Solubilization and purification of cytochrome a1 from Nitrosomonas

Cytochrome a₁ was solubilized with Triton X-100 from a membrane-envelope preparation of Nitrosomonas and partially purified by repeated fractionation with (NH₄)₂SO₄. The purified fraction of cytochrome a₁ was enriched over the crude extract by a factor of 16 and 300 with respect to protein and c-type cytochrome, respectively. The cytochrome was characterized as cytochrome a₁ on the basis of (a) reduced absorption maxima at 444 nm and 595 nm, (b) acid acetone extractibility and ether solubility of the heme and (c) absorption maximum of 587 nm of the ferro-hemochrome in alkaline pyridine. The α absorption band shifted from 600 nm to 595 nm upon solubilization of the cytochrome with Triton X-100. Spectral shifts were observed in the presence of cyanide and azide and the cytochrome changed with aging to a form with a reduced absorption band at 422 nm. Cytochrome a₁ was reduced anaerobically in the presence of reduced mammalian cytochrome c and was rapidly reoxidized in the presence of O₂. CO caused a shift in the soret peak of the reduced form but did not prevent reoxidation of cytochrome a₁ in the presence of CO-O₂ (95:5, v/v).     

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.     

Occurrence of cytochrome aa3 in Anacystis nidulans

The cytochrome content of membrane fragments prepared from the bluegreen alga (cyanobacterium) Anacystis nidulans was examined by difference spectrophotometry. Two b-type cytochromes and a hitherto unknown cytochrome a could be characterized. In the reduced-minus-oxidised difference spectra the a-type cytochrome showed an α-band at 605 nm and a γ-band at 445 nm. These bands shifted to 590 and 430 nm, respectively, in CO difference spectra. NADPH, NADH and ascorbate reduced the cytochrome through added horse heart cytochrome c as electron mediator. In presence of KCN the reduced-minus-oxidised spectrum showed a peak at 600 nm and a trough at 604 nm. Photoaction spectra of O₂ uptake and of horse heart cytochrome c oxidation by CO-inhibited membranes showed peaks at 590 and 430 nm. These findings are consistent with cytochrome aa₃ being the predominant respiratory cytochrome c oxidase in Anacystis nidulans.     

In Situ Raman Study of Redox State Changes of Mitochondrial Cytochromes in a Perfused Rat Heart

We developed a Raman spectroscopy-based approach for simultaneous study of redox changes in c-and b-type cytochromes and for a semiquantitative estimation of the amount of oxygenated myoglobin in a perfused rat heart. Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO₂ demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration. Conversely, transient hypoxia causes the reversible increase in the intensity of peaks assigned to cytochromes c and c1, reflecting electron stacking proximal to cytochrome oxidase due to the lack of terminal electron acceptor O₂. Intensities of peaks assigned to oxy- and deoxyhemoglobin were used for the semiquantitative estimation of oMb deoxygenation that was found to be of approximately 50 under hypoxia conditions.     

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.     

A sensitive method for the quantitative estimation of cytochromes a and a3 in tissues

A sensitive, low-temperature spectrophotometric method has been developed for independently estimating the quantities of cytochromes a and a₃ in a single, hemoglobin- and/or myoglobin-contaminated tissue homogenate or cell suspension. The concentration dependence of cytochrome absorption at liquid nitrogen temperature and the intensification of the cytochrome absorbance due to light scattering by the frozen hyperosmotic sucrose solution were used for detecting small amounts of cytochromes. The absorbance due to contaminated hemoglobin and myoglobin was cancelled in the difference spectrum by pretreating the sample with CO:O₂ (50:50) mixed gas. The resulting cytochrome $\text{a}{}_3{}^{\mathrm{2+}}\text{-}\text{CO}$ compound was irreversibly photolyzed at ?196°C, thus permitting us to obtain a fully reduced minus oxidized difference spectrum of the cytochromes. The method depends upon the non-Arrhenius behavior of carboxyhemoglobin and carboxymyoglobin which enables them to recombine with the CO molecule at the temperature of liquid nitrogen. Cytochrome a₃ does not exhibit this extreme non-Arrhenius behavior and hence does not recombine at the low temperature. Two to five picomoles of cytochromes could be estimated by this method.     

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 Δm_s = 2 EPR signals at g 4 and Δm_s = 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.     

