Optical properties and a new epr signal from type 3 copper in metal-depleted Rhus vernicifera laccase

Due to conflicting reports on the properties of Rhus laccase depleted in type 2 copper a further investigation of this protein derivative has been undertaken. In contrast to most other reports it is shown that the type 3 copper site retains its absorbance at 330 nm when type 2 copper is removed. The type 3 copper ions are oxidized in the resting protein and part of the type 3 Cu(II) can be made electron paramagnetic resonance (epr) detectable on reduction by ascorbate. This new epr signal is highly rhombic and the epr parameters are comparable to those found in other metalloproteins containing Cu(II) in binuclear sites. Certain preparations of type 2 deficient protein exhibit lower extinction coefficients at 330 nm. Since these protein derivatives have lost some type 3 copper, it is inferred that the absorbance at 330 nm is dependent on a native type 3 copper site. Also in contrast to other reports, it is found that the extinction coefficient at 614 nm of the type 1 Cu(II) decreases from 5700 to 4700 M^?1cm^?1 when type 2 copper is removed. The oxidized-reduced difference spectrum also shows a substantial decrease in the absorbance between 700 and 800 nm. The changes in absorbance above 600 nm are probably due to a modification of the type 1 Cu(II) site on removal of type 2 copper. The present results also suggest some explanations to the apparent discrepancies among the earlier reports.     

An unexpected formation of the spectroscopic Cu(I)-semiquinone radical by xenon-induced self-catalysis of a copper quinoprotein

Plant copper/quinone amine oxidases are homodimeric enzymes containing Cu(II) and a quinone derivative of a tyrosyl residue (2,4,5-trihydroxyphenylalanine, TPQ) as cofactors. These enzymes catalyze the oxidative deamination of primary amines by a classical ping-pong mechanism, i.e. two distinct half-reactions, enzyme reduction by substrate followed by its re-oxidation by molecular oxygen. In the first half-reaction two forms of the reduced TPQ have been observed, the colorless Cu(II)-aminoquinol and the yellow Cu(I)-semiquinolamine radical so that this enzyme may be referred to as a "protein-radical enzyme". The interaction of xenon, in aqueous solutions, with the copper/TPQ amine oxidase from lentil (Lens esculenta) seedlings has been investigated by NMR and optical spectroscopy. NMR data indicate that xenon binds to the protein. Under 10 atm gaseous xenon and in the absence of substrates more than 60% native enzyme is converted into Cu(I)-semiquinolamine radical species, showing for the first time that both monomers in the dimer can generate the radical. Under the same experimental conditions the copper-free lentil enzyme is able to generate an intermediate absorbing at about 360 nm, which is assigned to the product Schiff base quinolaldimine which, to the best of our knowledge, has never been observed during the catalytic mechanism of plant amine oxidases. A possible role of the lysine residue responsible for the formation of Cu(I)-semiquinolamine and quinolaldimine, is proposed.     

Preparation and characterization of met apo hemocyanin: a single copper (II) active site.

A new derivative of $\text{Busycon canaliculatum}$ hemocyanin has been prepared for which one copper has been removed from the binuclear active site of the holoprotein and the remaining copper has been oxidized with a variety of small molecule oxidizing agents. This met apo derivative [( )…Cu(II)] binds a number of ligands; EPR spectra of several forms are reported and compared to those obtained for a singly oxidized (half met-L) derivative [Cu(I)…Cu(II)L]. The site of the oxidized copper for both forms is found to be quite similar in structure but shows large differences in ligand binding ability.     

Interaction of nitric oxide with ceruloplasmin lacking an EPR-detectable type 2 copper.

