Active site intermediates in the reduction of O(2) by cytochrome oxidase, and their derivatives

The mechanism of dioxygen activation and reduction in cell respiration, as catalysed by cytochrome c oxidase, has a long history. The work by Otto Warburg, David Keilin and Britton Chance defined the dioxygen-binding heme iron centre, viz. das Atmungsferment, or cytochrome a(3). Chance brought the field further in the mid-1970's by ingenious low-temperature studies that for the first time identified the primary enzyme-substrate (ES) Michaelis complex of cell respiration, the dioxygen adduct of heme a(3), which he termed Compound A. Further work using optical, resonance Raman, EPR, and other sophisticated spectroscopic techniques, some of which with microsecond time resolution, has brought us to the situation today, where major principles of how O(2) reduction occurs in respiration are well understood. Nonetheless, some questions have remained open, for example concerning the precise structures, catalytic roles, and spectroscopic properties of the breakdown products of Compound A that have been called P, F (for peroxy and ferryl), and O (oxidised). This nomenclature has been known to be inadequate for some time already, and an alternative will be suggested here. In addition, the multiple forms of P, F and O states have been confusing, a situation that we endeavour to help clarifying. The P and F states formed artificially by reacting cytochrome oxidase with hydrogen peroxide are especially scrutinised, and some novel interpretations will be given that may account for previously unexplained observations.     

Spectroscopic identification of the catalytic intermediates of cytochrome c oxidase in respiring heart mitochondria

The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a₃-Cu_B binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respiration is commonly assigned to the fully reduced species of hemes a or a₃ (R). However, by analyzing the absorbance of isolated enzyme and mitochondria in the Soret (420–450 nm), alpha (560–630 nm) and red (630–700 nm) spectral regions, we demonstrate that the Peroxy (P) and Ferryl (F) intermediates of the binuclear center are observed during respiration, while the R form is only detectable under nearly anoxic conditions in which electrons also accumulate in the higher extinction coefficient low spin a heme. This implies that a large fraction of COX (>50 %) is active, in contrast with assumptions that assign spectral changes only to R and/or reduced heme a. The concentration dependence of the COX chromophores and reduced c-type cytochromes on the transmembrane potential (ΔΨ_m) was determined in isolated mitochondria during substrate or apyrase titration to hydrolyze ATP. The cytochrome c-type redox levels indicated that soluble cytochrome c is out of equilibrium with respect to both Complex III and COX. Thermodynamic analyses confirmed that reactions involving the chromophores we assign as the P and F species of COX are ΔΨ_m-dependent, out of equilibrium, and therefore much slower than the ΔΨ_m-insensitive oxidation of the R intermediate, which is undetectable due to rapid oxygen binding.     

The Protein Effect in the Structure of Two Ferryl-Oxo Intermediates at the Same Oxidation Level in the Heme Copper Binuclear Center of Cytochrome c Oxidase

Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O₂ activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O₂ with the mixed valence form (Cu_A²⁺, heme a³⁺, heme a₃²⁺-Cu_B¹⁺) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm⁻¹ at the peroxy oxidation level (P_M). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the P_M intermediate is also formed in the reaction of Y167F with O₂. These results suggest that in the fully reduced enzyme, the proton pumping ν_Fe(IV)=O = 804 cm⁻¹ to ν_Fe(IV)=O = 790 cm⁻¹ transition (P→F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a₃ but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a₃. The implications of these results with respect to the role of an O=Fe(IV)-His-411-H-bonded form to the ring A propionate of heme a₃-Asp-399-H₂O site and, thus, to the exit/output proton channel (H₂O) pool during the proton pumping P→F transition are discussed. We propose that the environment proximal to the heme a₃ controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.     

