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.     

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.     

Multifrequency EPR evidence for a bimetallic center at the CuA site in cytochrome c oxidase

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in bovine heart cytochrome c oxidase (COX) and nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the existence of Cu-Cu interaction in both enzymes. C-band (4.5 GHz) proves to be a particularly good frequency complementing the spectra of COX and N2OR recorded at 2.4 and 3.5 GHz. Both the high and low field region of the EPR spectra show the presence of a well-resolved 7-line pattern consistent with the idea of a binuclear Cu center in COX and N2OR. Based on this assumption consistent g-values are calculated for gz and gx at four frequencies. No consistent g-values are obtained with the assumption of a 4-line pattern indicative for a mononuclear Cu site.     

M?ssbauer study of a bacterial cytochrome oxidase: cytochrome c1aa3 from Thermus thermophilus

Cytochrome c1aa3 from Thermus thermophilus has optical and EPR properties similar to bovine cytochrome c oxidase. We have studied 87Fe-enriched samples with M?ssbauer spectroscopy in the fully oxidized and fully reduced states and in the oxidized state complexed with cyanide. The cytochromes a and c1 yielded spectra quite similar to those reported for the cytochromes c and b5; in the oxidized state the spectra reflect noninteracting, low spin ferric hemes, whereas the a- and c1-sites of the reduced enzyme are typical of low spin ferrous hemochromes. The spectra of the reduced enzyme show that reduced cytochrome a3 is high spin ferrous, with M?ssbauer parameters quite similar to those of deoxymyoglobin. Upon addition of cyanide to the oxidized enzyme, the a3-site exhibits in the absence of an applied magnetic field and at temperatures down to 1.3 K a quadrupole doublet with parameters typical of low spin ferric heme-CN complexes. The low temperature spectra taken in applied magnetic fields show that the electronic ground state of the a3-CN complex has integer electronic spin, suggesting ferromagnetic coupling of the low spin ferric heme (S = 1/2) to Cu2+ (S = 1/2) to yield as S = 1 ground state. We have examined the oxidized enzyme from two different preparations. Both had good activity and identical optical and EPR spectra. The M?ssbauer spectra, however, revealed that the a3-site had a substantially different electronic structure in the two preparations. Neither configuration had properties in accord with the widely accepted spin-coupling model proposed for the bovine enzyme.     

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.     

An EPR study of the lineshape of copper in cytochrome c oxidase.

The EPR spectrum of copper in cytochrome c oxidase (EC 1.9.3.1) has been studied between 5 and 220 degreesK, and the spectral parameters have been determined for both forms of EPR-detectable copper by computer simulation methods. Numerical methods have been developed to separate the spectra of intrinsic copper and inactive copper. Evidence is presented to show that inactive copper is probably formed by denaturation. The EPR parameters for intrinsic copper were determined as gx = 1.99, gy = 2.03, gz = 2.185, / Ax(Cu) / = 0.0020 cm-1, / Ay(Cu) / = 0.0025 cm-1, / Az(Cu) / = 0.0030 cm-1. The principal values of the g tensor and the small value of /Az(Cu) / are interpreted in terms of mixing of 3d, 4s, and 4p metal orbitals. A flattened-tetrahedral stereochemistry about Cu2+ with an additional rhombic distrotion is in best agreement with all of the data. The peak-to-peak linewidth is found to be orientation dependent, and is described by a tensor with principal values deltaHx = 45G, deltaHy = 65 G, deltaHz = 85 G. A weak dipolar interaction with a low-spin ferric species stereochemistry for the copper ion is consistent with the electron transport function of the enzyme. Broad EPR signals with a very short spin-lattice relaxation time has been observed near g = 14 and g = 3 at 5 degrees K in oxidized cytochrome oxidase but not in the reduced or denatured enzyme. The possibility that these are due to the "EPR-undetectable" iron and copper is raised.     

