Anion-Specific Effects on the Alkaline State of Cytochrome c.

Specific effects of anions on the structure, thermal stability, and peroxidase activity of native (state III) and alkaline (state IV) cytochrome c (cyt c) have been studied by the UV-VIS absorbance spectroscopy, intrinsic tryptophan fluorescence, and circular dichroism. Thermal and isothermal denaturation monitored by the tryptophan fluorescence and circular dichroism, respectively, implied lower stability of cyt c state IV in comparison with the state III. The pKa value of alkaline isomerization of cyt c depended on the present salts, i.e., kosmotropic anions increased and chaotropic anions decreased pKa (Hofmeister effect on protein stability). The peroxidase activity of cyt c in the state III, measured by oxidation of guaiacol, showed clear dependence on the salt position in the Hofmeister series, while cyt c in the alkaline state lacked the peroxidase activity regardless of the type of anions present in the solution. The alkaline isomerization of cyt c in the presence of 8 M urea, measured by Trp59 fluorescence, implied an existence of a high-affinity non-native ligand for the heme iron even in a partially denatured protein conformation. The conformation of the cyt c alkaline state in 8 M urea was considerably modulated by the specific effect of anions. Based on the Trp59 fluorescence quenching upon titration to alkaline pH in 8 M urea and molecular dynamics simulation, we hypothesize that the Lys79 conformer is most likely the predominant alkaline conformer of cyt c. The high affinity of the sixth ligand for the heme iron is likely a reason of the lack of peroxidase activity of cyt c in the alkaline state.

Peroxidase activity of cytochrome c in its compact state depends on dynamics of the heme region.

Cytochrome c (cyt c) is a small globular hemoprotein with the main function as an electron carrier in mitochondrial respiratory chain. Cyt c possesses also peroxidase-like activity in the native state despite its six-coordinated heme iron. In this work, we studied the effect of increasing urea concentration in the range from 0 M to 6 M at pH 7 (pH value of the bulk solvent) and pH 5 (pH value close to negatively charged membrane) on peroxidase-like activity of cyt c. We show that peroxidase-like activity, measured by guaiacol oxidation and the ferrous oxidation in xylenol orange methods, correlates with the accessibility of the heme iron, which was assessed from the association rate constant of cyanide binding to cyt c. Cyt c peroxidase-like activity linearly increases in the pre-denaturational urea concentrations (0-4 M) at both studied pHs without an apparent formation of penta-coordinated state of the heme iron. Our results suggest that dynamic equilibrium among the denaturant-induced non-native coordination states of cyt c, very likely due to reversible unfolding of the least stable foldons, is pre-requisite for enhanced peroxidase-like activity of cyt c in its compact state. Dynamic replacement of the native sixth coordination bond of methionine-80 by lysines (72, 73, and 79) and partially also by histidines (26 and 33) provides an efficient way how to increase peroxidase-like activity of cyt c without significant conformational change at physiological conditions.

Inner mechanism of protection of mitochondrial electron-transfer proteins against oxidative damage. Focus on hydrogen peroxide decomposition.

It is generally recognized that the mitochondria are the major source of reactive oxygen species including hydrogen peroxide (H2O2). Although the local concentration of H2O2 near the electron-transfer chain is potentially quite high, the chain's components are rarely found to be significantly damaged by H2O2. Our experimental data, as well as the data published by others, suggest that mitochondrial electron-transfer proteins, which are in the first line to be harmed by ROS, are well prepared to defend themselves. One of such protection mechanism involves peroxidase/catalase-like activity of all major mitochondrial respiration chain players, which catalyze the decomposition of H2O2. Understanding the molecular mechanisms, by which mitochondrial electron-transfer proteins might defend themselves against an oxidative stress and therefore being a part of the mitochondrial antioxidant system, can help to clarify many controversial experimental data.

Role of cardiolipin in stability of integral membrane proteins.

Cardiolipin (CL) is a unique phospholipid with a dimeric structure having four acyl chains and two phosphate groups found almost exclusively in certain membranes of bacteria and of mitochondria of eukaryotes. CL interacts with numerous proteins and has been implicated in function and stabilization of several integral membrane proteins (IMPs). While both functional and stabilization roles of CL in IMPs has been generally acknowledged, there are, in fact, only limited number of quantitative analysis that support this function of CL. This is likely caused by relatively complex determination of parameters characterizing stability of IMPs and particularly intricate assessment of role of specific phospholipids such as CL in IMPs stability. This review aims to summarize quantitative findings regarding stabilization role of CL in IMPs reported up to now.

Delipidation of cytochrome c oxidase from Rhodobacter sphaeroides destabilizes its quaternary structure.

Delipidation of detergent-solubilized cytochrome c oxidase isolated from Rhodobacter sphaeroides (Rbs-CcO) has no apparent structural and/or functional effect on the protein, however affects its resistance against thermal or chemical denaturation. Phospholipase A2 (PLA2) hydrolysis of phospholipids that are co-purified with the enzyme removes all but two tightly bound phosphatidylethanolamines. Replacement of the removed phospholipids with nonionic detergent decreases both thermal stability of the enzyme and its resilience against the effect of chemical denaturants such as urea. In contrast to nondelipidated Rbs-CcO, the enzymatic activity of PLA2-treated Rbs-CcO is substantially diminished after exposure to high (>4 M) urea concentration at room temperature without an alteration of its secondary structure. Absorbance spectroscopy and sedimentation velocity experiments revealed a strong correlation between intact tertiary structure of heme regions and quaternary structure, respectively, and the enzymatic activity of the protein. We concluded that phospholipid environment of Rbs-CcO has the protective role for stability of its tertiary and quaternary structures.

The kinetic stability of cytochrome C oxidase: effect of bound phospholipid and dimerization.

Thermally induced transitions of the 13-subunit integral membrane protein bovine cytochrome c oxidase (CcO) have been studied by differential scanning calorimetry (DSC) and circular dichroism (CD). Thermal denaturation of dodecyl maltoside solubilized CcO proceeds in two consecutive, irreversible, kinetically driven steps with the apparent transition temperatures at âˆ¼ 51°C and ∼ 61°C (5µM CcO at scan rate of 1.5 K/min). The thermal denaturation data were analyzed according to the Lyubarev and Kurganov model of two consecutive irreversible steps. However, because of the limitation of the model to describe the complex mechanism of the thermal denaturation of CcO, the obtained results were utilized only for comparison purposes of kinetic stabilities of CcO under specific protein concentration (5µM) and scan rate (1.5 K/min). This enabled us to show that both the amphiphilic environment and the self-association state of CcO affect its kinetic stability. Kinetic stabilities of both steps are significantly decreased when all of the phospholipids are removed from CcO by phospholipase A2 (the half-life decreases at 37°C). Conversely, dimerization of CcO induced by sodium cholate significantly increases its kinetic stability of only the first step (the half-life increases at 37°C). Protein concentration-dependent nonspecific oligomerization also indicate mild stabilization of CcO. Both, reversed-phase high-performance liquid chromatography (HPLC) and SDS-PAGE subunit analysis reveal that the first step of thermal denaturation involves dissociation of subunits III, VIa, VIb, and VIIa, whereas the second step is less well defined and most likely involves global unfold and aggregation of the remaining subunits. Electron transport activity of CcO decreases in a sigmoidal manner during the first transition and this dependence is very well described by kinetic parameters for the first step of the thermal transition. Therefore, dissociation of subunit III and/or VIIa is responsible for temperature-induced inactivation of CcO because VIa and VIb can be removed from CcO without affecting the enzyme activity. These results demonstrate an important role of tightly bound phospholipids and oligomeric state (particularly the dimeric form) of CcO for kinetic stability of the protein.

Bound cardiolipin is essential for cytochrome c oxidase proton translocation.