Nitric oxide (NO) has previously been reported to modify the EPR spectrum of multicopper blue oxidases, disclosing a pure type 2 copper and inducing half-field transitions at g = 4. In the present work the reactivity of NO was reinvestigated with respect to ceruloplasmins having an apparently EPR-silent type 2 copper in their native state. The optical properties of NO-treated ceruloplasmin were independent of the initial redox state of the metal sites. Addition of NO caused the absorption at 600 nm to decrease in the case of oxidized ceruloplasmin and to increase when starting from the reduced proteins. In this latter case the absorbance at 330 nm was also restored, indicating that NO was able to reoxidize the reduced protein. In all cases the band at 600 nm leveled to ca. 60% of the intensity of the native untreated protein, and new bands below 500 nm appeared in the spectra. While the blue absorption band was restored by removal of NO, the absorbance below 500 nm remained higher even after dialysis. The EPR spectrum resulting from reaction of NO with either oxidized, partially reduced, or fully reduced ceruloplasmin consisted in all cases of a broad, structureless resonance around g = 2. NO caused the reversible disappearance of the type 1 copper EPR spectrum in oxidized ceruloplasmin. Also, the transient novel copper signal that arises during the anaerobic reduction process by ascorbate completely disappeared in the presence of NO and did not reappear upon removal of the gas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mavicyanin, a blue copper protein from Cucurbita pepo medullosa. Purification and characterization.

1. The copper protein mavicyanin has been isolated and purified from the green squash Cucurbita pepo medullosa. 2. Mavicyanin contains one type-1 copper/18000 Mr, which can be characterized by: intense absorption maximum at 600 nm (epsilon = 5000 M-1 cm-1/Cu, A280/A600 = 8.0 +/- 0.5, A600/A403 = 7.0 +/- 0.25, maximum of fluorescence emission at 335 nM. 3. In the oxidized state the copper of mavicyanin is 100% detectable by electron paramagnetic resonance (EPR). Computer simulation of the rhombic EPR signal gives gz = 2.287, gy = 2.077, gx = 2.025, Az = 3.5 mT, Ay = 2.9 mT and Ax = 5.7 mT. 4. Like other simple type-1 copper proteins, such as stellacyanin, azurin or plastocyanin, mavicyanin is readily reduced by hydroquinone or L-ascorbic acid. Its midpoint potential E'm was determined to be + 285 mV. The reduced protein reacts rather slowly with dioxygen, but is rapidly reoxidized by ferricyanide.     

Stellacyanin. Studies of the metal-binding site using x-ray absorption spectroscopy.

Stellacyanin is a mucoprotein of molecular weight approximately 20,000 containing one copper atom in a blue or type I site. The metal ion can exist in both the Cu(II) and Cu(I) redox states. The metal binding site in plastocyanin, another blue copper protein, contains one cysteinyl, one methionyl, and two imidazoyl residues (Colman et al. 1978. Nature [Lond.]. 272:319-324.), but an exactly analogous site cannot exist in stellacyanin as it lacks methionine. The copper coordination in stellacyanin has been studied by x-ray edge absorption and extended x-ray absorption fine structure (EXAFS) analysis. A new, very conservative data analysis procedure has been introduced, which suggests that the there are two nitrogen atoms in the first coordination shell of the oxidized [Cu(II)] protein and one in the reduced [Cu(I)] protein; these N atoms have normal Cu--N distances: 1.95-2.05 A. In both redox states there are either one or two sulfur atoms coordinating the copper, the exact number being indeterminable from the present data. In the oxidized state the Cu--S distance is intermediate between the short bond found in plastocyanin and those found in near tetragonal copper model compounds. Above -140 degree C, radiation damage of the protein occurs. At room temperature the oxidized proteins is modified in the x-ray beam at a rate of 0.25%/s.     

NMR evidence for perturbation of the copper coordination sphere upon chemical modification of arginine 141 in bovine Cu,Zn superoxide dismutase.

The reaction of the Cu,Co derivative of bovine Cu,Zn superoxide dismutase with phenylglyoxal or butanedione, which are known to inactivate the enzyme by selectively binding to Arg 141, has been studied by 1H NMR. Several 1H NMR lines of the copper-liganding histidine residues were perturbed, reproducing an effect so far observed only in the case of binding of anions to this protein. The room temperature EPR spectrum of the modified Cu,Zn protein was altered very slightly, indicating that the geometry of the copper site was not grossly affected by the modification. NMR and EPR changes were reversed by dialysis in the case of the reversible butanedione adduct. These data show that the coordination of the copper in Cu,Zn superoxide dismutase can be destabilized by modifications occurring at a neighboring but not a metal-liganding residue. It is suggested that part of the NMR effects seen on copper ligands in the case of anion binding are produced by interaction of anions with Arg 141, rather than by direct ligand replacement.     