Electron paramagnetic resonance studies of Pseudomonas cytochrome c peroxidase

The EPR spectrum at 15 K of Pseudomonas cytochrome c peroxidase, which contains two hemes per molecule, is in the totally ferric form characteristic of low-spin heme giving two sets of g-values with gz 3.26 and 2.94. These values indicate an imidazole-nitrogen : heme-iron : methionine-sulfur and an imidazole-nitrogen : heme-iron : imidazole-nitrogen hemochrome structure, respectively. The spectrum is essentially identical at pH 6.0 and 4.6 and shows only a very small amount of high-spin heme iron (g 5--6) also at 77 K. Interaction between the two hemes is shown to exist by experiments in which one heme is reduced. This induces a change of the EPR signal of the other (to gz 2.83, gy 2.35 and gx 1.54), indicative of the removal of a histidine proton from that heme, which is axially coordinated to two histidine residues. If hydrogen peroxide is added to the partially reduced protein, its EPR signal is replaced by still other signals (gz 3.5 and 3.15). Only a very small free radical peak could be observed consistent with earlier mechanistic proposals. Contrary to the EPR spectra recorded at low temperature, the optical absorption spectra of both totally oxidized and partially reduced enzyme reveal the presence of high-spin heme at room temperature. It seems that a transition of one of the heme c moieties from an essentially high-spin to a low-spin form takes place on cooling the enzyme from 298 to 15 K.     

EPR characterization of the cytochrome b-c1 complex from Rhodobacter sphaeroides

EPR characteristics of cytochrome c1, cytochromes b-565 and b-562, the iron-sulfur cluster, and an antimycin-sensitive ubisemiquinone radical of purified cytochrome b-c1 complex of Rhodobacter sphaeroides have been studied. The EPR specra of cytochrome c1 shows a signal at g = 3.36 flanked with shoulders. The oxidized form of cytochrome b-562 shows a broad EPR signal at g = 3.49, while oxidized cytochrome b-565 shows a signal at g = 3.76, similar to those of two b cytochromes in the mitochondrial complex. The distribution of cytochromes b-565 and b-562 in the isolated complex is 44 and 56%, respectively. Antimycin and 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB) have little effect on the g = 3.76 signal, but they cause a slight downfield and upfield shifts of the g = 3.49 signal, respectively. 5-Undecyl-6-hydroxyl-4,7-dioxobenzothiazole (UHDBT) shifts the g = 3.49 signal downfield to g = 3.56 and sharpens the g = 3.76 signal slightly. Myxothiazol causes an upfield shift of both g = 3.49 and g = 3.76 signals. EPR characteristics of the reduced iron-sulfur cluster in bacterial cytochrome b-c1 complex are: gx = 1.8 with a small shoulder at g = 1.76, gy = 1.89 and gz = 2.02, similar to those observed with the mitochondrial enzyme. The gx = 1.8 signal decreased and the shoulder increased concurrently as the redox potential decreased, indicating that the environment of the iron-sulfur cluster is sensitive to the redox state of the complex. UHDBT sharpens the gz and and shifts it downfield from g = 2.02 to 2.03, and shifts gx upfield from g = 1.80 to 1.78. UHDBT also causes an upfield shift of gy but to a much lesser extent compared to the other two signals. Addition of DBMIB causes a downfield shift of the gy from 1.89 to 1.94 and broadens the gx signal with an upfield to g = 1.75. Myxothiazol and antimycin show little effect on the gy and gz signals, but they broaden and shift the gx signal upfield to g = 1.74. However, the myxothiazol effect is partially reversed by UHDBT. An antimycin-sensitive ubisemiquinone radical was detected in the cytochrome b-c1 complex. At pH 8.4, the antimycin-sensitive ubisemiquinone radical has a maximal concentration of 0.66 mol per mol complex at 100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytochrome electron spin resonance line shapes, ligand fields, and components stoichiometry in ubiquinol-cytochrome c oxidoreductase

