations of the mitochondrial redox carriers II. Orientation of the mitochondrial chromophores with respect to the plane of the membrane in hydrated,oriented mitochondrial multilayers

Orientations of the active site chromophores of the mitochondrial redox carriers have been investigated in hydrated, oriented multilayers of mitochondrial membranes using optical and EPR spectroscopy. The hemes of cytochrome c oxidase, cytochrome c₁, and cytochromes b were found to be oriented in a similar manner, with the normal to their heme planes lying approximately in the plane of the mitochondrial membrane. The heme of cytochrome c was either less oriented in general or was oriented at an angle closer to the plane of the mitochondrial membrane than were the hemes of the “tightly bound” mitochondrial cytochromes. EPR spectra of the azide, sulfide and formate complexes of cytochrome c oxidase in mitochondria in situ obtained as a function of the orientation of the applied magnetic field relative to the planes of the membrane multilayers showed that both hemes of the oxidase were oriented in such a way that the angle between the heme normal and the membrane normal was approx. 90°.     

Proteomic alterations of distinct mitochondrial subpopulations in the type 1 diabetic heart: contribution of protein import dysfunction

Diabetic cardiomyopathy is associated with increased risk of heart failure in type 1 diabetic patients. Mitochondrial dysfunction is suggested as an underlying contributor to diabetic cardiomyopathy. Cardiac mitochondria are characterized by subcellular spatial locale, including mitochondria located beneath the sarcolemma, subsarcolemmal mitochondria (SSM), and mitochondria situated between the myofibrils, interfibrillar mitochondria (IFM). The goal of this study was to determine whether type 1 diabetic insult in the heart influences proteomic make-up of spatially distinct mitochondrial subpopulations and to evaluate the role of nuclear encoded mitochondrial protein import. Utilizing multiple proteomic approaches (iTRAQ and two-dimensional-differential in-gel electrophoresis), IFM proteomic make-up was impacted by type 1 diabetes mellitus to a greater extent than SSM, as evidenced by decreased abundance of fatty acid oxidation and electron transport chain proteins. Mitochondrial phosphate carrier and adenine nucleotide translocator, as well as inner membrane translocases, were decreased in the diabetic IFM (P < 0.05 for both). Mitofilin, a protein involved in cristae morphology, was diminished in the diabetic IFM (P < 0.05). Posttranslational modifications, including oxidations and deamidations, were most prevalent in the diabetic IFM. Mitochondrial heat shock protein 70 (mtHsp70) was significantly decreased in diabetic IFM (P < 0.05). Mitochondrial protein import was decreased in the diabetic IFM with no change in the diabetic SSM (P < 0.05). Taken together, these results indicate that mitochondrial proteomic alterations in the type 1 diabetic heart are more pronounced in the IFM. Further, proteomic alterations are associated with nuclear encoded mitochondrial protein import dysfunction and loss of an essential mitochondrial protein import constituent, mtHsp70, implicating this process in the pathogenesis of the diabetic heart.     

Enhanced formation of methane in plant cell cultures by inhibition of cytochrome c oxidase

The claim of methane (CH₄) formation in plants has caused much controversy and debate within the scientific community over the past 4 years. Here, using both stable isotope and concentration measurements, we demonstrate that CH₄ formation occurs in plant cell cultures that were grown in the dark under sterile conditions. Under non‐stress conditions the plant cell cultures produced trace amounts [0.3–0.6 ng g^?1 dry weight (DW) h^?1] of CH₄ but these could be increased by one to two orders of magnitude (up to 12 ng g^?1 DW h^?1) when sodium azide, a compound known to disrupt electron transport flow at the cytochrome c oxidase (complex IV) in plant mitochondria, was added to the cell cultures. The addition of other electron transport chain (ETC) inhibitors did not result in significant CH₄ formation indicating that a site‐specific disturbance of the ETC at complex IV causes CH₄ formation in plant cells. Our study is an important first step in providing more information on non‐microbial CH₄ formation from living plants particularly under abiotic stress conditions that might affect the electron transport flow at the cytochrome c oxidase in plant mitochondria.     

Bioenergetic consequences from xenotopic expression of a tunicate AOX in mouse mitochondria: Switch from RET and ROS to FET

Electron transfer from all respiratory chain dehydrogenases of the electron transport chain (ETC) converges at the level of the quinone (Q) pool. The Q redox state is thus a function of electron input (reduction) and output (oxidation) and closely reflects the mitochondrial respiratory state. Disruption of electron flux at the level of the cytochrome bc₁ complex (cIII) or cytochrome c oxidase (cIV) shifts the Q redox poise to a more reduced state which is generally sensed as respiratory stress. To cope with respiratory stress, many species, but not insects and vertebrates, express alternative oxidase (AOX) which acts as an electron sink for reduced Q and by-passes cIII and cIV. Here, we used Ciona intestinalis AOX xenotopically expressed in mouse mitochondria to study how respiratory states impact the Q poise and how AOX may be used to restore respiration. Particularly interesting is our finding that electron input through succinate dehydrogenase (cII), but not NADH:ubiquinone oxidoreductase (cI), reduces the Q pool almost entirely (>90%) irrespective of the respiratory state. AOX enhances the forward electron transport (FET) from cII thereby decreasing reverse electron transport (RET) and ROS specifically when non-phosphorylating. AOX is not engaged with cI substrates, however, unless a respiratory inhibitor is added. This sheds new light on Q poise signaling, the biological role of cII which enigmatically is the only ETC complex absent from respiratory supercomplexes but yet participates in the tricarboxylic acid (TCA) cycle. Finally, we delineate potential risks and benefits arising from therapeutic AOX transfer.     

Inhibition of membrane-bound cytochrome c oxidase by zinc ions: High-affinity Zn-binding site at the P-side of the membrane

In the presence of the uncoupler, external zinc ions inhibit rapidly turnover of cytochrome c oxidase reconstituted in phospholipid vesicles or bound to the membrane of intact mitochondria. The effect is promoted by electron leaks into the oxidase during preincubation with Zn²⁺. Inhibition of liposome-bound bovine cytochrome oxidase by external Zn²⁺ titrates with a K_i of 1 ± 0.3 μM. Presumably, the Zn²⁺-binding group at the positively charged side is not reactive in the oxidized enzyme, but becomes accessible to the cation in some partially reduced state(s) of the oxidase; reduction of Cu_B is tentatively proposed to be responsible for the effect.