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.     

Azide- and cyanide-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption, EPR and FTIR spectroscopies.

Cytochrome bd-type ubiquinol oxidase contains two hemes b (b(558) and b(595)) and one heme d as the redox metal centers. To clarify the structure of the reaction center, we analyzed Escherichia coli cytochrome bd by visible absorption, EPR and FTIR spectroscopies using azide and cyanide as monitoring probes for the exogenous ligand binding site. Azide-binding caused the appearance of a new EPR low-spin signal characteristic of ferric iron-chlorin-azide species and a new visible absorption band at 647 nm. However, the bound azide ((14)N(3)) anti-symmetric stretching infrared band (2, 010.5 cm(-1)) showed anomalies upon (15)N-substitutions, indicating interactions with surrounding protein residues or heme b(595) in close proximity. The spectral changes upon cyanide-binding in the visible region were typical of those observed for ferric iron-chlorin species with diol substituents in macrocycles. However, we found no indication of a low-spin EPR signal corresponding to the ferric iron-chlorin-cyanide complexes. Instead, derivative-shaped signals at g = 3.19 and g = 7.15, which could arise from the heme d(Fe(3+))-CN-heme b(595)(Fe(3+)) moiety, were observed. Further, after the addition of cyanide, a part of ferric heme d showed the rhombic high-spin signal that coexisted with the g(z) = 2.85 signal ascribed to the minor heme b(595)-CN species. This indicates strong steric hindrance of cyanide-binding to ferric heme d with the bound cyanide at ferric heme b(595).     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate.

Electron paramagnetic resonance (EPR) and optical spectra are used as probes of the heme and its ligands in ferric and ferrous leghemoglobin. The proximal ligand to the heme iron atom of ferric soybean leghemoglobin is identified as imidazole by comparison of the EPR of leghemoglobin hydroxide, azide, and cyanide with the corresponding derivatives of human hemoglobin. Optical spectra show that ferric soybean leghemoglobin near room temperature is almost entirely in the high spin state. At 77 K the optical spectrum is that of a low spin compound, while at 1.6 K the EPR is that of a low spin form resembling bis-imidazole heme. Acetate binds to ferric leghemoglobin to form a high spin complex as judged from the optical spectrum. The EPR of this complex is that of high spin ferric heme in a nearly axial environment. The complexes of ferrous leghemoglobin with substituted pyridines exhibit optical absorption maxima near 685 nm, whose absorption maxima and extinctions are strongly dependent on the nature of the substitutents of the pyridine ring; electron withdrawing groups on the pyridine ring shift the absorption maxima to lower energy. A crystal field analysis of the EPR of nicotinate derivatives of ferric leghemoblobin demonstrates that the pyridine nitrogen is also bound to the heme iron in the ferric state. These findings lead us to picture leghemoglobin as a somewhat flexible molecule in which the transition region between the E and F helices may act as a hinge, opening a small amount at higher temperature to a stable configuration in which the protein is high spin and can accommodate exogenous ligand molecules and closing at low temperature to a second stable configuration in which the protein is low spin and in which close approach of the E helix permits the distal histidine to become the principal sixth ligand.     

The active form of the ferric heme in neutrophil cytochrome b(558) is low-spin in the reconstituted cell-free system in the presence of amphophil.

The spin state of the heme in superoxide (O(2)(.)(-))-producing cytochrome b(558) purified from pig neutrophils was examined by means of room-temperature magnetic circular dichroism (MCD) under physiological conditions. Cytochrome b(558) with varying amounts of low-spin and high-spin heme was prepared by either pH adjustment or heat treatment, and the O(2)(.)(-)-forming activity in a cell-free system was found to correlate with the low-spin heme content. The possibility that the O(2)(.)(-)-forming activity results from a transient high-spin ferric heme form that is induced during activation by anionic amphophils has also been investigated. EPR spectra of cytochrome b(558) activated by either arachidonic acid or myristic acid, showed that a transient high-spin ferric species accounting for approximately 50% of the heme appeared in the presence of arachidonic acid, but not in the presence of myristic acid. Hence the appearance of a transient high-spin ferric heme species on activation with an amphophil does not afford a common activation mechanism in the NADPH oxidase system. The EPR results for cytochrome b(558) activated with arachidonic acid showed that the transient high-spin ferric heme can bind cyanide. However, the high-spin ferric heme does not contribute to the O(2)(.)(-) production of cytochrome b(558) in cell-free assays in the presence of cyanide.     

Novel heme ligation in a c-type cytochrome involved in thiosulfate oxidation: EPR and MCD of SoxAX from Rhodovulum sulfidophilum

The SoxAX complex of the bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that plays an essential role in photosynthetic thiosulfate and sulfide oxidation. The three heme sites of SoxAX have been analyzed using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism spectroscopies. Heme-3 in the ferric state is characterized by a Large g(max) EPR signal and has histidine and methionine axial heme iron ligands which are retained on reduction to the ferrous state. Hemes-1 and -2 both have thiolate plus nitrogenous ligand sets in the ferric state and give rise to rhombic EPR spectra. Heme-1, whose ligands derive from cysteinate and histidine residues, remains ferric in the presence of dithionite ion. Ferric heme-2 exists with a preparation-dependent mixture of two different ligand sets, one being cysteinate/histidine, the other an unidentified pair with a weaker crystal-field strength. Upon reduction of the SoxAX complex with dithionite, a change occurs in the ligands of heme-2 in which the thiolate is either protonated or replaced by an unidentified ligand. Sequence analysis places the histidine/methionine-coordinated heme in SoxX and the thiolate-liganded hemes in SoxA. SoxAX is the first naturally occurring c-type cytochrome in which a thiolate-coordinated heme has been identified.     

Anion binding to resting and half-reduced Pseudomonas cytochrome c peroxidase

The anion-binding characteristics of resting and half-reduced Pseudomonas cytochrome c peroxidase (ferrocytochrome c-551: hydrogen peroxide oxidoreductase, EC 1.11.1.5) have been examined by EPR and optical spectroscopy with cyanide, azide and fluoride as ligands. The resting enzyme was found to be essentially inaccessible for ligation, which indicates that it has a closed conformation. In contrast, the half-reduced enzyme has a conformation in which the low-potential heme is easily accessible for ligands, a behavior parallel to that towards the substrate hydrogen peroxide (R?nnberg, M., Araiso, T., Ellfolk, N. and Dunford, H.B. (1981) Arch. Biochem. Biophys. 207, 197-204). Cyanide and azide caused distinct changes in the low-potential heme c moiety, and the gz values of the two low-spin derivatives were 3.14 and 3.22, respectively. Fluoride binds to the same heme, giving rise to a high-spin signal at g = 6. The dissociation constants of the anions differ widely from each other, the values for the cyanide, azide and fluoride being 23 microM, 2.5 mM and 0.13 M, respectively. In addition, a partial shift of the low-spin peak at g = 2.84 of the half-reduced species to 3.24 was observed even at low concentrations of fluoride.     

Spectroscopic, ligand binding, and enzymatic properties of the spleen green hemeprotein. A comparison with myeloperoxidase

The bovine spleen green hemeprotein, a peroxidase which exhibits spectrophotometric properties similar to those of granulocyte myeloperoxidase, was purified using an improved method. The ligand affinity of the ferric enzyme was spectroscopically determined using chloride and cyanide as exogenous ligands. The pH dependence of the apparent dissociation constant of the enzyme-chloride complex showed the presence of a proton dissociable group with a pKa value of 4 on the enzyme; chloride binds to the enzyme when this group is protonated with a dissociation constant of 60 microM. The cyanide affinity of the enzyme is also regulated by the group with a pKa value of 4, but in this case cyanide binds to the unprotonated enzyme with a dissociation constant of 0.6 microM; only the protonated, uncharged form of cyanide reacts with the enzyme. Cyanide binding was competitively inhibited by chloride, and chloride binding was also competitively inhibited by cyanide. The EPR spectrum of the resting enzyme exhibited a rhombic high spin signal at g = 6.65, 5.28, and 1.97 with a low spin signal at g = 2.55, 2.32, and 1.82. Upon formation of the chloride complex, the spectrum was replaced with a new high spin EPR signal with g-values of 6.81, 5.04, and 1.95. The cyanide complex showed a low spin EPR signal with g-values of 2.83, 2.25, and 1.66. Examination of the enzymatic activity of the spleen green hemeprotein by following the chlorination of monochlorodimedon has indicated that the enzyme has the same chlorinating activity as myeloperoxidase; the spleen green peroxidase can catalyze the formation of hypochlorous acid from hydrogen peroxide and chloride ion. Comparison of the present data with those of myeloperoxidase has led to the conclusion that the structure of the iron center and its vicinity in spleen green hemeprotein is very similar, if not identical, to that of myeloperoxidase. The spleen enzyme can thus be used as a model to study the active center, and its environment, in myeloperoxidase.     

Characterization of the heme in human cystathionine beta-synthase by X-ray absorption and electron paramagnetic resonance spectroscopies

Human cystathionine beta-synthase is one of two key enzymes involved in intracellular metabolism of homocysteine. It catalyzes a beta-replacement reaction in which the thiolate of homocysteine replaces the hydroxyl group of serine to give the product, cystathionine. The enzyme is unusual in its dependence on two cofactors: pyridoxal phosphate and heme. The requirement for pyridoxal phosphate is expected on the basis of the nature of the condensation reaction that is catalyzed; however the function of the heme in this protein is unknown. We have examined the spectroscopic properties of the heme in order to assign the axial ligands provided by the protein. The heme Soret peak of ferric cystathionine beta-synthase is at 428 nm and shifts to approximately 395 nm upon addition of the thiol chelator, mercuric chloride. This is indicative of 6-coordinate low-spin heme converting to a 5-coordinate high-spin heme. The enzyme as isolated exhibits a rhombic EPR signal with g values of 2.5, 2.3, and 1.86, which are similar to those of heme proteins and model complexes with imidazole/thiolate ligands. Mercuric chloride treatment of the enzyme results in conversion of the rhombic EPR signal to a g = 6 signal, consistent with formation of the high-spin ferric heme. The X-ray absorption data reveal that iron in ferric cystathionine beta-synthase is 6-coordinate, with 1 high-Z scatterer and 5 low-Z scatterers. This is consistent with the presence of 5 nitrogens and 1 sulfur ligand. Together, these data support assignment of the axial ligands as cysteinate and imidazole in ferric cystathionine beta-synthase.     

Imidazole, the ligand trans to mercaptide in ferric cytochrome P-450. An EPR study of proteins and model compounds.

A crystal field analysis of EPR data for various low spin ferric cytochromes P-450 suggests that in all of them, regardless of source or method of induction, the heme ligands are a sulfur atom, presumably from cysteine, and an imidazole from histidine. The imidazole can be displaced in the ferric protein by cyanide, guanidine, or by an amine, analogous to its displacement by CO or NO in the ferrous protein. The resulting changes in the EPR parameters for the ferric protein are consistent with similar substitutions in heme thiol model compounds. The analysis of the latter can be understood on the basis of alterations of the electronic structure of the ligands to the heme iron.     

Proton NMR spectroscopy of Alcaligenes cytochrome c556

A high molecular-weight c-type cytochrome was purified from Alcaligenes faecalis ATCC 8750. Its weight was 40,000 daltons by sodium dodecyl sulfate-gel electrophoresis. Heme content was determined to be one heme per 40,000 daltons. Proton nuclear magnetic resonance-NMR-spectroscopy determined that the ferrous form is low spin. The detection of a methyl resonance at -3 ppm in the ferrous form indicated that methionine is a heme ligand in this state. The NMR spectrum of the ferric form at pH 7.2 revealed hyperfine shifted methyl resonances at 67.79, 63.17, 57.71, and 50.46 ppm. The large downfield shifts observed are indicative of high spin character. The ferric spectrum was pH-sensitive, indicating two pH-linked structural transitions with estimated pKs at 6.0 and 10.5. The first is interpreted as due to the ionization of a heme propionate. The second is interpreted as the acquisition of a strong field ligand and the subsequent conversion to a low spin ferric form. The ferricytochrome did not form complexes with cyanide, azide, or fluoride at pH 5.2 or 7.9.     

Spectroscopic Properties of Siroheme Extracted from Sulfite Reductases

Siroheme has been extracted from sulfite reductases and its properties in aqueous solution have been investigated by optical absorption, electron paramagnetic resonance (EPR), and magnetic circular dichroism (MDC) spectroscopy. The absorption spectrum of siroheme exhibits a marked pH dependence, and two pK values, 4.2 and 9.0, were determined by pH titration in the range 2–12. The first pK (4.2) is thought to correspond to the ionization of the carboxylic acid side-chains on the tetrapyrrole rings, and the second pK (9.0) is attributed to displacement of the axial ligand chloride by hydroxide. The binding of the strong field ligands, CO, NO, and cyanide, were investigated by UV-visible absorption and, in the case of the cyanide complex, by low-temperature EPR and MCD spectroscopies. CO and NO were able to reduce and bind to siroheme without additional reducing agent. The EPR spectrum of the isolated siroheme (chloride-ferrisiroheme) exhibits an axial signal with g_XXX = 6.0 and ^g= 2.0, typical of high-spin ferric hemes (S = 5/2), whereas the cyanide-complexed siroheme exhibits an approximately axial signal with g_XXX = 2.38 and _g = 1.76 that is indicative of a low-spin ferric heme (S = 1/2). The low-temperature MCD spectra and magnetization data for the as-isolated and cyanide-complexed ferrisiroheme are entirely consistent with the interpretation of the EPR spectra. The results for ferrosiroheme indicate that the siroheme remains high spin (S = 2) and low spin (S = 0) on reduction of the as-isolated and cyanide-complexed siroheme, respectively. The isolated siroheme expressed sulfite reductase activity but the assessable catalytic cycle was much less than that of the native enzyme, showing the importance of the protein environment.     

Perturbations at the high spin heme b center in the membrane-bound nitric oxide reductase

The effects of lowering pH from 7 to 5 on the absorption, circular dichroism (MCD) and EPR spectra were studied for Paracoccus halodenitrificans nitric oxide reductase (NOR). Intensities of the characteristic bands for the high spin heme b, that at 592 nm in the absorption spectrum and those at 591 (+) and 606 (-) in the MCD spectrum decreased considerably. Concomitant cryogenic EPR spectrum indicated a drastic increase in the signal intensity due to the high spin heme b at g approximately 6, of which less than 5% had been EPR detectable at pH 7. Cyanide (x40) bound to the high spin heme b center in the reduced NOR irrespective of pH, while a much larger amount of azide (x1000) was necessary to bind to the reduced NOR at an acidic pH, ca. 5. Based on these results the structure and function of the high spin heme b center as the active site of NOR was discussed.     

Axial ligand coordination in intestinal peroxidase

EPR spectra of intestinal peroxidase are reported for the first time. The resting state of intestinal peroxidase exhibits only a high spin EPR spectrum with pH-dependent rhombicity. Addition of chloride shifts the equilibrium between an acidic and a neutral form of the enzyme. In contrast, resting lactoperoxidase shows EPR spectra of both low spin and high spin species, indicating a different heme environment between these two peroxidases. The high spin signal of lactoperoxidase consists of multiple components; the major component exhibits pH-dependent rhombicity similar to intestinal peroxidase and the equilibrium between the acidic and the neutral forms is also shifted by chloride ion. EPR features of the low spin cyanide complex of intestinal peroxidase and lactoperoxidase are compared with those of other hemeproteins, whose proximal axial ligands are known to be histidine residues. The g-values of the cyanide adducts of the mammalian peroxidases are similar. The relationship between the g-value anisotropy and imidazolate character of the proximal histidine is discussed.     

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

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

Hemoglobins of the Lucina pectinata/bacteria symbiosis. II. An electron paramagnetic resonance and optical spectral study of the ferric proteins

We report an optical and EPR spectral study of three hemoglobins, Hb I, II, and III, from the gill of the clam Lucina pectinata. Hemoglobin I reacts much more avidly with hydrogen sulfide than do Hbs II and III. The proximal ligand to the heme iron of each hemoglobin is histidyl imidazole. The acid/alkaline transition of ferric Hb I occurs with pK 9.6; those of ferric Hbs II and III with pK 6.6 and 5.9, respectively. At their acid limits each ferric hemoglobin exists as aquoferric hemoglobin. Broadening of the g = 6 resonance suggests that the bound water enjoys great positional freedom. Ferric Hb I, at the alkaline limit (pH 11), exists as ferric hemoglobin hydroxide. Ferric Hbs II and III, at their alkaline limit (pH 7.5), each exist as equal mixtures of two species. The low spin species with optical maxima near 541 and 576 nm and g values of 2.61, 2.20, and 1.82, are identified as ferric hemoglobin hydroxide. The high spin species, with optical maxima near 486 and 603 nm and g values of 6.71, 5.87, and 5.06, resemble Dicrocoelium hemoglobin and hemoglobin MSaskatoon. Here we show that Hbs II and III resemble hemoglobin MSaskatoon in which a distal tyrosinate oxygen ligated to the ferric heme iron at alkaline pH is displaced by water at acid pH. The H2S product of ferric Hb I is identified as ferric hemoglobin sulfide.     

Ferric haem forms of Mycobacterium tuberculosis catalase-peroxidase probed by EPR spectroscopy: Their stability and interplay with pH

Low temperature EPR spectroscopy was used to characterise Mycobacterium tuberculosis catalase-peroxidase in its resting ferric haem state. Several high spin ferric haem forms and no low spin forms were found in the enzyme samples frozen in methanol on dry ice. The EPR spectra depended not only on the pH but also on the buffer type. As a general trend, the higher the pH, the greater the ‘rhombic’ fraction of the high spin ferric haem that was observed. The rhombic form was characterised by well separated two lines in the g = 6 region whereas in the ‘axial’ form the two lines overlap. This pH dependence of the equilibrium of axial and rhombic ferric haem forms is also seen in rapidly freeze-quenched samples. Different high spin ferric haem forms were monitored during a 3 week storage of the enzyme at 4 °C. For some forms, extremal dependences, i.e. those progressing via maxima or minima over storage time, were found. This indicates that the mechanism of the time-dependent transition from one high spin ferric haem form to another must be more complex than a simple single site oxidation.     

Electron paramagnetic resonance spectroscopy of thyroid peroxidase

Thyroid peroxidase was isolated from porcine thyroids by two methods. Limited trypsin proteolysis was employed to obtain a cleaved enzyme, and affinity chromatography was used to isolate intact thyroid peroxidase. Enzyme isolated by both methods was used in the examination of the heme site of native thyroid peroxidase and its complexes by EPR spectroscopy. Intact thyroid peroxidase showed a homogeneous high-spin EPR signal with axial symmetry, in contrast to the rhombic EPR signal of native lactoperoxidase. Reaction of cyanide or azide ion with native thyroid peroxidase resulted in the loss of the axial EPR signal within several hours. The EPR spectroscopy of the nitrosyl adduct of ferrous thyroid peroxidase exhibited a three-line hyperfine splitting pattern and indicated that the heme-ligand structure of thyroid peroxidase is significantly different from that of lactoperoxidase.     

An EPR study of myeloperoxidase in human granulocytes.

1. EPR spectra of human granulocytes (4 - 10(8) cells per ml) show an intense high-spin ferric heme signal with rhombic symmetry (gx = 6.90 and gy = 5.07) for the heme group. These g-values are identical to those of partially purified myeloperoxidase and thus the signal is derived from ferric myeloperoxidase. In chicken granulocytes, which contain little or no myeloperoxidase, only an axial type of heme iron signal, weak in intensity, can be detected at g = 6.0. 2. Upon phagocytosis of latex particles by human granulocytes the high-spin heme signal with rhombic symmetry is slowly converted into a signal with axial symmetry (gx = gy = 6.0), showing that the EPR signals of myeloperoxidase in the intact cell can be used to study the involvement of the enzyme in metabolic changes during phagocytosis.     

Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives.

Magnetic circular dichroism (MCD) spectra have been recorded for beef heart cytochrome oxidase and a number of its inhibitor complexes. The resting enzyme exhibits a derivate shape Faraday C term in the Soret region, characteristic of low spin ferric heme, which accounts for 50% of the total oxidase heme a. The remaining heme a (50%) is assigned to the high spin state. MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. Furthermore, the ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a33+. The Soret MCD of the reduced protein is interpreted as th sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a3(2+) and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a2+. Temperature studies of the Soret MCD intensity support this proposed spin state heterogeneity. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome a3(2+); however, the band associated with cytochrome a2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. This conclusion is used in conjunction with the fact that ferric, high spin heme exhibits weak MCD intensity to calculate the MCD spectra for the individual cytochromes of the oxidase as well as the spectra for some inhibitor complexes of cytochrome a3. The results are most simply interpreted using the model we have recently proposed to account for the electronic and magnetic properties of cytochrome (Palmer, G., Babcock, F.T., and Vcikery, L.E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210).