The binding of carbon monoxide to cytochrome c oxidase.

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

Oxygenation of carbon monoxide by bovine heart cytochrome c oxidase

Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1), as the terminal enzyme of the mammalian mitochondrial electron transport chain, has long been known to catalyze the reduction of dioxygen to water. We have found that when reductively activated in the presence of dioxygen, the enzyme will also catalyze the oxidation of carbon monoxide to its dioxide. Two moles of carbon dioxide is produced per mole of dioxygen, and similar rates of production are observed for 1- and 2-electron-reduced enzyme. If 13CO and O2 are used to initiate the reaction, then only 13CO2 is detected as a product. With 18O2 and 12CO, only unlabeled and singly labeled carbon dioxide are found. No direct evidence was obtained for a water-gas reaction (CO + H2O----CO2 + H2) of the oxidase with CO. The CO oxygenase activity is inhibited by cyanide, azide, and formate and is not due to the presence of bacteria. Studies with scavengers of partially reduced dioxygen show that catalase decreases the rate of CO oxygenation.     

Stoichiometry of carbon monoxide binding by cytochrome c oxidase.

The stoichiometry of carbon monoxide binding to beef heart cytochrome c oxidase has been reinvestigated both by titration of the reduced oxidase with CO and by measuring the amount of carboxyhemoglobin that is formed after adding oxyhemoglobin to a solution of the CO-enzyme complex. In the titration experiments the ratio of CO bounds to total heme a present was always less than 0.50 while in the experiments where oxyhemoglobin was added the results were variable and of lower accuracy. These observations do not agree with the recent conclusion of Volpe, J.A., O'Toole, M.C., and Caughey, W.S. (1975) Biochem. Biophys. Res. Commun. 62, 48-53 that CO is bound in a 1:1 ratio with heme a. An explanation for their results is suggested.     

The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide.

The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 X 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 X 10(4)M-1-s-1 and 1.6 X 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.     

Spectroelectrochemical study of the cytochrome a site in carbon monoxide inhibited cytochrome c oxidase

The reduction potential of the cytochrome a site in the carbon monoxide derivative of beef heart cytochrome c oxidase has been studied under a variety of conditions by thin-layer spectroelectrochemistry. The reduction potential exhibits no ionic strength dependence and only a 9 mV/pH unit dependence between pH 6.5 and 8.5. The weak pH dependence indicates that protonation of the protein is not stoichiometrically linked to oxidoreduction over the pH range examined. The temperature dependence of the reduction potential implies a relatively large standard entropy of reduction of cytochrome a. The measured thermodynamic parameters for reduction of cyctochrome a are (all relative to the normal hydrogen electrode) delta Go'(25 degrees C) = -6.37 kcal mol-1, delta Ho' = -21.5 kcal mol-1, and delta So' = -50.8 eu. When cytochrome c is bound to the oxidase, the reduction potential of cytochrome a and its temperature dependence are not measurably affected. Under all conditions studied, the cytochrome a site did not exhibit simple Nernstian n = 1 behavior. The titration behavior of the site is consistent with a moderately strong anticooperative interaction between cytochrome a and CuA [Wang, H., Blair, D. F., Ellis, W. R., Jr., Gray, H. B., & Chan, S. I. (1985) Biochemistry (following paper in this issue)].     

The equilibrium between cytochrome oxidase and carbon monoxide

An evolution argument which attempted to trace the development of hemoglobins from such respiratory pigments as cytochrome oxidase presupposed that the latter possesses, in addition to its high affinity for oxygen, an approximately hyperbolic equilibrium function, and little if any Bohr effect (decline in affinity for oxygen with rise in acidity). Since cytochrome oxidase, unlike hemoglobin, is irreversibly oxidized by oxygen, the present experiments examine its combination with carbon monoxide, with which, like hemoglobin, it yields a true equilibrium. In all known hemoglobins the form of the equilibrium function and the vigor of the Bohr effect are similar with carbon monoxide and with oxygen, so that observations involving the former gas are relevant to the relations of the latter. The equilibrium function of cytochrome oxidase with carbon monoxide—percentage saturation vs. partial pressure of CO—is slightly inflected (in the Hill equation n = 1.26; for a hyperbola, n = 1). No Bohr effect is present in the range of pH 7–8. The pressure of carbon monoxide at which half-saturation occurs (p₅₀) is about 0.17 mm. at 10–13°C. The affinity for carbon monoxide is therefore higher than commonly supposed. These properties are consistent with the evolution argument. They are important also for the physiological functioning of cytochrome oxidase, the nearly hyperbolic equilibrium function facilitating a high degree of saturation, and the lack of Bohr effect making this enzyme impervious to hyperacidity. The slight inflection of the equilibrium function shows that the Fe-porphyrin units of cytochrome oxidase interact to a degree, hence that the enzyme must contain more than one such unit per molecule. It is suggested that in cytochrome oxidase two Fe-porphyrin groups may unite with one oxygen in the manner Fe⁺⁺-O₂-Fe⁺⁺; and that the evolution of hemoglobins proceeded over a first stage in which the hemes were separated so that each combines with only one molecule of oxygen, so tending to remain reduced; to a further stage in which the separated hemes interact through the protein to facilitate one another in combining with oxygen.     

