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 196 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 appearance 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).     

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 EPT-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 (less than 50 ms) approx. 0.5 electron equivalent per heme 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 (less than 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=o; 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-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

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).     

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.     

An investigation by e.p.r. and optical spectroscopy of cytochrome oxidase during turnover.

Cytochrome oxidase (EC 1.9.3.1; ferrocytochrome c:oxygen oxidoreductase) was studied during steady-state by optical and e.p.r. methods. Starting with either the 'resting' or the 'pulsed' enzyme, oxidase, cytochrome c, ascorbate and O2 were mixed and the reaction monitored optically. Tetramethylphenylenediamine was used as mediator to poise the steady-state to the desired reduction level. After mixing, the reaction was quenched by the used of rapid-freeze techniques. The e.p.r. spectra of samples captured at increasing tetramethylphenylenediamine concentrations (i.e. higher electron flux) show decreasing g = 2 (Cu A) and g = 3 (cytochrome a) signals. No Cu B or g = 6 signals (high-spin cytochrome a3) could be found during the reaction. Also, the signal with peaks at g = 1.69, 1.78 and 5 as well as the g = 12 signal was hardly detectable at higher turnover rates. The only new signal appearing during turnover is a radical signal, which is discussed in terms of a protein radical. Finally, a scheme is presented, proposing a catalytic cycle for cytochrome oxidase with respect to the O2 binding Cu B-cytochrome a3 unit.     

Pulsed cytochrome c oxidase from the thermophilic bacterium PS3.

A caa3-type terminal cytochrome c oxidase (EC 1.9.3.1) from the thermophilic bacterium PS3 containing three subunits showed conversion from resting into pulsed form. Upon pulsing (reduction and re-oxidation), the cytochrome c oxidase activity increased over 10-fold. This enhanced activity of the pulsed enzyme gradually decayed. Addition of phospholipids, necessary for the enzyme activity, did not affect this decay process. Small changes in the absorption spectrum were observed for the resting-into-pulsed transition and for H2O2 ligation to the pulsed enzyme. The e.p.r. spectrum of the resting enzyme was very similar to that of mitochondrial enzyme, but the transient g = 5, 1.78 and 1.69 set of e.p.r. signals, associated with the pulsed bovine heart oxidase, were not observed in the case of pulsed bacterium-PS3 enzyme.     

The binding of carbon monoxide to cytochrome c oxidase.

The effect of CO on the optical absorbance spectrum of partially reduced cytochrome c oxidase has been studied. The changes at 432 and 590 nm suggest that the cytochrome alpha2/3+ - CO compound is formed preferentially and that concomitantly a second electron is taken up by the enzyme. From the CO-induced changes at 830 nm it is concluded that in the partially reduced enzyme addition of CO causes reoxidation of the copper component of cytochrome c oxidase. Addition of CO to partially reduced enzyme (2 electrons per 4 metal ions) also brings about a decrease in the intensities of electron paramagnetic resonance signals of high-spin heme iron near g = 6 and of the low-spin heme at g = 2.6. Concomitantly both the low-spin heme a signal at g = 3 and the copper signal at g = 2 increase in intensity. These results demonstrate that formation of the reduced diamagnetic cytochrome a3 - CO compound is accompanied by reoxidation of both the copper component detectable by electron paramagnetic resonance and possibly also by cytochrome a.     

The kinetic mechanism of beef kidney D-aspartate oxidase

The mechanism of action of the flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been investigated by steady-state and stopped flow kinetic studies using D-aspartate and O2 as substrates in 50 mM KPi, 0.3 mM EDTA, pH 7.4, 4 degrees C. Steady-state results indicate that a ternary complex containing enzyme, O2, and substrate (or product) is an obligatory intermediate in catalysis. The kinetic parameters are turnover number = 11.1 s-1, Km(D-Asp) = 2.2 x 10(-3) M, Km(O2) = 1.7 x 10(-4) M. Rapid reaction studies show that 1) the reductive half reaction is essentially irreversible with a maximum rate of reduction of 180 s-1; 2) the free reduced enzyme cannot be the species which is reoxidized during turnover since its reoxidation by oxygen (second order rate constant equal to 5.3 x 10(2) M-1 s-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a ligand rapidly and be reoxidized as a complex at a rate faster than that observed for the free reduced enzyme; 4) the rate of reoxidation of reduced enzyme by oxygen during turnover is dependent on both O2 and D-aspartate concentrations (second order rate constant of reaction between O2 and reduced enzyme-substrate complex equal to 6.2 x 10(4) M-1 s-1); and 5) the rate-limiting step in catalysis occurs after reoxidation of the enzyme and before its reduction in the following turnover. A mechanism involving reduction of enzyme by substrate, dissociation of product from reduced enzyme, binding of a second molecule of substrate to the reduced enzyme, and reoxidation of the reduced enzyme-substrate complex is proposed for the enzyme-catalyzed oxidation of D-aspartate.     

EPR signals from cytochrome c oxidase.

1. The major EPR signals from native and cytochrome c-reduced beef heart cytochrome c oxidase (EC 1.9.3.1) are characterized with respect to resonance parameters, number of components and total integrated intensity. A mistake in all earlier integrations and simulations of very anisotropic EPR signals is pointed out. 2. The so-called Cu2+ signal is found to contain at least three components, one "inactive" form and two nearly similar active forms. One of the latter forms, corresponding to about 20% of the total EPR detectable Cu, has not been observed earlier and can only be resolved in 35 GHz spectra. It is not reduced by cytochrome c and is thought to reflect some kind of inhomogeneity in the enzyme preparation. The 35 GHz spectrum of the cytochrome c reducible component shows a rhombic splitting and can be well simulated with g-values 2.18, 2.03 and 1.99. The origin of such a unique type of Cu2+ spectrum is discussed. 3. The low-spin heme signal in the oxidized enzyme (g = 3.03, 2.21, 1.45) is found to correspond closely to one heme and shows no signs of interaction with other paramagnetic centres. 4. The high-spin heme signals appearing in partly reduced oxidase are found to consist of at least three species, one axial and two rhombic types. An integration procedure is described that allows the determination of the total integral intensity of high-spin heme EPR signals only by considering the g = 6 part of the signals. In a titration with ascorbate and cytochrome c the maximum intensity of the g = 6 species corresponds to 23% of the enzyme concentration.     

Electron paramagnetic resonance of the tungsten derivative of rat liver sulfite oxidase.

Sulfite oxidase purified from livers of tungsten-treated rats has been used for EPR studies of tungsten substituted at the molybdenum site of the enzyme in a fraction of the molecules. The EPR signal of W(V) in sulfite oxidase is quite similar to that of Mo(V) in its line shape and in its sensitivity to the presence of anions such as phosphate and fluoride. Hyperfine interaction with a dissociable proton is also observed in both signals. The pH-dependent alteration in line shape exhibited by the Mo(V) EPR signal of the rat liver enzyme. Incomplete reduction of the tungsten center at pH 9 is indicated by attenuated signal intensity at this pH. The W(V) signal has g values lower than those of the Mo(V) signal, has a much broader resonance envelope, and is much less readily saturated by increasing microwave power. Kinetic studies on the reduction of the heme and tungsten centers of sulfite oxidase have shown that reduction of de-molybdo forms of sulfite oxidase by sulfite is catalyzed by the residual traces of native molybdenum-containing molecules. Reduction is accomplished by electron transfer involving intermolecular heme-heme interaction. The W(V) signal is generated only after all the heme centers are reduced. The rate and extent of heme reduction at pH 9 are the same as at pH 7. Studies on the reoxidation of W(V) and reduced heme by O2 and by cytochrome c suggest that the cytochrome b5 of sulfite oxidase is the site of electron transfer to cytochrome c, whereas oxidase activity is the property of the molybdenum center. It appears that the tungsten center in sulfite oxidase is incapable of oxidizing sulfite.     

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.     

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.     

A new copper(II) electron paramagnetic resonance signal in two laccases and in cytochrome c oxidase

A new EPR signal from Cu2+ has been discovered in reductive experiments with type 2 copper-depleted laccase from Polyporus versicolor. A novel EPR signal has also been found in native laccase from Rhus vernicifera on oxidation of the reduced protein with H2O2. In reoxidation experiments with cytochrome c oxidase from beef heart, a new Cu2+ signal has been observed. With Rhus laccase, the new signal is shown to originate from one of the copper ions that are nondetectable in the resting enzyme, and evidence is presented for the signals in Polyporus laccase and cytochrome c oxidase also stemming from the metal pairs that are antiferromagnetically coupled in the oxidized enzymes. The new signals show strong rhombic character, and the EPR parameters place them in a category different from the signals of type 1 as well as of type 2 Cu2+ ions.     