Imidazolate bridged heterobinuclear complexes of zinc(II)tetraphenylporphyrin. Modeling cytochrome c oxidase

The reaction of a copper(II) or nickel(II) imidazolate complex (M[CBP-PHEN-4-CHO-Im]) with zinc(II)tetraphenylporphyrin (TPP) in toluene results in the formation of an imidazolate bridged heterobinuclear axial adduct. Conversion of the four-coordinated Zn(TPP) to the five-coordinated species is followed in the visible region between 700 and 500 nm. Isosbestic behavior is exhibited at 523, 556, 588, and 638 nm by solutions of Zn(TPP) to which varying amounts of the metal imidazolate complex are added, indicating the existence of an equilibrium between Zn(TPP) and its axial adduct. The products exhibit maxima beta and alpha bands at 566 and 606 nm, respectively, which are red-shifted from 548 and 588 nm for Zn(TPP) and yield epsilon alpha/epsilon beta ratios of 0.57 and 0.55 for the Ni(II) and Cu(II) adducts, respectively. The binding of the metal imidazolate complexes is thought to closely resemble that of N-methylimidazole, N-CH3Im, rather than imidazolate, owing to the close spectral similarities with the adduct of the former and significant differences from the latter. Formation constants were determined using the 548-nm beta band of Zn(TPP) in the 293-308 K range by the method of Rose and Drago. At 25 degrees C, K = 152,000 M-1 and 110,000 M-1 for the copper and nickel adducts, respectively. Comparison of these values to that of 54,100 M-1 for N-CH3Im indicates that the metal-imidazolate complexes are considerably more reactive. Van't Hoff plots for the two series are very similar with enthalpies of -41.9 and -43.3 kJ/mole respectively. The structural core of these complexes is similar to the imidazolate bridged model of cytochrome c oxidase in that they contain a metal imidazolate axially adducted to a metalloporphyrin.     

An NMR view of the unfolding process of rusticyanin: Structural elements that maintain the architecture of a beta-barrel metalloprotein

The unfolding process of the blue copper protein rusticyanin (Rc) as well as its dynamic and D(2)O/H(2)O exchange properties in an incipient unfolded state have been studied by heteronuclear NMR spectroscopy. Titrations of apo, Cu(I), and Cu(II)Rc with guanidinium chloride (GdmCl) show that the copper ion stabilizes the folded species and remains bound in the completely unfolded state. The oxidized state of the copper ion is more efficient than the reduced form in this respect. The long loop of Rc (where the first ligand of the copper ion is located) is one of the most mobile domains of the protein. This region has no defined secondary structure elements and is prone to exchange its amide protons. In contrast, the last loop (including a short alpha-helix) and the last beta-strand (where the other three ligands of the metal ion are located) form the most rigid domain of the protein. The results taken as a whole suggest that the first ligand detaches from the metal ion when the protein unfolds, while the other three ligands remain bound to it. The implications of these findings for the biological folding process of Rc are also discussed.     

Cytochrome aa3 from the aerobic photoheterotroph Erythrobacter longus: purification, and enzymatic and molecular features

Cytochrome c oxidase (cytochrome aa3-type) [EC 1.9.3.1] was purified from Erythrobacter longus to homogeneity as judged by polyacrylamide gel electrophoresis, and some of its properties were studied. The spectral properties of the oxidase closely resembled those of mitochondrial and other bacterial cytochromes aa3. The enzyme showed absorption peaks at 430 and 598 nm in the oxidized form, and at 444 and 603 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 432 and 600 nm. The enzyme oxidized eukaryotic ferrocytochromes C more rapidly than E. longus ferrocytochrome c. The reactions catalyzed by the enzyme were 50% inhibited by 0.7 microM KCN. The enzyme contained 1 g atom of copper and 1 g atom of magnesium per mol of heme a. The enzyme molecule seemed to be composed of two identical subunits, each with a molecular weight of 43,000.     