In vivo binding of carbon monoxide to cytochrome c oxidase in rat brain

The possibility of binding of CO to cytochrome c oxidase (cytochrome a,a3) in brain cortex has been examined in vivo by reflectance spectrophotometry. During ventilation with CO-containing gases, cytochrome a,a3 absorption at 605 nm increased in the parietal cortex of anesthetized rats during carboxyhemoglobin (HbCO) formation. HbCO levels, measured by changes in absorption at 569-586 nm in vivo, correlated positively with arterial HbCO by CO oximetry. Arterial blood pressure and calculated O2 content varied inversely with HbCO. During CO exposure, decreases in blood pressure, O2 content, and cytochrome a,a3 oxidation level could be reversed partly at constant HbCO by compression to 3 atmospheres absolute (ATA). After removing CO from inspired gas at 3 ATA, optical and physiological parameters recovered completely to control values except for minor persistent elevations of HbCO. Difference spectra from parallel experiments at constant HbCO revealed absorption minima at 588-592 nm and 600-605 nm as a result of hyperbaric exposure. Spectral analysis of these components was consistent with partial dissociation of a cytochrome a3-CO complex and cytochrome a reoxidation with increasing dissolved O2 in hyperbaric conditions.     

The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes

Cytochrome c oxidase forms tight binding complexes with the cytochrome c analog, porphyrin cytochrome c. The behaviour of the reduced and pulsed forms of the oxidase with porphyrin cytochrome c have been followed as functions of ionic strength; this behaviour has been compared with that of the resting oxidase [Kornblatt, Hui Bon Hoa and English (1984) Biochemistry 23, 5906-5911]. All forms of the cytochrome oxidase studied bind one porphyrin cytochrome c per 'functional' cytochrome oxidase (two heme a); it appears as though porphyrin cytochrome c and cytochrome c compete for the same site on the oxidase. The resting enzyme binds cytochrome c 8 times more strongly than porphyrin cytochrome c; the reduced enzyme, in contrast, binds the two with almost equal affinity. In all three cases, resting, pulsed and reduced, the heme-to-porphyrin distance is estimated to be about 3 nm. The tight-binding complexes formed between cytochrome oxidase and porphyrin cytochrome c can be dissociated by salt. Debye-Hückel analysis of salt titrations indicate that the resting enzyme and the reduced enzyme are similar in that the product of the interaction charges on the two proteins is about -14. The product of the charges for the pulsed enzyme is -25, indicating that on average another positive and negative charge take part in the interaction of the two proteins. While there is one tight binding site for cytochrome c per two heme a, cytochrome c is able to 'communicate' with four heme a. In the absence of cytochrome c, electron transfer from tetramethylphenylenediamine to the oxidase to oxygen results in the conversion of the resting form to the 'oxygenated'; in the presence of cytochrome c, the same electron transfer results in the appearance of the 'pulsed' form. Cytochrome c titrations of the enzyme show that a ratio of only one cytochrome c to four heme a is sufficient to convert all the oxidase to the 'pulsed' form. Porphyrin cytochrome c, like cytochrome c, catalyzes the same conversion with the same stoichiometry. The binding data and salt effects indicate that major structural alterations occur in the oxidase as it is converted from the resting to the partially reduced and subsequently to the pulsed form.     

Effects of inhibitory ligands on the aerobic carbon monoxide complex of cytochrome c oxidase.

1. In the presence of both CO and O2, ox heart cytochrome c oxidase forms a 607 nm-peak intermediate distinct from both the cytochrome a2+a3 2+CO and the cytochrome a3+a3 2+CO ('mixed-valence') CO complexes. 2. This aerobic CO compound is stable towards ferricyanide addition, but decomposed on treatment with ferric cytochrome a2 ligands such as formate, cyanide and azide. 3. Addition of formate or cyanves rise to a complex with alpha-peak at 598 nm, not identical with any azide complex of the free enzyme, but possibly a cytochrome a3 2+NO complex produced by oxidative attack of partially reduced O2 on the azide. 4. The results support the idea that although the initial reaction of oxygen is with cytochrome a3 2+, the next step is not an oxidation of the ferrous cytochrome a3, but a transfer of O2 to a neighbouring group, such as Cu+, to give Cu2+O2- or similar complexes. 5. The aerobic CO complex is then identified as a3+a3 2+COCu2+O2-; a similar compound ('Compound C') is formed by photolysis of a3+a3 2+CO (the 'mixed-valence' CO complex) in the presence of oxygen at low temperatures.     