Biochemical and biophysical studies on cytochrome c oxidase XVI. Circular dichroic study of cytochrome c oxidase and its ligand complexes

1. CD spectra of cytochrome c oxidase have been determined both in the absence and presence of the extrinsic ligands CO, NO, cyanide and azide.2. CO and NO affect the CD spectrum of cytochrome c oxidase in a similar way.3. Cyanide and azide also affect the CD spectrum of cytochrome c oxidase in a similar way, but distinctly different from CO and NO.4. From the CD spectra of the oxidized and reduced enzyme, in the presence and absence of extrinsic ligands, CD difference spectra (reduced minus oxidized) are calculated for the so-called cytochrome a and cytochrome a₃ moieties of the enzyme.5. These spectra are largely dependent on the extrinsic ligand used. It is therefore concluded that these spectra do not represent independent cytochrome a and cytochrome a₃ difference spectra, but that heme-heme interactions occur within the cytochrome c oxidase molecule, in such a way that binding of a ligand to one of the heme a groups of cytochrome c oxidase affects the spectral properties of the other heme a group.6. As a consequence, ligand-binding studies cannot give information as to the pre-existence of separate cytochrome a and cytochrome a₃ moieties in the absence of extrinsic ligands.     

Biochemical and biophysical studies on cytochrome c oxidase. XVIII. Potentiometric titrations of cytochrome c oxidase followed by circular dichroism

1. Potentiometric circular dichroism titrations of cytochrome c oxidase, carried out in the absence of cytochrome c, confirm the potentiometric equivalence of the two heme a groups of cytochrome c oxidase. In the presence of cytochrome c, two different midpoint potentials are found for the two heme a groups of cytochrome c oxidase.2. Circular dichroism difference spectra (reduced minus oxidized) of the two heme a components of cytochrome c oxidase have been obtained by means of this potentiometric titration. On reduction of the first heme a group a circular dichroism difference spectrum is obtained with peaks at 425, 442 and 602.5 nm; the second heme a group shows difference peaks at 434, 447 and 608 nm. Whereas both heme a groups contribute about equally to the absorbance difference spectrum, the second heme a group reduced contributes about twice as much to the circular dichroism difference spectrum as does the first heme a group.3. From these spectral and circular dichroism differences it is concluded that, on reduction of or ligand binding to cytochrome c oxidase, conformational changes occur which affect the symmetry of the environments of the heme a groups.     

Kinetic studies on cytochrome c oxidase by combined EPR and reflectance spectroscopy after rapid freezing

1. Techniques and experiments are described concerned with the millisecond kinetics of EPR-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O₂ or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy.2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (< 50 ms) approx. 0.5 electron equivalent per hame a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (> 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a.3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O₂ in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O₂ side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates.4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms, whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO.5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C. R., Hansen, R. E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477–2481). Both the low-spin (g = 3; 2.2; 1.5) and slowly appearing high-spin (g = 6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a₃, when it is present in a state of interaction with EPR-undetectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

Some properties of the redox components of cytochrome c oxidase and their interactions.

The electron paramagnetic resonance (epr) properties of cytochrome c oxidase have been examined with special attention to the effect of added ligands and of interactions between the redox components. The fully oxidized preparations have a very small g6 signal which increases greatly as the redox potential is made more negative, a process exactly paralleling the disappearance of the g3 signal. The potential for half appearance or disappearance (E_m), respectively, is 380 mV at pH 7.0 and 300 mV at pH 8.5. This identifies the changes as accompanying reduction of cytochrome a₃ because the E_m of the “invisible copper” is 340 mV and pH independent. Nitric oxide (NO) binds reduced cytochrome a₃ to form a paramagnetic species. This resulting epr signal is strongly dependent on the redox state of cytochrome a, another expression of heme-heme interaction in cytochrome oxidase. The NO compound is also unique in that under the appropriate conditions three of the four redox components (cytochrome a₃, cytochrome a, and the “visible” copper) are epr active. In potentiometric titrations in the presence of azide the formation of the azide compound responsible for the g2.9 signal appears to require reduction of both cytochrome a₃ and the “invisible copper.” An internal sulfur compound is present which, at alkaline pH values, can bind the heme responsible for the g6 signal and change it to a low-spin sulfur compound with a signal at approximately g2.6. Evidence is also presented for the cytochrome c oxidase in situ being an equilibrium mixture of two different conformational states.     