The proton pumping activity of bovine heart cytochrome c oxidase (CcO) is completely inhibited when all of the cardiolipin (CL) is removed from the enzyme to produce monomeric CcO containing only 11 subunits. Only dimeric enzyme containing all 13 subunits and 2-4 cardiolipin per CcO monomer exhibits a "normal" proton translocating stoichiometry of ∼1.0 H(+) per/e(-) when reconstituted into phospholipid vesicles. These fully active proteoliposomes have high respiratory control ratios (RCR = 7-15) with 75-85% of the CcO oriented with the cytochrome c binding sites exposed to the external medium. In contrast, reconstitution of CL-free CcO results in low respiratory control ratios (RCR < 5) with the enzyme randomly oriented in the vesicles, i.e., ∼50 percent oriented with the cytochrome c binding site exposed on the outside of the vesicle. Addition of exogenous CL to the CL-free enzyme completely restores electron transport activity, but restoration of proton pumping activity does not occur. This is true whether CL is added to CL-free CcO prior to reconstitution into phospholipid vesicles, or whether CL is included in the phospholipid mixture that is used to form the vesicles. Another consequence of CL removal is the inability of the 11-subunit, CL-free enzyme to dimerize upon exposure to either cholate or the cholate/PC/PE/CL mixture used during proteoliposome formation (monomeric, 13-subunit, CL-containing CcO completely dimerizes under these conditions). Therefore, a major difference between reconstitution of CL-free and CL-containing CcO is the incorporation of monomeric, rather than dimeric CcO into the vesicles. We conclude that bound CL is necessary for proper insertion of CcO into phospholipid vesicles and normal proton translocation.

Dual effect of heparin on Fe²âº-induced cardiolipin peroxidation: implications for peroxidation of cytochrome c oxidase bound cardiolipin.

The effect of heparin on peroxidation of cardiolipin (CL) initiated by ferrous iron was studied in vitro using detergent-solubilized CL, liposomal CL, or CL bound to isolated cytochrome c oxidase (CcO). Heparin increased both the rate and the extent of CL peroxidation for detergent-solubilized CL and for CcO-bound CL. The effect of heparin was time- and concentration-dependent as monitored by the formation of conjugated dienes or thiobarbituric acid reactive substances. The results showed great similarity between the effect of heparin and the effect of certain iron chelators, such as ADP, on phospholipid peroxidation. Heparin increased the peroxidation of CcO-bound CL only when tertiary butyl hydroperoxide was also present. The enzyme activity of the resulting CcO complex decreased 25 %, in part due to peroxidation of functionally important CL. In contrast to peroxidation of detergent-solubilized CL, peroxidation of liposomal CL was inhibited by heparin, suggesting that the effect of heparin and ferrous iron depends on their proximity to the acyl chains of CL.

Elucidating the mechanism of ferrocytochrome c heme disruption by peroxidized cardiolipin.

The interaction of peroxidized cardiolipin with ferrocytochrome c induces two kinetically and chemically distinct processes. The first is a rapid oxidation of ferrocytochrome c, followed by a slower, irreversible disruption of heme c. The oxidation of ferrocytochrome c by peroxidized cardiolipin is explained by a Fenton-type reaction. Heme scission is a consequence of the radical-mediated reactions initiated by the interaction of ferric heme iron with peroxidized cardiolipin. Simultaneously with the heme c disruption, generation of hydroxyl radical is detected by EPR spectroscopy using the spin trapping technique. The resulting apocytochrome c sediments as a heterogeneous mixture of high aggregates, as judged by sedimentation analysis. Both the oxidative process and the destructive process were suppressed by nonionic detergents and/or high ionic strength. The mechanism for generating radicals and heme rupture is presented.

Susceptibility of mitochondrial electron-transport complexes to oxidative damage. Focus on cytochrome c oxidase.

Reactive oxygen species (ROS) are associated with a number of mitochondrial disorders. These include: ischemia/reperfusion injury, Parkinson's disease, Alzheimer's disease, neurodegenerative diseases, and other age-related degenerative changes. ROS can be generated at numerous sites within the cell, but the mitochondrial electron transport chain is recognized as the major source of intracellular ROS. Two mitochondrial electron-transfer complexes are major sources of ROS: complex I and complex III. Oxidative damage to either of these complexes, or to electron transport complexes that are in close proximity to these ROS sources, e.g., cytochrome c oxidase, would be expected to inhibit electron transport. Such inhibition would lead to increased electron leakage and more ROS production, much like the well-known effect of adding electron transport inhibitors. Recent studies reveal that ROS and lipid peroxidation products are effective inhibitors of the electron-transport complexes. In some cases, inactivation of enzymes correlates with chemical modification of only a small number of unusually reactive amino acids. In this article, we review current knowledge of ROS-induced alterations within three complexes: (1) complex IV; (2) complex III; and (3) complex I. Our goal is to identify "hot spots" within each complex that are easily chemically modified and could be responsible for ROS-induced inhibition of the individual complexes. Special attention has been placed on ROS-induced damage to cardiolipin that is tightly bound to each of the inner membrane protein complexes. Peroxidation of the bound cardiolipin is thought to be particularly important since its close proximity and long residence time on the protein make it an especially effective reagent for subsequent ROS-induced damage to these proteins.

Ferricytochrome c protects mitochondrial cytochrome c oxidase against hydrogen peroxide-induced oxidative damage.

An excess of ferricytochrome c protects purified mitochondrial cytochrome c oxidase and bound cardiolipin from hydrogen peroxide-induced oxidative modification. All of the peroxide-induced changes within cytochrome c oxidase, such as oxidation of Trp(19,IV) and Trp(48,VIIc), partial dissociation of subunits VIa and VIIa, and generation of cardiolipin hydroperoxide, no longer take place in the presence of ferricytochrome c. Furthermore, ferricytochrome c suppresses the yield of H(2)O(2)-induced free radical detectable by electron paramagnetic resonance spectroscopy within cytochrome c oxidase. These protective effects are based on two mechanisms. The first involves the peroxidase/catalase-like activity of ferricytochrome c, which results in the decomposition of H(2)O(2), with the apparent bimolecular rate constant of 5.1±1.0M(-1)s(-1). Although this value is lower than the rate constant of a specialized peroxidase, the activity is sufficient to eliminate H(2)O(2)-induced damage to cytochrome c oxidase in the presence of an excess of ferricytochrome c. The second mechanism involves ferricytochrome c-induced quenching of free radicals generated within cytochrome c oxidase. These results suggest that ferricytochrome c may have an important role in protection of cytochrome c oxidase and consequently the mitochondrion against oxidative damage.

Removal of bound Triton X-100 from purified bovine heart cytochrome bc1.

Cytochrome bc(1) isolated from Triton X-100-solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nanomole of the enzyme. Purified cytochrome bc(1) is fully active; however, protein-bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc(1) was accomplished by incubation with Bio-Beads SM-2 in the presence of sodium cholate. Sodium cholate is critical because it does not interfere with the adsorption of protein on the hydrophobic surface of the beads. The resulting Triton X-100-free cytochrome bc(1) retained nearly full activity, absorption spectra, subunit, and phospholipid composition.

Glutathione peroxidase 4 differentially regulates the release of apoptogenic proteins from mitochondria.

Glutathione peroxidase 4 (Gpx4) is a unique antioxidant enzyme that repairs oxidative damage to biomembranes. In this study, we examined the effects of Gpx4 on the release of various apoptogenic proteins from mitochondria using transgenic mice overexpressing Gpx4 [Tg(GPX4(+/0))] and mice deficient in Gpx4 (Gpx4+/- mice). Diquat exposure triggered apoptosis that occurred through an intrinsic pathway and resulted in the mitochondrial release of cytochrome c (Cyt c), Smac/DIABLO, and Omi/HtrA2 in the liver of wild-type (Wt) mice. Liver apoptosis and Cyt c release were suppressed in Tg(GPX4(+/0)) mice but exacerbated in Gpx4+/- mice; however, neither the Tg(GPX4(+/0)) nor the Gpx4+/- mice showed any alterations in the levels of Smac/DIABLO or Omi/HtrA2 released from mitochondria. Submitochondrial fractionation data showed that Smac/DIABLO and Omi/HtrA2 existed primarily in the intermembrane space and matrix, whereas Cyt c and Gpx4 were both associated with the inner membrane. In addition, diquat exposure induced cardiolipin peroxidation in the liver of Wt mice; the levels of cardiolipin peroxidation were reduced in Tg(GPX4(+/0)) mice but elevated in Gpx4+/- mice. These data suggest that Gpx4 differentially regulates apoptogenic protein release owing to its inner membrane location in mitochondria and its ability to repair cardiolipin peroxidation.

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 microg protein) or preparative (>250 microg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.

Multidomain initiation factor 2 from Thermus thermophilus consists of the individual autonomous domains.

The three-dimensional chalice-like crystal structure of initiation factor 2 IF2/eIF5B from Methanobacterium thermoautotrophicum represents a novel fold and domain architecture in which the N-terminal G domain and the C-terminal C domain are separated by an approximately 40 A alpha-helix. Homologous Thermus thermophilus initiation factor 2 (IF2wt), G (IF2G), and C (IF2C) domains were successfully overexpressed and purified which enabled us to perform a thermodynamic analysis and to asses the role of the domain architecture in this atypical fold. Circular dichroism in the far-UV region demonstrated that the proteins are well-folded and that the secondary structure content resembles that of IF2 from M. thermoautotrophicum. IF2wt and IF2G are monomeric proteins, while IF2C has a tendency to form dimeric species as shown by sedimentation velocity studies on analytical ultracentrifugation and differential scanning calorimetry scan analysis. Thermal denaturation studies of multidomain IF2wt reveals an exceptionally high reversibility (>90%) of the transition with a melting temperature of 94.5 degrees C. Melting temperature of IF2wt may be further increased in the presence of its physiological ligand GDP and the GTP analogue, GppNHp. The high reversibility of denaturation is achieved by the modular structure of the protein and by the high reversibility of the thermal denaturation of IF2G. On the other hand, hydrophobic IF2C aggregates during the thermal transition, and the aggregation is suppressed by guanidine hydrochloride. Isothermal denaturation demonstrates that both IF2G and IF2C have comparable stabilities of 46 and 33 kJ/mol, respectively. The apparent cooperative unfolding of the full-length protein has an unusually small denaturant m value. This together with the phase diagram method of analysis indicates the presence of intermediate(s) due to the independent unfolding of IF2G and IF2C. Despite an absence of apparent interactions between the domains in vitro, IF2G plays a role in IF2C reversibility in thermal denaturation. In conclusion, interactions between the domains of folded IF2wt in vivo are likely mediated by their alpha-helix connection and/or by a conformational change on the ribosome.

Differential stability of dimeric and monomeric cytochrome c oxidase exposed to elevated hydrostatic pressure.

Detergent-solubilized dimeric and monomeric cytochrome c oxidase (CcO) have significantly different quaternary stability when exposed to 2-3 kbar of hydrostatic pressure. Dimeric, dodecyl maltoside-solubilized cytochrome c oxidase is very resistant to elevated hydrostatic pressure with almost no perturbation of its quaternary structure or functional activity after release of pressure. In contrast to the stability of dimeric CcO, 3 kbar of hydrostatic pressure triggers multiple structural and functional alterations within monomeric cytochrome c oxidase. The perturbations are either irreversible or slowly reversible since they persist after the release of high pressure. Therefore, standard biochemical analytical procedures could be used to quantify the pressure-induced changes after the release of hydrostatic pressure. The electron transport activity of monomeric cytochrome c oxidase decreases by as much as 60% after exposure to 3 kbar of hydrostatic pressure. The irreversible loss of activity occurs in a time- and pressure-dependent manner. Coincident with the activity loss is a sequential dissociation of four subunits as detected by sedimentation velocity, high-performance ion-exchange chromatography, and reversed-phase and SDS-PAGE subunit analysis. Subunits VIa and VIb are the first to dissociate followed by subunits III and VIIa. Removal of subunits VIa and VIb prior to pressurization makes the resulting 11-subunit form of CcO even more sensitive to elevated hydrostatic pressure than monomeric CcO containing all 13 subunits. However, dimeric CcO, in which the association of VIa and VIb is stabilized, is not susceptible to pressure-induced inactivation. We conclude that dissociation of subunit III and/or VIIa must be responsible for pressure-induced inactivation of CcO since VIa and VIb can be removed from monomeric CcO without significant activity loss. These results are the first to clearly demonstrate an important structural role for the dimeric form of cytochrome c oxidase, i.e., stabilization of its quaternary structure.

Tryptophan 334 oxidation in bovine cytochrome c oxidase subunit I involves free radical migration.

A single tryptophan (W(334(I))) within the mitochondrial-encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W(334(I)) is converted to hydroxytryptophan as identified by reversed-phase HPLC-electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS-PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W(334(I)) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear-encoded subunits, W(48(IV)) and W(19(VIIc)), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003-1009). Two aromatic-rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface-exposed tryptophans, where they produce hydroxytryptophan.

Contribution of peroxidized cardiolipin to inactivation of bovine heart cytochrome c oxidase.

The lipid-soluble peroxides, tert-butyl hydroperoxide and peroxidized cardiolipin, each react with bovine cytochrome c oxidase and cause a loss of electron-transport activity. Coinciding with loss of activity is oxidation of Trp19 and Trp48 within subunits VIIc and IV, and partial dissociation of subunits VIa and VIIa. tert-Butyl hydroperoxide initiates these structural and functional changes of cytochrome c oxidase by three mechanisms: (1) radical generation at the binuclear center; (2) direct oxidation of Trp19 and Trp48; and (3) peroxidation of bound cardiolipin. All three mechanisms contribute to inactivation since blocking a single mechanism only partially prevents oxidative damage. The first mechanism is similar to that described for hydrogen peroxide [Biochemistry43:1003-1009; 2004], while the second and third mechanism are unique to organic hydroperoxides. Peroxidized cardiolipin inactivates cytochrome c oxidase in the absence of tert-butyl hydroperoxide and oxidizes the same tryptophans within the nuclear-encoded subunits. Peroxidized cardiolipin also inactivates cardiolipin-free cytochrome c oxidase rather than restoring full activity. Cardiolipin-free cytochrome c oxidase, although it does not contain cardiolipin, is still inactivated by tert-butyl hydroperoxide, indicating that the other oxidation products contribute to the inactivation of cytochrome c oxidase. We conclude that both peroxidized cardiolipin and tert-butyl hydroperoxide react with and triggers a cascade of structural alterations within cytochrome c oxidase. The summation of these events leads to cytochrome c oxidase inactivation.

Influence of reduction of heme a and Cu(A) on the oxidized catalytic center of cytochrome c oxidase: insight from organic solvents.

Purified bovine heart cytochrome c oxidase (CcO) has been extracted from aqueous solution into hexane in the presence of phospholipids and calcium ions. In extracts, CcO is in the so-called "slow" form and probably situated in reverse micelles. At low water:phospholipid molar ratios, electron transfer from reduced heme a and Cu(A) to the catalytic center is inhibited and both heme a3 and Cu(B) remain in the oxidized state. The rate of binding of cyanide to heme a3 in this oxidized catalytic center is, however, dependent on the redox state of heme a and Cu(A). When heme a and Cu(A) are reduced, the rate is increased 20-fold compared to the rate when these two centers are oxidized. The enhanced rate of binding of cyanide to heme a3 is explained by the destabilization of an intrinsic ligand, located at the catalytic site, that is triggered by the reduction of heme a and Cu(A).