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⁺.     

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.     

CO-binding pigments and the functional terminal oxidase of the trypanosomatid hemoflagellate Crithidia fasciculata

CO-difference absorbance spectra of both intact cells and of mitochondrial preparations isolated from Crithidia fasciculata were obtained after anaerobiosis was attained either with substrates or with dithionite. Under both sets of conditions, the CO-difference spectrum of cytochrome a₃, with difference absorbance maxima at 430 and 589 nm and minima at 443 and 612 nm, was readily identified in both the intact cells and in the mitochondria. In addition to the difference absorbance bands of cytochrome a₃-CO, three difference absorbance maxima at 417, 538 and 570 nm and a minimum at 556 nm were observed. The magnitude of the maximum at 570 nm relative to the maximum of cytochrome a₃-CO at 589 nm was less for mitochondria rendered anaerobic with substrate than for mitochondria rendered anaerobic with dithionite. This difference was taken to define operationally two groups of mitochondrial CO-binding pigments: Group I is that group observed on anaerobiosis with substrate: Group II is the additional group observed on anaerobiosis with dithionite. The Group I CO-binding pigments were virtually absent from submitochondrial particles derived by sonication, but the Group II pigments remained.Photochemical action spectra were obtained with isolated mitochondria and intact cells to ascertain if cytochrome o was present. These action spectra, obtained in CO plus O₂ atmospheres, had maxima only at 432, 550 and 588 nm, attributable to the photodissociation of cytochrome a₃-CO. Even after suppression of cytochrome a₃ activity to 10% of the normal value, no contribution of cytochrome o activity to the photochemical action spectrum was observed. Cytochrome a₃ is therefore the only functional terminal oxidase present in the mitochondria of Crithidia fasciculata.     

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.     

A temperature-induced absorption band centred in the region of 666 nm related to the configuration of the active site in frozen cytochrome oxidase

The existence of a temperature-induced absorption band centred in the region of 666 nm is demonstrated for both membrane-bound and soluble cytochrome oxidase in the fronze state.The 666 nm band is generated solely by an increase in temperature of both fully reduced and mixed valence state cytochrome oxidase in the presence of CO or O₂ within the ‘pocket’ containing the active site; it is not formed in the absence of both CO and O₂ from the sample.The formation of the 666 nm band is entirely reversible when the temperature is decreased again and its formation is not dependent on the presence of liganded CO at the sixth coordination site of haem a₃ in the low temperature range (below ?120°C) prior to photolysis.The shape and intensity of the 666 nm band are not affected by the extent of CO recombination following flash and photolysis and temperature increase and are not affected by changes in the valence states of the four metal centres when the O₂ reaction is in progress.     

Mitochondrial respiratory chain of Tetrahymena pyriformis. The thermodynamic and spectral properties

The mitochondria isolated from the ciliate protozoon Tetrahymena pyriformis carry an oxidative phosphorylation with P/O ratio of 2 for succinate oxidation and P/O ratio of 3 for the oxidation of the NAD-linked substrates. The respiration is more than 90% inhibited with 1 mM cyanide while antimycin A and rotenone inhibit at concentrations of 1000-fold higher than those effective in mammalian mitochondria.Using a combination of spectral studies and potentiometric titrations, the components of the respiratory chain were identified and characterized with respect to the values of their half-reduction potentials. In the cytochrome bc₁ region of the chain a cytochrome c was present with an E_m7.2 of 0.225 V and two components with absorption maxima at 560 nm and the half-reduction potential values of ?0.065 and ?0.15 V at pH 7.2. The cytochrome with the more positive half-reduction potential was identified as the analogue of the cytochrome(s) b present in mitochondria of higher organisms, while the cytochrome with the more negative half-reduction potential was tentatively identified as cytochrome o. In addition ubiquinone was present at a concentration of approx. 4 nmol per mg mitochondrial protein.In the spectral region where cytochromes a absorb at least three cytochromes were found. A cytochrome with an absorption maximum at 593 nm and a midpoint potential of ?0.085 V at pH 7.2 was identified as cytochrome a₁. The absorption change at 615–640 nm, attributed usually to cytochrome a₂ was resolved into two components with E_m7.2 values of 0.245 and 0.345 V. It is concluded that the terminal oxidase in Tetrahymena pyriformis mitochondria is cytochrome a₂ which in its two-component structure resembles cytochrome aa₃.     

The cytochromes of mitochondria from Tetrahymena pyriformis strain ST

1. Mitochondria of the obligately aerobic ciliate protozoon, Tetrahymena pyriformis strain ST, are unusual in that they possess a cytochrome oxidase system that does not react with reduced mammalian cytochrome c; the presence of cytochromes a₆₀₃+a₃ is masked in the α-band region of spectra by the broad absorption band of cytochrome a₆₂₀. 2. Other haemoproteins present include cytochromes b₅₆₀, b₅₅₆, c₅₅₃ and c₅₄₉. 3. The reaction of reduced cytochrome a₃ with CO is reversed by flash photolysis, and in the presence of O₂ the subsequent oxidation of this cytochrome is followed by that of cytochrome a₆₀₃. 4. Cytochromes a₆₂₀ and b₅₆₀ also react with CO and with KCN; the latter cytochrome corresponds with that designated cytochrome o by other workers. 5. The contribution of cytochrome a₆₀₃ to difference spectra is revealed by making use of the fact that it does not react with KCN. 6. Cytochrome a₆₂₀ is unstable, and its α-absorption band is lost from spectra of mitochondria which have been aged or treated with ultrasound, detergents or organic solvents. 7. Possible pathways of electron transport via the several different terminal oxidases in Tetrahymena mitochondria are proposed.     

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.     

Reaction of Mixed Valence State Cytochrome Oxidase with Oxygen in Plant Mitochondria: A STUDY BY LOW TEMPERATURE FLASH PHOTOLYSIS AND RAPID WAVELENGTH SCANNING OPTICAL SPECTROMETRY

The reaction of mixed valence state cytochrome oxidase (Cu_A²⁺a³⁺ · Cu_B⁺a₃²⁺) with O₂ at 173 K has been investigated in purified potato mitochondria by low temperature flash photolysis and rad wavelength scanning optical spectrometry in the visible region. The kinetics of the reaction have been analyzed simultaneously at six wavelength pairs (586-630, 590-630, 594-630, 604-630, 607-630, and 610-630 nanometers) by nonlinear optimization techniques, and found to proceed by a two-species sequential mechanism. The “pure” difference spectra of the two species, I_M and II_M, relative to unliganded mixed valence state cytochrome oxidase have been obtained. The difference spectrum of species I_M is characterized by a peak at 591 nanometers, with a shoulder at 584 nanometers and a trough at 602 nanometers, and that of species II_M by an α band split into a prominent peak at 607 nanometers and a small side peak at 594 nanometers. Evidence is presented to suggest that these two bands arise from O₂⁻ → Cu_B²⁺ and O₂⁻ → a₃²⁺ charge transfer transitions which would imply that O₂⁻ forms a bridging ligand between Cu_B and the iron atom of cytochrome a₃ in species II_M. The kinetics of the reaction and the spectral characteristics of species I_M and II_M obtained with the potato mitochondrial system are compared and contrasted with data in the literature on the beef heart mitochondrial system.     

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1 I. Spectral properties and redox potentials

1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl₁ which lacks cytochrome aa₃. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied.2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the α peaks of b-type cytochrome(s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two α peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochrome c₁. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl₁ in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa₃ was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm.3. Cytochrome aa₃ was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl₁. Cytochrome aa₃ was more susceptible to heat than cytochromes b and c,c₁.4. CO difference spectra at 77 K revealed two different Co-cytochrome complexes. The first, found only in wild type mitochondria, was a typical CO-cytochrome a₃ complex characterized by peaks at 596 and 435 nm and troughs at 613 and 450 nm. The second, found both in mitochondria of the wild type and the mutant, was a CO-cytochrome b complex with peaks at 567, 539 and 420 nm and a trough at 558-549 nm. Both complexes are photo-dissociable.5. Spectral evidence was obtained for interaction of cyanide with the a-type cytochrome (shift of the α peak at 77 K from 608 to 605 nm), but not with the b-type cytochrome.6. The mid-point potentials of the different cytochromes at neutral pH are as follows: cytochrome aa₃ 235 and 395 mV, cytochrome c,c₁ 233 mV, cytochromes b 120 mV.     

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.