Effect of calcium ions on electron transfer between hemes <Emphasis Type="Italic">a</Emphasis> and <Emphasis Type="Italic">a</Emphasis><Subscript>3</Subscript> in cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

Kinetics of the reduction of the hemes in cytochrome c oxidase in the presence of high concentration of ruthenium(III)hexaammine chloride was examined using a stopped-flow spectrophotometer. Upon mixing of the oxidized enzyme with dithionite and Ru(NH₃) ₆ ³⁺ , three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a ₃ reduction. The difference spectrum of heme a ₃ reduction in the visible region is characterized by a maximum at ～612 nm, rather than at 603 nm as was believed earlier. It is shown that in the case of bovine heart cytochrome c oxidase containing a special cation-binding site in which reversible binding of calcium ion occurs, heme a ₃ reduction is slowed down by low concentrations of Ca²⁺. The effect is absent in the case of the bacterial cytochrome oxidase in which the cation-binding site contains a tightly bound Ca²⁺ ion. The data corroborate the inhibition of the cytochrome oxidase enzymatic activity by Ca²⁺ ions discovered earlier and indicate that the cation affects intramolecular electron transfer.     

Single-electron photoreduction of the PM intermediate of cytochrome c oxidase

The P_M → F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P_M state of the oxidase was generated by exposing the oxidized enzyme to CO plus O₂. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a₃ with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is ∼ 5-fold more rapid than the rate of electron transfer from heme a to heme a₃ in the F → O transition, but is significantly slower than formation of the F state from the P_R intermediate in the reaction of the fully reduced enzyme with O₂ to form state F (70–90 μs). The ∼ 0.3 ms P_M → F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P_M → F transition is generated much later, with a time constant of 1.3 ms. In addition, the P_M → F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with τ values of 0.18 and 0.85 ms.     

Blocking the K-pathway still allows rapid one-electron reduction of the binuclear center during the anaerobic reduction of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides

The K-pathway is one of the two proton-input channels required for function of cytochrome c oxidase. In the Rhodobacter sphaeroides cytochrome c oxidase, the K-channel starts at Glu101 in subunit II, which is at the surface of the protein exposed to the cytoplasm, and runs to Tyr288 at the heme a₃/Cu_B active site. Mutations of conserved, polar residues within the K-channel block or inhibit steady state oxidase activity. A large body of research has demonstrated that the K-channel is required to fully reduce the heme/Cu binuclear center, prior to the reaction with O₂, presumably by providing protons to stabilize the reduced metals (ferrous heme a₃ and cuprous Cu_B). However, there are conflicting reports which raise questions about whether blocking the K-channel blocks both electrons or only one electron from reaching the heme/Cu center. In the current work, the rate and extent of the anaerobic reduction of the heme/Cu center were monitored by optical and EPR spectroscopies, comparing the wild type and mutants that block the K-channel. The new data show that when the K-channel is blocked, one electron will still readily enter the binuclear center. The one-electron reduction of the resting oxidized (“O”) heme/Cu center of the K362M mutant, results in a partially reduced binuclear center in which the electron is distributed about evenly between heme a₃ and Cu_B in the R. sphaeroides oxidase. Complete reduction of the heme/Cu center requires the uptake of two protons which must be delivered through the K-channel.     