The oxidation of cytochrome c oxidase by hydrogen peroxide

The reaction of H2O2 with mixed-valence and fully reduced cytochrome c oxidase was investigated by photolysis of fully reduced and mixed-valence carboxy-cytochrome c oxidase in the presence of H2O2 under anaerobic conditions. The results showed that H2O2 reacted rapidly (k = (2.5-3.1) X 10(4) M-1 X s-1) with both enzyme species. With the mixed-valence enzyme, the fully oxidised enzyme was reformed. On the time-scale of our experiments, no spectroscopically detectable intermediate was observed. This demonstrates that mixed-valence cytochrome c oxidase is able to use H2O2 as a two-electron acceptor, suggesting that cytochrome c oxidase may under suitable conditions act as a peroxidase. Upon reaction of H2O2 with the fully reduced enzyme, cytochrome a was oxidised before cytochrome a3. From this observation it was possible to estimate that the rate of electron transfer from cytochrome a to a3 is about 0.5-5 s-1.     

Electron paramagnetic resonance observations on the cytochrome c-containing nitrous oxide reductase from Wolinella succinogenes.

Nitrous oxide reductase from Wolinella succinogenes, an enzyme containing one heme c and four Cu atoms/subunit of Mr = 88,000, was studied by electron paramagnetic resonance (EPR) at 9.2 GHz from 6 to 80 K. In the oxidized state, low spin ferric cytochrome c was observed with gz = 3.10 and an axial Cu resonance was observed with g parallel = 2.17 and g perpendicular = 2.035. No signals were detected at g values greater than 3.10. For the Cu resonance, six hyperfine lines each were observed in the g parallel and g perpendicular regions with average separations of 45.2 and 26.2 gauss, respectively. The hyperfine components are attributed to Cu(I)-Cu(II) S = 1/2 (half-met) centers. Reduction of the enzyme with dithionite caused signals attributable to heme c and Cu to disappear; exposure of that sample to N2O for a few min caused the reappearance of the g = 3.10 component and a new Cu signal with g parallel = 2.17 and g perpendicular = 2.055 that lacked the simple hyperfine components attributed to a single species of half-met center. The enzyme lost no activity as the result of this cycle of reduction and reoxidation. EPR provided no evidence for a Cu-heme interaction. The EPR detectable Cu in the oxidized and reoxidized forms of the enzyme comprised about 23 and 20% of the total Cu, respectively, or about one spin/subunit. The enzyme offers the first example of a nitrous oxide reductase which can have two states of high activity that present very different EPR spectra of Cu. These two states may represent enzyme in two different stages of the catalytic cycle.     

Effect of changing the detergent bound to bovine cytochrome c oxidase upon its individual electron-transfer steps

The influence of the detergent environment upon individual electron-transfer rates of cytochrome c oxidase was investigated by stopped-flow spectrophotometry. The effects of three detergents were studied: lauryl maltoside, which supports a high turnover number (TN = 350 s-1), n-dodecyl octaethylene glycol monoether (C12E8), which supports an intermediate TN (150 s-1), and Triton X-100 in which oxidase is nearly inactive (TN = 2-3 s-1). Under limited turnover conditions (cytochrome c:cytochrome c oxidase ratio = 1:1 to 8:1), the rate of oxidation of cytochrome c was measured and compared with the fast reduction of cytochrome a and its relatively slow reoxidation. Two reducing equivalents of cytochrome c were rapidly oxidized in a burst phase; the remaining two to six equivalents were oxidized more slowly, concurrent with the reoxidation of cytochrome a; i.e., the percent reduced cytochrome a reflects the percent reduced cytochrome c. With the resting enzyme, the bimolecular reaction between reduced cytochrome c and cytochrome a was rapid, was insensitive to the detergent environment, and was not the rate-limiting step in the presence of any detergent. The rate of internal electron transfer from cytochrome a to cytochrome a3 in the resting enzyme was slow and only slightly affected by the detergent environment: 1.0-1.1 s-1 in Triton X-100, 5-7 s-1 in C12E8, and 5-12 s-1 in lauryl maltoside. With the pulsed enzyme, the intramolecular electron transfer between cytochrome a and cytochrome a3 increased 4-5-fold in the lauryl maltoside enzyme but did not increase in the Triton X-100 enzyme (intermediate values were obtained with the C12E8 enzyme). We conclude that cytochrome c oxidase acquires the pulsed conformation only in those detergents that support high TN's, e.g., lauryl maltoside and C12E8, but it is locked in the resting conformation in those detergents which result in low TN's, e.g., Triton X-100.     

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 represent a3+(3) (Beinert, H. and Shaw, R.W.(1977) Biochim. Biophys. Acta 462, 12u--130), and can be detected and quantitatively determined by EPR. We have now used out 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 less than 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.     

Biochemical and biophysical studies on cytochrome c oxidase. XX. Reaction with sulphide.

1.Upon addition of sulphide to oxidized cytochrome c oxidase, a low-spin heme sulphide compound is formed with an EPR signal at gx = 2.54, gy = 2.23 and gz = 1.87. Concomitantly with the formation of this signal the EPR-detectable low-spin heme signal at g = 3 and the copper signal near g = 2 decrease in intensity, pointing to a partial reduction of the enzyme by sulphide. 2. The addition of sulphide to cytochrome c oxidase, previously reduced in the presence of azide or cyanide, brings about a disappearance of the azido-cytochrome c oxidase signal at gx = 2.9, gy = 2.2, and gz = 1.67 and a decrease of the signal at g = 3.6 of cyano-cytochrome c oxidase. Concomitantly the sulphide-induced EPR signal is formed. 3. These observations demonstrate that azide, cyanide and sulphide are competitive for an oxidized binding site on cytochrome c oxidase. Moreover, it is shown that the affinity of cyanide and sulphide for this site is greater than that of azide.     

Low-spin ferric forms of cytochrome a3 in mixed-ligand and partially reduced cyanide-bound derivatives of cytochrome c oxidase.

Optical-absorption-, e.p.r.- and m.c.d. (magnetic-circular-dichroism)-spectroscopic measurements were made on liganded derivatives of oxidized and partially reduced cytochrome c oxidase. When NO was added to oxidized cyanide-bound cytochrome c oxidase, no changes occurred in the optical-absorption difference spectrum. In contrast, NO induced reduction of cytochrome a3 and formation of the nitrosylferrohaem species when the oxidized resting enzyme was the starting material. E.p.r. spectroscopy of the NO-treated oxidized cyanide-bound enzyme revealed the presence of a low-spin haem signal at g = 3.40, whereas the g = 3.02 and g = 2.0 signals of the oxidized enzyme remained unchanged. Both haem groups in this species are e.p.r.-detectable simultaneously. Examination of an identical sample by m.c.d. spectroscopy in the near-i.r. region identified two distinct low-spin species at 1565 and 1785 nm. Irradiation with white light of the NO-treated cyanide-bound sample at 10K resulted in the disappearance of the g = 3.40 e.p.r. signal and the m.c.d. signal at 1785 nm, whereas a band at 1950nm increased in intensity. When the photolysed sample was warmed to 50K and held in the dark for 15 min, the original spectrum returned. Magnetization studies of the 1785nm m.c.d. band support the assignment of this signal to the same metal centre that gives rise to the g = 3.40 e.p.r. signal. The effect of NO on the oxidized cyanide-bound enzyme was compared with that obtained when the oxidized cyanide-bound species was taken to the partially reduced state. Cytochrome a3 is e.p.r.-detectable with a g-value of 3.58 [Johnson, Eglinton, Gooding, Greenwood & Thomson (1981) Biochem. J. 193, 699-708]. Its near-i.r. m.c.d. spectrum shifts from 1950nm in the oxidized cyanide-bound enzyme to 1545nm on addition of reductant. A scheme is advanced for the structure of the cytochrome a3-CuB site that allows for cyanide binding to Fea3 and NO binding to CuB. Cyanide is the bridging ligand in the ferromagnetically coupled cytochrome a3-CuB pair of oxidized cyanide-bound cytochrome c oxidase. The bridged structure and the magnetic interaction are broken when the enzyme is partially reduced. However, when NO binds to CuB the cyanide bridge remains intact, but now the odd spins of NO and CuB are magnetically coupled.     

Identification of a ferryl intermediate of Escherichia coli cytochrome d terminal oxidase by resonance Raman spectroscopy.

The 680-nm-absorbing "peroxide state" of the Escherichia coli cytochrome d terminal oxidase complex, obtained by addition of excess hydrogen peroxide to the enzyme, is shown to be a ferryl intermediate in the catalytic cycle of the enzyme. This ferryl intermediate is also created by aerobic oxidation of the fully reduced enzyme. Resonance Raman spectra with 647.1-nm excitation show an FeIV = O stretching band at 815 cm-1, a higher frequency than noted in any other ferryl-containing enzyme to date. The band shows an 16O/18O frequency shift of -46 cm-1, larger than that observed for any porphyrin ferryl species. The FeIV = O formulation was unambiguously established by oxidations of the reduced enzyme with 16O2, 18O2, and 16O18O. Only the use of a mixed-isotope gas permitted discrimination between a ferryl and a peroxo structure. A catalytic cycle for the cytochrome d terminal oxidase complex is proposed, and possible reasons for the high v(Fe = O) frequency are discussed.