Temperature dependence of the reduction potential of CuA in carbon monoxide inhibited cytochrome c oxidase

The temperature dependence of the reduction potential of the CuA site in carbon monoxide inhibited cytochrome c oxidase has been measured with a spectroelectrochemical method adapted to the relatively weak near-infrared absorption of this copper ion. These measurements, together with parallel measurements on the 604-nm absorption due to Fea, indicate that an interaction between CuA and Fea causes the reduction potential for one of these sites to be decreased by approximately 40 mV upon reduction of the other. The temperature dependence of the CuA reduction potential indicates a relatively large and negative standard entropy of reduction of CuA (delta So' = -48.7 +/- 2.3 eu). Possible implications of the intersite redox interaction and the large standard entropy of reduction of the CuA site are discussed.     

Myocardial cytochrome oxidase activity is decreased following carbon monoxide exposure

Carbon monoxide (CO) inhalation often leads to cardiac dysfunction, dysrhythmias, ischemia, infarction, and death. However, the underlying mechanism of CO toxicity is poorly understood. We hypothesize that inhaled CO interrupts myocardial oxidative phosphorylation by decreasing the activity of myocardial cytochrome oxidase (CcOX), the terminal oxidase of the electron transport chain. Male C57Bl6 mice were exposed to either 1000 ppm (0.1%) CO or air for 3 h. Cardiac ventricles were harvested and mitochondria were isolated. CcOX kinetics and heme aa(3) content were measured. V(max), K(m), and turnover number were determined. Levels of CcOX subunit I message and protein were evaluated. Carboxyhemoglobin (COHb) levels were measured and tissue hypoxia was assessed with immunohistochemistry for pimonidazole hydrochloride. CO significantly decreased myocardial CcOX activity and V(max) without altering K(m). Heme aa(3) content and CcOX I protein levels significantly decreased following CO exposure while enzyme turnover number and CcOX I mRNA levels remained unchanged. CO exposure increased COHb levels without evidence of tissue hypoxia as compared to sham and hypoxic controls. Decreased CcOX activity following CO inhalation was likely due to decreased heme aa(3) and CcOX subunit I content. Importantly, myocardial CcOX impairment could underlie CO induced cardiac dysfunction.     

Characterization of two low Em forms of cytochrome a3 and their carbon monoxide complexes in mammalian cytochrome c oxidase.

Evidence is presented for the existence of two forms of low-potential cytochrome a3. One appears to be similar to the low-spin form reported by Nicholls, P., and V. Hildebrandt (1978 Biochem. J. 173:65-72) and Wrigglesworth, J. M., J. Elsden, A. Chapman, N. Van der Water, and M. F. Grahn (1988. Biochim. Biophys. Acta. 936:452-464). It has a reduced Soret peak near 428 nm and a prominent alpha peak near 602 nm. This form is seen when the enzyme is either supplemented with lipoprotein or incorporated into a liposomal membrane, preexposed to a voltage greater than 400 mV for at least 30 min, and titrated in the presence of approximately 1 mM K3Fe(CN)6. The other form has a reduced Soret peak near 446 nm, and no prominent alpha peak. The 428-nm form has an Em near 175 mV and forms a CO complex with an Em near 225 mV. The 446-nm form has an Em near 200 mV and forms a CO complex with an Em near 335 mV.     

The cytochrome c binding site on cytochrome c oxidase.

Cytochrome $\text{c}$ was chemically coupled to cytochrome $\text{c}$ oxidase using the reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) which couples amine groups to carboxyl residues. The products of this reaction were analyzed on 2.5–27% polyacrylamide gradient gels electrophoretically. Since cytochrome $\text{c}$ binds to cytochrome oxidase electrostatically in an attraction between certain of its lysine residues and carboxyl residues on the oxidase surface, EDC is an especially appropriate reagent probe for binding-subunit studies. Coupling of polylysine to cytochrome oxidase using EDC was also performed, and the products of this reaction indicate that polylysine, an inhibitor of the cytochrome $\text{c}$ reaction with oxidase, binds to the same oxidase subunit as does cytochrome $\text{c}$, subunit IV in the gel system used.     

Differential carbon monoxide sensitivity of cytochrome C oxidase in the leaves of c(3) and c(4) plants

CO sensitivity of cytochrome a₃ in the leaves of a number of C₃ and C₄ plants was monitored by the nitrate reductase assay under differing CO to O₂ ratios. All the C₃ plants were relatively insensitive to CO and required a high CO to O₂ ratio of 40 to promote significant nitrate reductase activity. However, when treated with 2 millimolar 2,4-dinitrophenol, these leaves readily responded to CO even at low CO to O₂ ratios of 10 or less. On the other hand, the leaves of all C₄ plants tested, belonging to the three subgroups, were highly sensitive to CO, even at CO to O₂ ratios of 5 or less. In these leaves, the uncoupler was without any effect, probably because the mitochondria, either from mesophyll or bundle sheath cells or both, lacked tight respiratory control.