On the identity of the high spin heme components of cytochrome c oxidase

In oxidized, resting cytochrome c oxidase (EC 1.9.3.1) and under most conditions of partial reduction ? 50% of the heme components are detected by EPR spectroscopy. When the enzyme is fully reduced in the presence of equimolar quantities of cytochrome c, anaerobic reoxidation by an excess of a chemical oxidant (ferricyanide, porphyrexide) produces intense high and low spin heme signals simultaneously. The time range in which maximal high spin signals are observed is 0.1–2 s after mixing. Under these conditions 35–50% of the total heme a is accounted for by the low spin heme signal and 35–40% by the high spin signals, with the rhombic component accounting for 30–35% of the total heme. It is concluded that under these conditions, the major portion of both heme components must be EPR detectable. Thus, if the generally accepted assignment of the low spin signal to cytochrome a is adopted, it follows that in the experiments described, cytochrome a₃ is represented in the rhombic high spin signal. The quantities of heme represented in the axial high spin signal are too small for a definitive assignment; these signals could originate from either heme. When after formation of high spin signals as described, O₂ is admitted, the rhombic signal is eliminated within 4 ms. In the presence of the strongest rhombic high spin signals, the absorption band at 655 nm is only ? 25% developed. The implications of these findings are discussed in the context of present hypotheses concerning the state and interactions of cytochrome c oxidase components during oxidation-reduction.     

The oxygen reactions of reduced cytochrome c oxidase Position of a form with an unusual EPR signal in the sequence of early intermediates

The order of appearance of intermediates in the reoxidation of reduced cytochrome c oxidase by oxygen has been examined. Particular emphasis was placed on determining where the intermediate with the EPR signal at g = 5, 1.78, 1.69 (Shaw, R.W., Hansen, R.E. and Beinert, H. (1978) J. Biol. Chem. 253, 6637–6640) appears in the sequence of events during reoxidation. Flash photolysis of reduced, CO-complexed samples of cytochrome c oxidase in the presence of oxygen in a buffer containing 30% (v/v) ethylene glycol at 77 K and 195 K has been used to generate states of partial reoxidation. The intermediate with the EPR signal at g = 5, 1.78, and 1.69 can be detected as a product of the photolysis and subsequent oxidation but does not appear until the photolyzed sample is incubated at temperatures well above 195 K. In the course of the reoxidation, the intermediate characterized by the g = 5, 1.78, 1.69 signal occurs in the reaction sequence after the states referred to as ‘Compound A’ and ‘Compound B’ (Chance, B., Saronio, C., and Leigh, J.S. (1975) J. Biol. Chem. 250, 9226–9237). Its apperance is within the time range reported for the formation of ‘oxygenated’ cytochrome c oxidase (Orii, Y. (1979) in Cytochrome Oxidase (King. T.E., Orii, Y., Chance, B. and Okunuki, K., eds.), pp. 331–340, Elsevier/North-Holland Biomedical Press, Amsterdam).     

The influence of pH on the EPR and redox properties of cytochrome c oxidase in detergent solution and in phospholipid vesicles

The EPR signals of oxidized and partially reduced cytochrome oxidase have been studied at pH 6.4, 7.4, and 8.4. Isolated cytochrome oxidase in both non-ionic detergent solution and in phospholipid vesicles has been used in reductive titrations with ferrocytochrome c.The g values of the low- and high-field parts of the low-spin heme signal in oxidized cytochrome oxidase are shown to be pH dependent. In reductive titrations, low-spin heme signals at g 2.6 as well as rhombic and nearly axial high-spin heme signals are found at pH 8.4, while the only heme signals appearing at pH 6.4 are two nearly axial g 6 signals. This pH dependence is shifted in the vesicles.The g 2.6 signals formed in titrations with ferrocytochrome c at pH 8.4 correspond maximally to 0.25–0.35 heme per functional unit (aa₃) of cytochrome oxidase in detergent solution and to 0.22 heme in vesicle oxidase. The total amount of high-spin heme signals at g 6 found in partially reduced enzyme is 0.45–0.6 at pH 6.4 and 0.1–0.2 at pH 8.4. In titrations of cytochrome oxidase in detergent solution the g 1.45 and g 2 signals disappear with fewer equivalents of ferrocytochrome c added at pH 8.4 compared to pH 6.4.The results indicate that the environment of the hemes varies with the pH. One change is interpreted as cytochrome a₃ being converted from a high-spin to a low-spin form when the pH is increased. Possibly this transition is related to a change of a liganded H₂O to OH^? with a concomitant decrease of the redox potential. Oxidase in phosphatidylcholine vesicles is found to behave as if it experiences a pH, one unit lower than that of the medium.     

