EPR studies of the photodissociation reactions of cytochrome c oxidase-nitric oxide complexes

Three complexes of NO with cytochrome c oxidase are described which are all photodissociable at low temperatures as measured by EPR. The EPR parameters of the cytochrome a2+(3)-NO complex are the same both in the fully reduced enzyme and in the mixed-valence enzyme. The kinetics of photodissociation of cytochrome a2+(3)-NO and recombination of NO with cytochrome a2+(3) (in the 30-70 K region) revealed no differences in structure between cytochrome a2+(3) in the fully reduced and the mixed-valence states. The action spectrum of the photodissociation of cytochrome a2+(3)-NO as measured by EPR has maxima at 595, 560 and 430 nm, and corresponds to the absorbance spectrum of cytochrome a2+(3)-NO. Photodissociation of cytochrome a2+(3)-NO in the mixed-valence enzyme changes the EPR intensity at g 3.03, due to electron transfer from cytochrome a2+(3) to cytochrome a3+. The extent of electron transfer was found to be temperature dependent. This suggests that a conformational change is coupled to this electron transfer. The complex of NO with oxidized cytochrome c oxidase shows a photodissociation reaction and recombination of NO (in the 20-40 K region) which differ completely from those observed in cytochrome a2+(3)-NO. The observed recombination occurs at a temperature 15 K lower than that found for the cytochrome a2+(3)-NO complex. The action spectrum of the oxidized complex shows a novel spectrum with maxima at 640 and below 400 nm; it is assigned to a Cu2+B-NO compound. The triplet species with delta ms = 2 EPR signals at g 4 and delta ms = 1 signals at g 2.69 and 1.67, that is observed in partially reduced cytochrome c oxidase treated with azide and NO, can also be photodissociated.     

Electron-transfer processes in carboxy-cytochrome c oxidase after photodissociation of cytochrome a2+3 · CO

Under continuous illumination the CO binding curve of reduced carboxy-cytochrome c oxidase maintains the shape of the binding curve in the dark. The apparent dissociation constant calculated from the binding curves at various light intensities is a linear function of the light intensity.Marked differences are observed between the light-induced difference spectra of the fully reduced carboxy-cytochrome c oxidase and the mixed-valence carboxy-cytochrome c oxidase. These differences are enhanced in the presence of ferricyanide as an electron acceptor and are explained by partial oxidation of cytochrome a₃ in the mixed-valence enzyme after photodissociation.Upon addition of CO to partially reduced formate cytochrome c oxidase (a²⁺a³⁺₃ · HCOOH) the cytochrome a²⁺₃ · CO compound is formed completely with a concomitant oxidation of cytochrome a and the Cu associated with cytochrome a. During photodissociation of the CO compound the formate rebinds to cytochrome a₃ and cytochrome a and its associated Cu are simultaneously reduced. These electron transfer processes are fully reversible since in the dark the a³⁺₃ · HCOOH compound is dissociated slowly with a concomitant formation of the a²⁺₃ · CO compound and oxidation of cytochrome a.When these experiments are carried out in the presence of cytochrome c, both cytochrome c and cytochrome a are reduced upon illumination of the mixed-valence carboxy-cytochrome c oxidase. In the dark both cytochrome c and cytochrome a are reoxidized when formate dissociates from cytochrome a₃ and the a²⁺₃ · CO compound is formed back. Thus, in this system we are able to reverse and to modulate the redox state of the different components of the final part of the respiratory chain by light.     

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a³⁺a²⁺₃-CO) complex and the mixed-valence carboxycytochrome c oxidase (a³⁺a²⁺₃-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a²⁺₃-CO complex. This would confirm that the spectral properties of cytochrome a₃ are not affected by the redox state of cytochrome a and Cu_A. When the absorbance changes after photolysis of cytochrome a²⁺₃-CO with a laser flash were followed on a time scale from 1 μs to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7·10³s^?1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a₃. Concomitantly, a decrease of the 830 nm band was observed due to reduction of Cu_A. This demonstrates that in the partially reduced enzyme a pathway is present between Cu_A and the cytochrome a₃-Cu_B pair, via which electrons are transferred rapidly.     

Heme-heme interaction in cytochrome c oxidase: the cooperativity of the hemes of cytochrome c oxidase as evidenced in the reaction with CO

Isolated and purified cytochrome c oxidase from beef heart muscle mitochondria (Kuboyama et al. (1972) J. Biol. Chem.247, 6375–6383) is shown to be very similar to the hemoprotein in situ with respect to its EPR absorption properties and the half-reduction potentials of the hemes and copper. The half-reduction potentials of cytochromes a and a₃ in the purified cytochrome c oxidase are 205 mV and 360 mV, respectively, and these values are the same in the presence and absence of cytochrome c.Low-temperature EPR spectra show that the binding of CO to reduced cytochrome a₃ changes the oxidized cytochrome a from high spin (g 6) to low spin (g 3). In samples at 5–8 °K the photodissociation of the reduced cytochrome a₃CO compound shifts the spectrum of the oxidized low-spin cytochrome a to a lower g value and converts approximately 5% of the low-spin form to a high-spin form. The heme-heme interaction demonstrated in this reaction is very fast as evidenced by the fact that even at 5 °K the measured change in oxidized cytochrome is complete within 5 msec.     

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.     

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.     

An EPR signal from the "invisible" copper of cytochrome oxidase.

Sulfide is both an inhibitor and a slow reductant of oxidized cytochrome $\text{c}$ oxidase. When the enzyme is exposed to sulfide for short times (one minute or less) and frozen, the resultant electron paramagnetic resonance (EPR) signals show clearly: low spin heme a, low spin heme a₃, the usual “EPR detectable” Cu²⁺ signal (g_⊥ = 2.17, g_⊥ = 2.03), and a new Cu²⁺ signal superimposed on the same region, with (g_⊥ ～ 2.19, g_⊥ = 2.05). This new signal presumably arises because the antiferromagnetic coupling postulated to exist between the iron atom of heme a₃ and this copper is disrupted when heme a₃ is driven to a low spin state by sulfide. The implications of this result with respect to models of the O₂-binding site and redox geometry of oxidase are briefly discussed.     

Individual heme a and heme a3 contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase

Bovine cytochrome c oxidase (CcO) contains two hemes, a and a₃, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa₃-type cytochrome c oxidase consists of overlapping bands of the hemes a²⁺ and a₃²⁺. It shows a peak at ～444 nm and a distinct shoulder at ～425 nm. However, attribution of individual spectral lineshapes to hemes a²⁺ and a₃²⁺ in the Soret is controversial. In the present work, we characterized spectral contributions of hemes a²⁺ and a₃²⁺ using two approaches. First, we reconstructed bovine CcO heme a²⁺ spectrum using a selective Ca²⁺-induced spectral shift of the heme a²⁺. Second, we investigated photobleaching of the reduced Thermus thermophilus ba₃- and bovine aa₃-oxidases in the Soret induced by femtosecond laser pulses in the Q-band. The resolved spectra show splitting of the electronic B_0x-, B_0y-transitions of both reduced hemes. The heme a²⁺ spectrum is shifted to the red relative to heme a₃²⁺ spectrum. The ～425 nm shoulder is mostly attributed to heme a₃²⁺.     

Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with K_i values of 4.5, 91, 200 and 330 μM, respectively.2. The inhibition constant K_i of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme.3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k₁ of 33 M^?1 · s^?1 and dissociation constant K_d of 3.9 mM.4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M^?1 · s^?1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes.5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome a₃-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

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.     

Cytochrome c oxidase. Time dependence of optical and EPR spectral changes related to the ‘oxygen-pulsed’ form

Time-dependent changes in the optical spectrum (450–920 nm) of cytochrome c oxidase, following oxidation with oxygen of the stoichiometrically reduced form, have been investigated and where possible, attempts have been made to correlate our observations with variations in the EPR spectrum over a parallel time course at 2°C. In this regard, particular emphasis has been placed on establishing absorption features related to the presence of EPR resonances at g 5, 1.78 and 1.69, which have been tentatively assigned to a spin-coupled state involving cytochrome a₃ and ‘EPR-undetectable Cu’ (Beinert, H., Shaw, R.W., Dunham, R.W. and Sands, R.H. (1982) in Oxidases and Related Redox Systems (King, T.E., Mason, H.S. and Morrison, M., eds.), Pergamon Press, Oxford, in the press). For optical studies we have used a versatile rapid-scanning spectrophotometer to obtain well resolved spectra down to 2 ms reaction time. Concomitant with the appearance (within 10 ms) of EPR signals at g 5, 1.78 and 1.69 is the presence of an enhanced absorption (Δε = 0.25 mM (heme a)^?1·cm^?1) at 660 nm, with a trough (relative to following spectra) at 580 nm. In our hands, this feature disappears in a first-order process with a half-life of 46 s at pH 7.2 and 2°C. The effect of this spectral transformation is to decrease considerably the acuteness of the 655 nm absorption band, previously suggested as representing a state of the enzyme in which ferric cytochrome a₃ is coupled to oxidised EPR-undetectable Cu (Beinert, H., Hansen, R.E. and Hartzell, C.R. (1976) Biochim. Biophys. Acta 423, 339–355). This observation can be correlated satisfactorily with a small field shift of the high-field resonances at g 1.78 and 1.69 and a broadening at g 1.78. Support for this and further correlative assignments arises from parallel experiments using cytochrome c oxidase purified via an alternative procedure, which displays different kinetic behavior. Further transformations of the oxidized enzyme are evident through an approx. 10% decrease in absorbance at 600 nm together with small changes centered at 640 and 665 nm (which serve to restore the sharpness of the 655 nm band). The kinetics, as analyzed by the Guggenheim procedure using the absorbance at 597 nm, indicate approx. 50% first-order linearity (half-life 40 min) with additional species contributing at longer times, while over a parallel time course (0–3 h) the EPR resonances at g 5, 1.78 and 1.69 virtually disappear. These novel signals can also be seen at a lower intensity in samples of cytochrome c oxidase anaerobically reoxidized by porphyrexide and frozen after a 6 min incubation period at 4°C. This observation, along with the establishment of similar optical changes over the time course of 1 min to 3 h, suggests that aerobic and anaerobic reoxidation produce common forms of the enzyme. Comparison of the g 1.78 and 1.69 resonances between samples rapidly aerobically reoxidized in the presence of H₂¹⁶O and H₂¹⁷O yielded no evidence for the presence of any labile oxygen ligand (including OH^?, H₂O) in the coordination sphere of the species involved.     

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.     

Biochemical and biophysical studies on cytochrome c oxidase. XVII. An epr study of the photodissociation of cytochrome a32+ · CO

1. The photodissociation reaction of the cytochrome c oxidase-CO compound was studied by EPR at 15 °K. Illumination with white light at both room and liquid N₂ temperatures of the partially reduced cytochrome c oxidase (2 electrons per 4 metals) in the presence of CO, causes the appearance of a rhombic (g_x = 6.60, g_y = 5.37) high-spin heme signal.This signal disappears completely upon darkening of the sample and reappears upon illumination at room temperature; accordingly the photolytic process is reversible. Under these conditions, no great changes in the intensities are observed, neither of the copper signal at g = 2, nor of the low-spin heme signal at g = 3, 2.2 and 1.5.2. In the presence of ferricyanide (2 mM) and CO, both the low-spin heme signal (g = 3.0, 2.2 and 1.5) and the copper signal of the partially reduced enzyme have intensities about equal to those of the completely oxidized enzyme in the absence of CO. Upon illumination of the carboxy-cytochrome c oxidase in the presence of ferricyanide, it was found that the rhombic high-spin heme signal appears without affecting appreciably the copper of low-spin heme signals. Thus, in the presence of ferricyanide the EPR-detectable paramagnetism of the illuminated carboxy-cytochrome c oxidase is higher than in the untreated oxidized enzyme.3. The membrane-bound cytochrome c oxidase reduced with NADH in the presence of CO and subsequently oxidized with ferricyanide shows a similar rhombic high-spin heme signal (g_x = 6.62, g_y = 5.29) upon illumination at room temperature. This signal disappears completely upon darkening and reappears upon illumination at room temperature.     

Responses of the a3 component of cytochrome c oxidase to substrate and ligand addition

We have previously described a transient high spin ferric heme species in cytochrome c oxidase (EC 1.9.3.1) which represents a³⁺₃ (Beinert, H. and Shaw, R.W. (1977) Biochim. Biophys. Acta 462, 121–130), and can be detected and quantitatively determined by EPR. We have now used our ability to generate this species to study reactions of a³⁺₃ with substrates and ligands and also responses to pH changes. This was accomplished by multiple rapid mixing and freezing techniques in conjunction with low temperature EPR and optical reflectance spectroscopies. The substrates used were O₂ and ferrocytochrome c and the ligands cyanide, sulfide, azide and carbon monoxide. Contrary to the oxidized, resting form of the enzyme, the transient high spin species of a³⁺₃ reacts within <10 ms stoichiometrically with cyanide and sulfide and at a slower rate with azide. The transient a³⁺₃ species responds to O₂ and CO by changes in signal size or shape, although no oxidoreduction is involved, indicating that a³⁺₃ registers the presence of these gases. The high spin signal of the transient species is readily abolished by ferrocytochrome c or on raising the pH. Decreasing the pH induces a shift from the rhombic towards the axial component of the signal. Since the responses to CO and pH are analogous for the rhombic transient species to those observed with the rhombic high spin ferric heme species produced on partial reduction, it is suggested that the rhombic signals represent a³⁺₃ in either case. In all these experiments, in which EPR detectable a³⁺₃ was observed in large yield, no extra signals for copper or correspondingly increased intensity in the copper signal at g = 2 were seen. The relationship is discussed of the obviously reactive transient species of a³⁺₃ to other ‘activated’ species that have been reported and to the oxidized resting form of the enzyme, which is known to react only slowly with ligands and to respond sluggishly to substrate.     

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.     

The identity of a new copper(II) electron paramagnetic resonance signal in cytochrome c oxidase

In reoxidation experiments with cytochrome c oxidase (EC 1.9.3.1) in the presence of both reducing substrate and molecular oxygen, a new EPR signal from Cu²⁺ has been observed. The new signal corresponds to 0.45 Cu per functional unit. It is concluded that the new EPR signal originates from Cu²⁺_B, the copper which is EPR-nondetectable in the resting enzyme.Optical absorption changes in the 500–700 nm region accompanies the decay of the new Cu²⁺ EPR signal.Based on the results in this investigation a catalytic cycle for cytochrome oxidase is proposed.     

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.     

Transient Binding of CO to CuB in Cytochrome c Oxidase Is Dynamically Linked to Structural Changes around a Carboxyl Group: A Time-Resolved Step-Scan Fourier Transform Infrared Investigation

The redox-driven proton pump cytochrome c oxidase is that enzymatic machinery of the respiratory chain that transfers electrons from cytochrome c to molecular oxygen and thereby splits molecular oxygen to form water. To investigate the reaction mechanism of cytochrome c oxidase on the single vibrational level, we used time-resolved step-scan Fourier transform infrared spectroscopy and studied the dynamics of the reduced enzyme after photodissociation of bound carbon monoxide across the midinfrared range (2300-950 cm⁻¹). Difference spectra of the bovine complex were obtained at -20°C with 5 μs time resolution. The data demonstrate a dynamic link between the transient binding of CO to Cu_B and changes in hydrogen bonding at the functionally important residue E(I-286). Variation of the pH revealed that the pK_a of E(I-286) is >9.3 in the fully reduced CO-bound oxidase. Difference spectra of cytochrome c oxidase from beef heart are compared with those of the oxidase isolated from Rhodobacter sphaeroides. The bacterial enzyme does not show the environmental change in the vicinity of E(I-286) upon CO dissociation. The characteristic band shape appears, however, in redox-induced difference spectra of the bacterial enzyme but is absent in redox-induced difference spectra of mammalian enzyme. In conclusion, it is demonstrated that the dynamics of a large protein complex such as cytochrome c oxidase can be resolved on the single vibrational level with microsecond Fourier transform infrared spectroscopy. The applied methodology provides the basis for future investigations of the physiological reaction steps of this important enzyme.     

Reduction of oxygen-pulsed cytochrome c oxidase by cytochrome c and other electron donors

1. Stopped-flow experiments were performed in which solutions containing dithionite were mixed with air-saturated buffer. Cytochrome c oxidase present in the dithionite-containing syringe is fully oxidized within the mixing time and the oxygen-pulsed form of the oxidase is produced.2. The reduction of this form by dithionite, by dithionite plus cytochrome c and by dithionite plus methyl viologen or benzyl viologen was followed and compared with the corresponding reduction reactions of the ‘resting’ oxidized enzyme. Reduction by dithionite is relatively slow, but the rate of reduction is greatly increased by addition of cytochrome c or the viologens, which are even more effective than cytochrome c on a molar basis.3. Profound differences between the transient kinetics of the reduction of the two oxidized oxidase derivatives were observed. The results are consistent with a direct reduction of cytochrome a followed by an intramolecular electron transfer to cytochrome a₃ (k^obs₁ = 7.5 s^?1 for the oxygen-pulsed oxidase).4. The spectrum of the oxygen-pulsed oxidase formed within 5 ms of the mixing closely resembles that of the ‘oxygenated’ compound, but there were small differences between the two spectra.