The EPR spectra of the cytochromes in ubiquinol-cytochrome c oxidoreductase (Complex III) have peaks at g = 3.78 (cytochrome b566) g = 3.45 (cytochrome b562) and g = 3.35 (cytochrome c1). The highly asymmetric peak of cytochrome b566 has been simulated using an arbitrary gaussian distribution of crystal field parameters. The asymmetry is due to the nonlinear relationship between field position and crystal field. The results suggest that the b cytochromes have bis-imidazole ligation. The gz peak of cytochrome c1 was also found to be asymmetric; simulations suggest histidine-methionine ligation. No other important cytochrome components were needed to simulate the spectrum of the oxidized complex; these results are consistent with 1:1:1 stoichiometry of components. These results argue against any asymmetric dimer model for Complex III.     

Angular dependences of perpendicular and parallel mode electron paramagnetic resonance of oxidized beef heart cytochrome c oxidase

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The "slow" form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu(B). The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (|J| approximately 1 cm(-1)), with the axial zero-field splitting (D approximately 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.     

The effect of phospholipid depletion on the EPR behavior of cytochrome oxidase

Phospholipids are essential components for electron transport activity of cytochrome oxidase. Recently, we have found that the removal of phospholipids from the oxidase affected the copper and low-spin heme signals, and conceivably other paramagnetic centers as demonstrated by EPR spectroscopy. At 4.2–30 °K, the signal amplitudes and power saturation behaviors were studied at approximately g = 2.0 for the copper signal, and in the neighborhood of g = 3.0 for the low-spin heme signal. After depletion of phospholipids the amplitude of the copper signal decreased 25–30% at 12–30 °K and below 12 °K 40–50% under nonsaturating conditions. The amplitude of the low-spin heme signal decreased 60–70% at 4.2–20 °K. Below 14 °K both signals became more resistant to power saturation, but the copper signal was more readily saturated above this temperature, compared to the oxidase with about 25% lipid. After removal of phospholipids, the spectral features of the copper signal remained essentially the same, but the low-spin heme signal broadened and became very asymmetric to show two signals as revealed by the second harmonic EPR spectra. These findings may explain, at least partially, the wide variations in percentage of EPR detectable copper and heme of cytochrome oxidase reported by different laboratories. Unequivocally, the EPR behavior of cytochrome oxidase is not only affected by the protein moiety, but also by the associated phospholipids of the enzyme.     

The EPR spectrum for CuB in cytochrome c oxidase

Incubation of cytochrome c oxidase (CcO) in its resting state in saturated ammonium sulfate, at room temperature overnight, gave EPR signals characteristic of a single Cu(II) center. From the g// and A// values it is concluded that this is a square-planar type 2 copper center, and superhyperfine splitting shows the presence of three nearly equivalent 14N nuclei in the plane. It is suggested that this center, also formed by incubating the enzyme in 10% methanol followed by direct irradiation, must be the CuB center. This type 2 copper EPR spectrum is identical to the EPR spectrum of CuB reported for the isolated cytochrome bo3 complex from Escherichia coli; and to the EPR spectrum reported for the sulfobetaine 12 heat-treated cytochrome c oxidase complex. It is argued that a small perturbation in the system causes decoupling of the magnetic coupling of the heme a3-CuB binuclear center and the appearance of the type 2 EPR signal.     