Comparative Analysis on the Effect of <Emphasis Type="Italic">Plantago</Emphasis> Species Aqueous Extracts on Tissue Trace Element Content in Rats

The interaction between iron and copper has been discussed in association with human health and diseases for many years. Ceruloplasmin, a multi-copper oxidase, is mainly involved in iron metabolism and its genetic defect, aceruloplasminemia (ACP), shows neurological disorders and diabetes associated with excessive iron accumulation, but little is known about the state of copper in the brain. Here, we investigated localization of these metals in the brains of three patients with ACP using electron microscopes equipped with an energy-dispersive x-ray analyzer. Histochemically, iron deposition was observed mainly in the basal ganglia and dentate nucleus, and to lesser degree in the cerebral cortex of the patients, whereas copper grains were not detected. X-ray microanalysis identified two types of iron-rich particles in their brains: dense bodies, namely hemosiderins, and their aggregated inclusions. A small number of hemosiderins and most inclusions contained a significant amount of copper which was enough for distinct Cu x-ray images. These copper-containing particles were observed more frequently in the putamen and dentate nucleus than the cerebral cortex. Coexistence of iron and copper was supported by good correlations in the molecular ratios between these two metals in iron-rich particles with Cu x-ray image. Iron-dependent copper accumulation in iron-rich particles may suggest that copper recycling is enhanced to meet the increased requirement of cuproproteins in iron overload brain. In conclusion, the iron-rich particles with Cu x-ray image were found in the ACP brain.     

Role of tryptophan in the spectral and catalytic properties of the copper enzyme, galactose oxidase.

Previous results indicate that a tryptophan residue(s) may interact with the sugar substrate and Cu(II) atom of galactose oxidase (Ettinger, M. J., and Kosman, D. J. (1974), Biochemistry 13, 1248). We now show that N-bromosuccinimide (NBS) reduces enzymatic activity to 2% as two tryptophans are oxidized; only four residues are easily oxidized in the holoenzyme. An enzymatic activity vs. number of residues oxidized profile suggests that this inactivation is probably associated with only one of the first 2 residues oxidized. There is no evidence for chain cleavage or modification of amino acids other than tryptophan. While substrate protection is not afforded by the sugar substrate, the activity-related tryptophan is placed within the active-site locus by spectral evidence. NBS oxidation of two tryptophans results in a marked diminution of the large copper optical-activity transition at 314 nm. Under some reaction conditions, a doubling of ellipticity in the 600-nm region of copper CD is also observed. The effects of the NBS oxidation on the CD spectra of galactose oxidase permit the assignment of the 314-nm CD band to a charge-transfer transition and the 229-nm extremum to a specific tryptophan contribution. The AZZ parameter from electron spin resonance spectra is also markedly reduced by the NBS oxidation. Moreover, while cyanide binds to the native enzyme without reducing the Cu(II) atom, cyanide rapidly reduces the Cu(II) atom to Cu(I) in the NBS-oxidized enzyme. These CD and ESR results are taken to suggest that one aspect of the inactivation by NBS oxidation may be a conversion of the pseudosquare planar copper complex in the native enzyme to a more distorted, towards tetrahedral, complex in the inactivated enzyme. Since the inactivation can be accomplished without affecting binding of the sugar substrate, tryptophan oxidation must affect catalysis per se.     

Investigation of the active site of Escherichia coli Cu,Zn superoxide dismutase reveals the absence of the copper-coordinated water molecule. is the water molecule really necessary for the enzymatic mechanism?

The active site of the Cu,Zn superoxide dismutase from Escherichia coli in the oxidized Cu(II) state has been studied by nuclear magnetic relaxation dispersion (NMRD), optical and nuclear magnetic resonance spectroscopy. The orientation of some metal ligands is different with respect to all the other Cu,Zn superoxide dismutases. Moreover, NMRD measurements demonstrate the lack of a copper-coordinated water molecule. In spite of these differences the enzymatic activity is still high. Azide also binds copper with normal affinity and induces modifications in the active site comparable to those previously observed in the eukaryotic enzymes. Our results suggest that, in this enzyme, the copper-coordinated water molecule appears not necessary for the enzymatic reaction. A role for the copper-coordinated water molecule is discussed in the light of recent crystallographic studies.     

Optical properties of japanese-lacquer-tree (Rhus vernicifera) laccase depleted of type 2 copper(II). Involvement of type-2 copper(II) in the 330nm chromophore.