Concerted involvement of cooperative proton–electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu_A and heme a₃/Cu_B are cooperatively linked to proton transfer at acid/base groups in the enzyme. H⁺/e⁻ cooperative linkage at Fe_a3/Cu_B is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H⁺/e⁻ linkage at heme a (and Cu_A). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H₂O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu_A and heme a₃/Cu_B in the soluble oxidase; (iii) H⁺ release in the external phase (i.e. H⁺ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H⁺/e⁻ linkage at heme a/Cu_A and heme a₃/Cu_B with acid/base clusters, C₁ and C₂ respectively, and protonmotive steps of the reduction of O₂ to water are involved in proton pumping.     

Reconstruction of absolute absorption spectrum of reduced heme a in cytochrome c oxidase from bovine heart

This paper presents a new experimental approach for determining the individual optical characteristics of reduced heme a in bovine heart cytochrome c oxidase starting from a small selective shift of the heme a absorption spectrum induced by calcium ions. The difference spectrum induced by Ca²⁺ corresponds actually to a first derivative (differential) of the heme a ²⁺ absolute absorption spectrum. Such an absolute spectrum was obtained for the mixed-valence cyanide complex of cytochrome oxidase (a ²⁺ a ₃ ³⁺ -CN) and was subsequently used as a basis spectrum for further procession and modeling. The individual absorption spectrum of the reduced heme a in the Soret region was reconstructed as the integral of the difference spectrum induced by addition of Ca²⁺. The spectrum of heme a ²⁺ in the Soret region obtained in this way is characterized by a peak with a maximum at 447 nm and half-width of 17 nm and can be decomposed into two Gaussians with maxima at 442 and 451 nm and half-widths of ～10 nm (589 cm^?1) corresponding to the perpendicularly oriented electronic π→π* transitions B _0x and B _0y in the porphyrin ring. The reconstructed spectrum in the Soret band differs significantly from the “classical” absorption spectrum of heme a ²⁺ originally described by Vanneste (Vanneste, W. H. (1966) Biochemistry, 65, 838–848). The differences indicate that the overall γ-band of heme a ²⁺ in cytochrome oxidase contains in addition to the B _0x and B _0y transitions extra components that are not sensitive to calcium ions, or, alternatively, that the Vanneste’s spectrum of heme a ²⁺ contains significant contribution from heme a ₃ ²⁺ . The reconstructed absorption band of heme a ²⁺ in the α-band with maximum at 605 nm and half-width of 18 nm (850 cm^?1) corresponds most likely to the individual Q _0y transition of heme a, whereas the Q _0x transition contributes only weakly to the spectrum.     

Enhanced cytochrome oxidase activity and modification of lipids in heart mitochondria from hyperthyroid rats

In order to further investigate the mechanism regulating the control of mitochondrial respiration by thyroid hormones, the effect of the hyperthyroidism on the kinetic characteristics of cytocrome c oxidase in rat heart mitochondria was studied. Mitochondrial preparations from both control and hyperthyroid rats had equivalent K_m values for cytochrome c, while the maximal activity of cytochrome oxidase was significantly increased (by around 30%) in mitochondrial rats. This enhanced activity of cytochrome oxidase was associated to a parallel increases in mitochondrial State 3 respiration. The hormone treatment resulted in a decrease in the flux control coefficient of the oxidase. The enhanced activity of cytochrome oxidase in hyperthyroid rats does not appear to be dependent on an increases in the mass of this enzyme complex in that the heme aa₃ content was equivalent in both hyperthyroid and control preparations. The Arrhenius plot characteristics differ for cytochrome oxidase activity in mitochondria from hyperthyroid rats as compared with control rats in the breakpoint of the biphasic plot is shifted to a lower temperature. Cardiolipin content was significantly increased in mitochondrial preparations from hyperthyroid rats, while there were no significant alterations in the fatty acid composition of cardiolipin of control and hyperthyroid preparations. The results support the conclusion that the enhanced cytochrome oxidase activity in heart mitochondrial preparations from hyperthyroid rats is due to a specific increase in the content of cardiolipin.     