Metal site cooperativity within cytochrome oxidase

Low temperature (9-15 K) EPR of isolated bovine heart cytochrome oxidase titrated potentiometrically in the presence of azide reveals the formation of two distinct species of low-spin cytochrome a3(III)-azide which differ in redox properties and g values. Both species are formed with characteristic midpoint potentials during the course of oxidative titration and disappear at higher potentials. The signal appearing at lower potential has principal g values 2.88, 2.19, and 1.64; that appearing at higher potential has g values 2.77, 2.18, and 1.74. A good fit to the experimental data (per cent of cytochrome present in a given paramagnetic state versus oxidation potential) was obtained with a model whereby the gz = 2.88 species arises from cytochrome a3(III)-azide with cytochrome a reduced, which is converted to the gz = 2.77 species upon oxidation of cytochrome a. Potentiometric titration of cytochrome oxidase in the presence of cyanide produces two low-spin heme EPR signals attributable to cytochrome a3(III)-cyanide which are incompletely resolved, but are distinguishable nonetheless. The low-potential signal has peak amplitude at gz = 3.63 and a long high-field tail; this resonance has been seen by other workers in the partially reduced enzyme (DerVartanian, D. V., Lee, I. Y., Slater, E. C., and van Gelder, B. F. (1974) Biochim. Biophys. Acta 347, 321-327). The high-potential signal is much more symmetric about its peak amplitude, which is at approximately 10 G higher field with gz = 3.61. As with the azide complex, the titration behavior in the presence of 2 mM KCN is adequately simulated by assuming that the appearance of the two species is a function of the oxidation state of cytochrome a. Like the a3-azide signals, the a3-cyanide signals disappear upon further oxidation with some characteristic midpoint potential. If the disappearance of the a3-ligand signals with increasing potential is assumed to be the result of antiferromagnetic (or ferromagnetic) coupling of a3(III) (S = 1/2) to CuB(II) (S = 1/2), then cooperativity between cytochrome a and CuB is implied. The data are consistent with the hypothesis that oxidation of cytochrome a raises the midpoint potential of CuB by 55 +/- 10 mV.     

Studies on the origin of the near-infrared (800-900 nm) absorption of cytochrome c oxidase

Data are presented which were collected in the course of the past ten years and bear on the correlation of absorbance at 800 nm and the EPR signal at g = 2 ('copper signal') of cytochrome c oxidase in various states of oxidation and ligation. Both EPR and optical reflectance spectra were obtained at low temperature (-170 to -190 degrees C). For some sets of samples spectra were recorded in the range 500-1100 nm. A particular efFort was made to study this correlation with what are called 'mixed valence' states (Greenwood, C., Wilson, M.T. and Brunori, M. (1974) Biochem. J. 137, 205-215), when cytochrome a and the EPR-detectable copper are thought to be oxidized and the other components reduced and vice versa. These data show no evidence that the copper component of cytochrome oxidase which has so far not been detected by EPR makes a contribution to the absorption between 800 and 900 nm exceeding 10-15% of the total, which is close to or within the error of the respective measurements. For the various states of the oxidase examined in this work the 700-800 nm region did not appear to be more useful than the 800-900 nm region for determining the state of the EPR-undetectable copper in a reliable way. These conclusions are in agreement with results presented previously from other laboratories concerning the relationship of optical (approx. 800 nm) and EPR spectroscopic (g = 2) data obtained with the enzyme.     

Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E. coli cytochrome o quinol oxidase complex.

The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.     

Biochemical and biophysical studies on cytochrome c oxidase XVI. Circular dichroic study of cytochrome c oxidase and its ligand complexes

1. CD spectra of cytochrome c oxidase have been determined both in the absence and presence of the extrinsic ligands CO, NO, cyanide and azide.2. CO and NO affect the CD spectrum of cytochrome c oxidase in a similar way.3. Cyanide and azide also affect the CD spectrum of cytochrome c oxidase in a similar way, but distinctly different from CO and NO.4. From the CD spectra of the oxidized and reduced enzyme, in the presence and absence of extrinsic ligands, CD difference spectra (reduced minus oxidized) are calculated for the so-called cytochrome a and cytochrome a₃ moieties of the enzyme.5. These spectra are largely dependent on the extrinsic ligand used. It is therefore concluded that these spectra do not represent independent cytochrome a and cytochrome a₃ difference spectra, but that heme-heme interactions occur within the cytochrome c oxidase molecule, in such a way that binding of a ligand to one of the heme a groups of cytochrome c oxidase affects the spectral properties of the other heme a group.6. As a consequence, ligand-binding studies cannot give information as to the pre-existence of separate cytochrome a and cytochrome a₃ moieties in the absence of extrinsic ligands.     

Circular dichroism spectra of cytochrome c oxidase

Circular dichroism spectra of bovine heart aa(3)-type cytochrome c oxidase have been studied with a major focus on the Soret band π → π* transitions, B(0(x,y)), in the two iron porphyrin groups of the enzyme. The spectra of the fully reduced and fully oxidized enzyme as well as of its carbon monoxide and cyanide complexes have been explored. In addition, CD spectra of the reduced and oxidized ba(3)-type cytochrome c oxidase from Thermus thermophilus were recorded for comparison. An attempt is made to interpret the CD spectra of cytochrome c oxidase with the aid of a classical model of dipole-dipole coupled oscillators taking advantage of the known 3D crystal structure of the enzyme. Simultaneous modeling of the CD and absorption spectra shows that in the bovine oxidase, the dipole-dipole interactions between the hemes a and a(3), although contributing significantly, cannot account either for the lineshape or the magnitude of the experimental spectra. However, adding the interactions of the hemes with 22 aromatic amino acid residues located within 12 ? from either of the two heme groups can be used to model the CD curves for the fully reduced and fully oxidized oxidase with reasonable accuracy. Interaction of the hemes with the peptide bond transition dipoles is found to be insignificant. The modeling indicates that the CD spectra of cytochrome oxidase in both the reduced and oxidized states are influenced significantly by interaction with Tyr244 in the oxygen-reducing center of the enzyme. Hence, CD spectroscopy may provide a useful tool for monitoring the redox/ionization state of this residue. The modeling confirms wide energy splitting of the orthogonal B(x) and B(y) transitions in the porphyrin ring of heme a.     

Two sites of interaction of anions with cytochrome a in oxidized bovine cytochrome c oxidase

An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.     

A carbon monoxide irreducible form of cytochrome c oxidase and other unusual properties of the “monomeric” shark enzyme

Contrary to previous reports, the functional and spectral properties of “monomeric” shark cytochrome c oxidases are not entirely similar to those of the “dimeric” beef enzyme. Most significantly, unlike the behavior of beef oxidase, the fully oxidized shark enzyme is not reducible by carbon monoxide. Also, preparations of the shark enzyme, isolated at pH 7.8-8.0, lead to more than 60% of the sample always being obtained in a resting form, whereas similarly prepared beef oxidase is very often obtained, both by ourselves and others, exclusively in the pulsed form. Although the electronic absorption, magnetic circular dichroism and electron paramagnetic resonance (EPR) spectra of cytochrome c oxidase obtained from several shark species are similar to those of the beef enzyme, there are some significant differences. In particular, the Soret maximum is at 422 nm in the case of the fully oxidized resting shark oxidases at physiological pH and not 418 nm as commonly found for the beef enzyme. Moreover, the resting shark oxidases do not necessarily exhibit a “g = 12” signal in their EPR spectra. The turnover numbers of recent preparations of the shark enzyme are higher than previously reported and, interestingly, do not differ within experimental uncertainty from those documented for several beef isoenzymes assayed under comparable conditions.     

Nitric oxide and cytochrome oxidase: substrate,inhibitor or effector?

Endogenously produced nitric oxide (NO) controls oxygen consumption by inhibiting cytochrome c oxidase, the terminal electron acceptor of the mitochondrial electron transport chain. The oxygen-binding site of the enzyme is an iron/copper (haem a3/CuB) binuclear centre. At high substrate (ferrocytochrome c) concentrations, NO binds reversibly to the reduced iron in competition with oxygen. At low substrate concentrations, NO binds to the oxidized copper. Inhibition at the haem iron site is relieved by dissociation of the NO from the reduced iron. Inhibition at the copper site is relieved by oxidation of the bound NO and subsequent dissociation of nitrite from the enzyme. Therefore, NO can be a substrate, inhibitor or effector of cytochrome oxidase, depending on cellular conditions.