1. Spectroscopic and functional properties of Japanese-lacquer-tree (Rhus vernicifera) laccase were re-investigated, with special emphasis on the relationships between the different types of copper centres (Types 1, 2, and 3). 2. On removal of the Type 2 Cu(II), a decrease of absorbance occurred in the wavelength region above 650 nm (delta epsilon 750 = 300 M-1 . cm-1) and around 330 nm (delta episom 330 up to 2200 M-1 . cm-1). 3. Reductive titrations with ascorbic acid or ferrocyanide showed that the electron-accepting capacity of the partial apoprotein is one electron-equivalent lower than that of the native protein, i.e. the protein two-electron acceptor is present in the oxidized state in spite of absorbance loss at 330 nm. 4. The 330 nm chromophore apparently depends on the presence of both the Type 2 and the Type 3 copper in the oxidized state. 5. This finding may have implications in the relative location of Type 2 and 3 copper centres and on the redox behaviour of laccase.     

Crystallographic structures of bovine copper-zinc superoxide dismutase reveal asymmetry in two subunits: functionally important three and five coordinate copper sites captured in the same crystal

A key feature of the generally accepted catalytic mechanism of CuZn superoxide dismutase (CuZnSOD) is the breakage of the imidazolate bridge between copper and zinc and the loss of a coordinated water molecule from copper on reduction from Cu(II) to Cu(I). Crystal structures exist for the enzyme from a number of sources in the oxidised, five coordinate copper form. For the reduced form two structures from different sources have been determined only recently but provide contradictory results. We present crystal structures of bovine CuZnSOD (BSOD) in two different space groups. The structure of the P212121 form (pBSOD), at 1.65 A resolution clearly shows one subunit with Cu in the five coordinate, oxidised form, and the other with Cu in the three coordinate form expected for the reduced state. This mixed state of pBSOD is confirmed by XANES data of these crystals. The pBSOD structure has thus captured each subunit in one of the two oxidation state conformations and thus provides direct crystallographic evidence for the superoxide dismutase mechanism involving the breakage of the imidazole bridge between Cu and Zn. A shift in the position of copper in subunit A poises the catalytic centre to undergo the first stage of catalysis via dissociation of Cu from His61 with a concomittant movement of the coordinated water molecule towards His61, which rotates by approximately 20 degrees, enabling it to form a hydrogen bond to the water molecule. The Cu-Zn separation in the reduced site is increased by approximately 0.5 A. In contrast the 2.3 A resolution structure in space group C2221 (cBSOD) shows both of the Cu atoms to be in the five coordinate, oxidised form but in this space group the whole of subunit A is significantly more disordered than subunit B. An examination of published structures of "oxidised" SODs, shows a trend towards longer Cu-Zn and Cu-His61 separations in subunit A, which together with the structures reported here indicate a potential functional asymmetry between the subunits of CuZnSODs. We also suggest that the increased separation between Cu and Zn is a precursor to breakage of His61.     

Copper binding traps the folded state of the SCO protein from Bacillus subtilis

The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu(A) center. BsSCO has been proposed to play various roles in Cu(A) assembly including, the direct delivery of copper ions to the Cu(A) site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu(A). BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.     

Conformational variability of the Cu site in one subunit of bovine CuZn superoxide dismutase: the importance of mobility in the Glu119-Leu142 loop region for catalytic function

The structure of the catalytic site in one subunit of bovine CuZn superoxide dismutase is shown to be highly variable. A series of crystal structures at approximately 1.7 A have been determined using data collected from different crystals. Several conformations are observed for the copper site from one of the subunits. These conformations lie between those expected for the Cu(II) and Cu(I) forms of the enzyme and may represent a slow positional rearrangement of the Cu site during the crystallisation process due to the presence of a trace reductant in the mother liquor. These states perhaps indicate some functionally relevant structural steps that ultimately result in the breakage of the imidazolate bridge between the two metal sites.This behaviour is not observed for the second subunit of the dimeric enzyme, which remains in the five-coordinate, distorted square planar geometry in all cases. We suggest that this asymmetric behaviour may be caused by the lack of mobility for the Glu119-Leu142 loop region in the second subunit caused by crystal contacts. This region forms one wall of the active-site cavity, and its mobility has been suggested, via molecular dynamics studies, to be important for the catalytic mechanism.     

Type 2-depleted fungal laccase.

Although copper is quantitatively removed from fungal laccase (Polyporus versicolor) by extended dialysis against high concentrations of cyanide, we have been unable to reconstitute the protein by addition of Cu(I) ions. However, two new methods for reversibly removing the type 2 Cu centre have been developed. The visible absorption at 610 nm, which is attributable to type 1 Cu, is unaffected by the procedure, but the absorbance of the type 3 Cu at 330 nm is decreased by 60 +/- 10%. The decrease is due, at least in part, to partial reduction of the binuclear type 3 centre, although there may be some change in the molar absorptivity of the oxidized chromophore as well. The change in the c.d. spectrum that occurs at approx. 350 nm may be explained in the same way, but it may also reflect the loss of a signal due to the type 2 Cu. Upon removal of the type 2 Cu an absorbance increase appears at approx. 435 nm, and it is assigned to the semi-reduced form of the type 3 pair. In the e.p.r. spectrum of the type 2-depleted enzyme the type 1 Cu signal exhibits well-resolved ligand hyperfine splitting, which can be simulated on the basis of contributions from two N and two H nuclei (AH congruent to AN congruent to 25 MHz). The H atoms are assumed to be attached to the beta-carbon of the covalently bonded cysteine ligand. A signal from a semi-reduced form(s) of the type 3 site can also be resolved in the spectrum of the type 2-depleted enzyme, and on the basis of the second integral of the e.p.r. spectrum 40% of the type 3 pairs are believed to be in a partially reduced state. The semi-reduced type 3 site is remarkably stable and is not readily oxidized by H2O2 or IrCl6(2-) or reduced by Fe(CN)6(4-). Intramolecular electron transfer is apparently quite slow in at least some forms of the type 2-depleted enzyme, and this may explain why the activity is at best 5% of that of the native enzyme. Full activity returns when type 2 copper is restored.     

A proposal for the metal geometry in yeast superoxide dismutase based on results from EXAFS spectroscopy

Extended x-ray absorption fine structure (EXAFS) spectra have been recorded at the Cu edge and Zn edge in native yeast superoxide dismutase and at the Cu edge and Cd edge in the yeast superoxide dismutase derivative, where Zn has been substituted with Cd. Two different metal ligand distances in the range 1.9-2.0 A and 2.3-2.4 are determined for the Cu and Zn metals. For Cd in the Zn site two different metal ligand distances about 2.2 A and 2.6 A, respectively, were found. The striking feature is the similarity between the amplitude and radii determined for both the Cu and Zn sites. The increased distances for Cd can be explained by the increased ionic radius of Cd relative to Cu and Zn. Based on these EXAFS results and other relevant knowledge about the metal geometries, we propose that histidine 61 (63) positioned between the Cu and Zn metals are in one subunit bound to Zn and in the other to Cu. This model explains the recently observed difference between the two metal sites in each subunit.     

Characterization of the partially reduced cyanide-inhibited derivative of cytochrome c oxidase by optical, electron-paramagnetic-resonance and magnetic-circular-dichroism spectroscopy.

Optical. e.p.r. and near-infrared low-temperature m.c.d. (magnetic-circular-dichroism) spectroscopy were used to characterize the partially reduced cyanide-inhibited derivative of cytochrome c oxidase produced by anaerobic reductive titration with dithionite. The reductions of cytochrome a3+ and Cu2+a were followed by observation of the e.p.r. signals at g = 3.03, 2.21 and 1.5 and at g = 2.18, 2.03 and 1.99. As reduction proceeds new e.p.r. signals (g = 3.58 and 1.56) appear that quantify to give one haem per enzyme unit when a small excess of dithionite has been titrated in. The e.p.r. signal of the Cu2+a titrates in parallel with the disappearance of the band and 820nm in the optical absorption spectrum. The near-infrared m.c.d. spectrum shows the presence of the low-spin ferric haem, a3+, in the oxidized state of the enzyme, as a well-resolved positive peak at 1650nm. As reduction proceeds this band is replaced by one at 1550nm due to haem a3+(3)--CN in the partially reduced state. Hence as haem a3+(3)--CN becomes e.p.r.-detectable it also shows a near-infrared m.c.d. spectrum characteristic of a low-spin ferric haem. It is concluded that the partially reduced state of cyanide-inhibited cytochrome c oxidase contains a2+ . Cu+a . a3+(3)--CN . Cu+a3.