Studies on the orientations of the mitochondrial redox carriers I. Orientation of the hemes of cytochrome c oxidase with respect to the plane of a cytochrome oxidase-lipid model membrane

The liganded derivatives of mitochondrial cytochrome c oxidase have been prepared in hydrated oriented multilayers of membranous cytochrome c oxidase. The optical spectra of the liganded derivatives recorded at an angle of 45° between the incident light beam and the normal to the planes of the membranes in the multilayers show dichroic ratios of almost 2 in the visible region and 1.2–1.4 in the Soret region. The dichroic ratios were found to be similar for both cytochromes a and a₃. Electron paramagnetic resonance spectra of the azide, sulfide, and formate complexes of cytochrome c oxidase obtained as a function of the orientation of the applied magnetic field relative to the planes of the membranes in the multilayer confirm the optical data and demonstrate that both hemes of cytochrome c oxidase are oriented such that the angle between the heme normal and the membrane normal is approximately 90°.     

Nitric oxide and peroxynitrite cause irreversible increases in the Km for oxygen of mitochondrial cytochrome oxidase: in vitro and in vivo studies

Mitochondrial cytochrome oxidase is competitively and reversibly inhibited by inhibitors that bind to ferrous heme, such as carbon monoxide and nitric oxide. In the case of nitric oxide, nanomolar levels inhibit cytochrome oxidase by competing with oxygen at the enzyme's heme-copper active site. This raises the K_m for cellular respiration into the physiological range. This effect is readily reversible and may be a physiological control mechanism. Here we show that a number of in vitro and in vivo conditions result in an irreversible increase in the oxygen K_m. These include: treatment of the purified enzyme with peroxynitrite or high (μM) levels of nitric oxide; treatment of the endothelial-derived cell line, b.End5, with NO; activation of astrocytes by cytokines; reperfusion injury in the gerbil brain. Studies of cell respiration that fail to vary the oxygen concentration systematically are therefore likely to significantly underestimate the degree of irreversible damage to cytochrome oxidase.     

Studies of the orientation of the mitochondrial redox carriers III. Orientation of the gx and gy axes of the hemes of cytochrome oxidase with respect to the plane of the membrane in oriented membrane multilayers

The EPR absorption properties of the hemes of cytochrome oxidase and their liganded derivatives were examined in oriented multilayers from isolated oxidase, mitochondrial membranes and membrane fragments of a bacterium, Paracoccus denitrificans. The hemes of the oxidase in all the systems investigated were oriented normal to the plane of the multilayers. The directions of the g signals corresponding to the g_x and g_y axes of the g tensor were found to be different in low-spin ferric heme in fully oxidized oxidase and in half-reduced liganded oxidase. It is suggested that this different orientation of g_x and g_y in fully oxidized oxidase and half-reduced liganded oxidase arises because the respective EPR signals belong to two different hemes, those of cytochrome a and a₃.     

Copper and manganese electron spin resonance studies of cytochrome c oxidase from Paracoccus denitrificans

The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms. However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II). The pure Mn(II) spectrum is observed at ?40°C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P. denitrificans cells that had been grown in a Mn(II)-depleted medium. This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart. Manganese is apparently not an essential component of P. denitrificans cytochrome c oxidase since it is present in substoichiometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c. However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase. In contrast, manganese added to the yeast oxidase or to the manganese-free P. denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme. This suggests that the manganese normally associated with P. denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme.