Evidence for a ferryl Fea3 in oxygenated cytochrome c oxidase

Evidence is reported which shows that a reactive ferryl Fea3/cupric CuB binuclear couple is present at the dioxygen reduction site in "oxygenated" cytochrome c oxidase; when the fully reduced enzyme is reoxidized at low temperatures; and when partially reduced cytochrome c oxidase is reoxidized with dioxygen at room temperature.     

The conformational states of cytochrome oxidase in the mitochondrion.

THE Soret spectrum of "resting" cytochrome oxidase in cytochrome-c depleted mitochondria has been determined. The spectrum obtained is dependent on the rate at which the oxidase is turning over. In the least active preparations, the spectrum is almost pure "oxidized" oxidase. With increasing activity the spectrum is converted to a mixture of "oxidized" and "oxygenated" oxidases. It is concluded that the same conformational differences between the two non-reduced forms that are found in the purified enzyme also occur in these cytochrome-c depleted mitochondria.     

"Peroxidatic" form of cytochrome oxidase as studied by X-ray absorption spectroscopy

X-ray absorption spectroscopy shows pulsed oxidase to be similar to resting oxidase but to lack the sulfur bridge between iron and copper of active sites (Powers, L., Y. Ching, B. Chance, and B. Muhoberac, 1982, Biophys. J., 37[2, Pt. 2]: 403a. [Abstr.] ) The first shell ligands and bond lengths of the pulsed oxidase active site heme most clearly fit the ferric peroxidases from horseradish and yeast, and the pulsed oxidase cyanide compound resembles the low spin hemoprotein cyanide compounds. The structural results are consistent with an aquo or a peroxo form for pulsed oxidase as is also observed by optical studies. These structural and chemical data are consistent with a role for the pulsed forms in a cyclic peroxidatic side reaction in which the pulsed and pulsed peroxide compounds act as peroxide scavengers. The peroxidatic role of cytochrome oxidase in the nonsulfur bridged form suggests the renaming of the "oxygenated" or "pulsed" forms on a functional basis as "peroxidatic" forms of cytochrome oxidase.     

Spectroscopic and quantitative analysis of the oxygenated and peroxy states of the purified cytochrome d complex of Escherichia coli

Oxygenated and peroxy states of the cytochrome d complex of Escherichia coli have been proposed as intermediates in the reaction mechanism of this ubiquinol oxidase. In this report, several stable states of the purified enzyme were examined spectroscopically at room temperature. As purified, the cytochrome d complex exists in an oxygenated state characterized by an absorbance band at 650 nm. Removal of oxygen results in loss of absorbance at this wavelength, which is restored upon the return of oxygen. The presence of one oxygen molecule in the oxygenated state was quantified by measuring oxygen released when excess hydrogen peroxide was added to the oxygenated state by passage of argon generates a "partially reduced" state with an absorbance peak at 628 nm, apparently due to reduced cytochrome d. Addition of equimolar hydrogen peroxide to the fully oxidized state produces the peroxy state. This peroxy state is also formed upon addition of excess hydrogen peroxide to the oxygenated state via a stable intermediate termed "peroxy intermediate." It is likely that 1) the oxygenated state consists of one molecule of oxygen bound to reduced heme d, and 2) there are at least two stable states that have bound peroxide at room temperature, the peroxy state and a newly discovered peroxy intermediate.     

On the reaction of cyanide with an oxygenated form of cytochrome oxidase.

The reaction of an oxygenated form of cytochrome oxidase [EC 1.9.3.1] with cyanide was examined under conditions where spontaneous decay was prevented. The equilibrium and kinetic constants for the reaction agreed well with those for the normally operating enzyme, indicating that the oxygenated form is one of the active intermediates of the cytochrome oxidase reaction.     

Structure and reactivity of multiple forms of cytochrome oxidase as evaluated by X-ray absorption spectroscopy and kinetics of cyanide binding

The extended X-ray absorption fine structure (EXAFS) data show differences between the active site structures of different cytochrome oxidase preparations. In the resting (as isolated) state of the Yonetani preparation, the bridging atom between Fe3+a3 and Cu2+a3 is present [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465], whereas in another preparation (e.g., Hartzell-Beinert), this atom seems to be bound only to Fe3+a3 in a significant fraction of the molecules. Both preparations bind cyanide in a multiphasic fashion, suggesting that the resting cytochrome oxidase is not homogeneous but rather is a mixture of several forms. The proportion of these forms as detected by cyanide binding kinetics differs for different preparations. However, upon reduction and reoxidation (conversion to the "oxygenated" form) the cyanide binding kinetics become monophasic and all preparations of the oxygenated form bind cyanide at the same rate. Thus, a combination of structural and kinetic approaches seems necessary for evaluation of the nature of the active site of cytochrome oxidase in its various forms.     

Redox-cycled oxidase. One of the reaction products of reduced cytochrome c, cytochrome c oxidase, and dioxygen

Pulsed and oxygenated forms of cytochrome c oxidase are believed to be variants of the oxidized enzyme. They were produced as a consequence of one or more reduction-oxidation cycles of the resting form and are characterized by an increase of the alpha band intensity and a red-shift of the Soret absorption band to 428 nm. The rate of decay of these species back to the resting enzyme varies appreciably and appears to depend on the nature of the reductant and/or oxidant used in their preparation. Here we report that if resting oxidase is incubated with either reduced or oxidized cytochrome c and then exposed to dioxygen, an activated form is rapidly produced which appears to be more oxidized than the starting material. This finding suggest some degree of partial reduction of the resting enzyme, but this by itself cannot explain the extent of activation. Our results further question the significance of the optical spectral "signature" of the oxygenated (Okunuki, K., and Sekuzu, I. (1954) Seitaino Kagaka 5, 265-272), pulsed (Antonini, E., Brunori, M., Colosimo, A., Greenwood, C., and Wilson, M. T. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3128-3132), and "420 nm" species (Kumar, C., Naqui, A., and Chance, B. (1984) J. Biol. Chem. 259, 2073-2076, 11668-11671), which are thought to be activated forms of oxidized cytochrome c oxidase.     

Reduction of oxygen-pulsed cytochrome c oxidase by cytochrome c and other electron donors

1. Stopped-flow experiments were performed in which solutions containing dithionite were mixed with air-saturated buffer. Cytochrome c oxidase present in the dithionite-containing syringe is fully oxidized within the mixing time and the oxygen-pulsed form of the oxidase is produced. 2. The reduction of this form by dithionite, by dithionite plus cytochrome c and by dithionite plus methyl viologen or benzyl viologen was followed and compared with the corresponding reduction reactions of the "resting" oxidized enzyme. Reduction by dithionite is relatively slow, but the rate of reduction is greatly increased by addition of cytochrome c or the viologens, which are even more effective than cytochrome c on a molar basis. 3. Profound differences between the transient kinetics of the reduction of the two oxidized oxidase derivatives were observed. The results are consistent with a direct reduction of cytochrome a followed by an intramolecular electron transfer to cytochrome a3 (k1obs = 7.5 s-1 for the oxygen-pulsed oxidase). 4. The spectrum of the oxygen-pulsed oxidase formed within 5 ms of the mixing closely resembles that of the "oxygenated" compound, but there were small differences between the two spectra.     

The use of a maleimido spin label to detect conformational changes in cytochrome c oxidase.

Spin labeling with a maleimido spin label has been used to investigate conformational changes of bovine cytochrome c oxidase. These experiments show that the spin label is immobilized to a lesser degree when the enzyme is in the “oxygenated” form than it is in the oxidized state and support the view that the oxygenated form is a conformational variant. Experiments in which the maleimido spin-labeled cytochrome c oxidase was titrated with H₂O₂ reveal that the peroxide-treated enzyme, although possessing an absorption spectrum similar to that of the oxygenated form, has an electron paramagnetic resonance (epr) spectrum that is different from that of either the oxygenated form or the oxidized state. Extremes of pH cause a marked decrease in the degree of immobilization of maleimido spin labels bound to the oxidase. Alterations in the epr spectrum are reversible if the pH is held between 5.3 and 10.2 but are irreversible outside that range. Urea and guanidine hydrochloride also decrease the immobilization of the spin labels bound to the oxidase. The nature of the epr spectra indicates that under these conditions the enzyme assumes a more open conformation. Exposure to concentrations of sodium dodecyl sulfate as high as 10% does not result in as much loss of the immobilization as with urea or guanidine. Detergents such as cholate, Tween 80, and Triton X-100 have no significant effect on the epr spectrum of maleimido spin-labeled cytochrome c oxidase.     

On the nature of biochemically generated hydroxyl radicals. Studies using the bleaching of p-nitrosodimethylaniline as a direct assay method.

An efficient scavenger for radiolytically generated hydroxyl (OH) radicals, p-nitrosodimethylaniline, was used to try to substantiate the presence of this oxygen radical species in several biochemical systems. Most of these systems which were investigated had previously been assumed to generate OH radicals, e.g. the autoxidation of 6-hydroxydopamine, the hydroxylating system NADH/phenazine methosulfate, and the oxidation of xanthine or acetaldehyde by xanthine oxidase. We did not observe inhibition of the bleaching of p-nitrosodimethylaniline in oxygenated solutions by other scavengers of OH radicals nor, in the case of xanthine/xanthine oxidase, by catalase and superoxide dismutase. We therefore conclude that, under biochemical conditions as opposed to radiolysis or photolysis, no freely diffusable OH radicals are formed. Rather, a strongly oxidizing OH-analogous complex is considered to represent the p-nitrosodimethylaniline-detectable species formed under these conditions.     

Kinetic studies on the binding of cyanide to oxygenated cytochrome c oxidase.

The reaction of cyanide with oxygenated cytochrome c oxidase was followed by means of flow-flash techniques. The oxygenated form, produced after photolysis of the partially reduced CO complex in the presence of cyanide and O2, shows cyanide-binding properties distinct from those of both the oxidized and the reduced forms of the protein. The binding is a single process (k = 22M-1-S-1) linearly dependent on cyanide concentration to as high as 75 mM. It is suggested that the oxygenated form is a conformational variant of the oxidized protein.     

The role of aeration and agitation in the production of glucose oxidase in submerged culture

The influence of agitation and aeration on growth and on production of glucose oxidase of Asp. niger has been studied. It was found that both rate of growth and glucose oxidase production was higher at an agitation speed of 700 rpm than at 460 rpm. Further increase in speed of agitation resulted in neither a higher rate of growth nor a higher glucose oxidase activity. Total glucose oxidase activity was highest in a medium containing 5% sugar (at an agitation speed of 700 rpm) and did not get higher when the sugar concentration of the medium was increased to 7%. When pure oxygen was bubbled through the culture the rate of growth of the culture (in the linear phase) was 95 mg. mycelial dry wt./100 ml./hr., and only 61 mg. when air was applied. The glucose oxidase activity of oxygenated culture was double the activity of aerated culture. Viscosity of the homogenized culture became higher with higher concentration of mycelia. The viscosity of oxygenated culture was found to be lower than that of aerated culture.     

pH-induced alterations in the structure and activity of cytochrome c oxidase

The effects of pH on the activity and structure of beef heart cytochrome c oxidase have been studied in the pH range 5.0-7.6. (i) A group with pK of approximately 5.45 has been readily detected in the pH vs. activity curve. This group must be deprotonated to achieve maximal activity. (ii) A group with a similar pK (5.45) has been detected and contributes to the spectral character of the reduced oxidase. Over the range pH 5.0-7.6 no other acid-sensitive group contributes to the spectrum of the reduced oxidase. (iii) The oxidized oxidase shows at least three acid-sensitive groups contributing to the spectrum. One occurs in the pH 7 range and another in the pH 5.6 range; below pH 5.2 additional pH-sensitive groups are apparent. Accurate estimation of the pK's of the groups responsible for the spectral changes in the oxidized oxidase has not been possible. (iv) The spectrum of the "oxygenated" (428 nm) conformer of the oxidized protein is invariant over the range ph 5.5-7. (v) The changes occurring in the spectrum of the purified oxidase also occur in the protein contained in phospholipid vesicles. (vi) The data are discussed in terms of the mechanism by which the oxidase, during its in situ catalytic cycle, may give rise to the primary events in energy coupling.     

Two-electron reduction is required for rapid internal electron transfer in resting, pulsed and oxygenated cytochrome c oxidase

The resting as well as the 420 nm and 428 nm forms of cytochrome oxidase have been studied in kinetic experiments with an excess of enzyme over reduced cytochrome c. No difference was found in the behavior of the two activated forms. With all three forms, a fraction of cytochrome a was reoxidized with a rate which was much lower than kcat. This suggests that intramolecular transfer to the dioxygen-reducing site occurs only if both cytochrome a and CuA are reduced. An initial rapid phase in the oxidation of cytochrome a in the pulsed and oxygenated enzymes is related to the presence of a three-electron-reduced dioxygen intermediate. The increased catalytic activity of pulsed and oxygenated oxidase can be explained on the basis of a shift in the redox equilibrium between cytochrome a and CuA.     

Nitric oxide, cytochrome c oxidase and myoglobin: competition and reaction pathways

It is relevant to cell physiology that nitric oxide (NO) reacts with both cytochrome oxidase (CcOX) and oxygenated myoglobin (MbO(2)). In this respect, it has been proposed [Pearce, L.L., et al. (2002) J. Biol. Chem. 277, 13556-13562] that (i) CcOX in turnover out-competes MbO(2) for NO, and (ii) NO bound to reduced CcOX is "metabolized" in the active site to nitrite by reacting with O(2). In contrast, rapid kinetics experiments reported in this study show that (i) upon mixing NO with MbO(2) and CcOX in turnover, MbO(2) out-competes the oxidase for NO and (ii) after mixing nitrosylated CcOX with O(2) in the presence of MbO(2), NO (and not nitrite) dissociates from the enzyme causing myoglobin oxidation.     

A CO-Binding Hemoprotein Derived from Tetrahymena pyriformis

A CO-binding hemoprotein was purified from Tetrahymena pyriformis and some of its properties were studied.

The hemoprotein possessed protoheme, its molecular weight was about 11,000, and its isoelectric point was at pH 8.2. The oxidized form of the hemoprotein showed the Soret band at 406 nm and had no distinct peaks in the region of α- and β-bands, while the reduced form showed the peaks at 426, 527 and 560 nm. The hemoprotein reacted with CO resulting in shift of the Soret band from 426 to 420 nm. The CO-compound showed a broad band from 537 to 573 nm. The hemoprotein was not autoxidizable or oxygenated either. It did not show either of the cytochrome oxidase, peroxidase and NADH oxidase activities.

It should be carefully determined whether or not cytochrome o is functioning as the terminal oxidase in T. pyriformis, as the CO-binding hemoprotein which does not react with molecular oxygen exists in the organism.     

A novel electron paramagnetic resonance signal of "oxygenated" cytochrome c oxidase.

It had been observed previously that a pair of transient EPR resonances (g = 1.78 and 1.69) appears within less than 5 ms on reoxidation of reduced cytochrome c oxidase by O2. Since the location of other lines that are part of the same signal was not known, the quantity of the paramagnetic species involved, and thus the significance of the observed resonances, remained questionable. We have now found a broad resonance at g = 5 which is obviously associated with those at g = 1.78 and 1.69. The width of the signal (approximately 250 mT) at the observed intensity suggests that it represents a significant fraction of one of the components of the enzyme. The signal disappears within less than 5 ms on addition of cyanide or sulfide but only within several hundred milliseconds after addition of ferrocytochrome c. This behavior suggests that it originates from the a3 component of the enzyme. It is suggested that the species represented in the signal is either identical with or part of what has been named collectively the "oxygenated" form and recently described "activated" forms of the enzyme. On reoxidation of reduced oxidase with oxygen enriched 90% in 17O, no change of signal shape was seen.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Mechanism of "uncoupled" tetrahydropterin oxidation by phenylalanine hydroxylase

Phenylalanine hydroxylase can catalyze the oxidation of its tetrahydropterin cofactor without concomitant substrate hydroxylation. We now report that this "uncoupled" tetrahydropterin oxidation is mechanistically distinct from normal enzyme turnover. Tetrahydropterins are oxygenated to 4a-carbinolamines only during catalytic events involving substrate hydroxylation. In the absence of hydroxylation tetrahydropterins are oxidized directly to quinonoid dihydropterins. Stoichiometry studies define a ratio of two tetrahydropterins oxidized per O2 consumed in uncoupled enzyme turnover, thus indicating the complete reduction of O2 to H2O. Complementary results establish the lack of H2O2 production by the enzyme when uncoupled and define a tetrahydropterin oxidase activity for the enzyme. Thus, the hydroxylating intermediate of phenylalanine hydroxylase may be discharged in two ways, by substrate hydroxylation or by electron abstraction. A mechanism is proposed for the uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase, and the significance of these findings is discussed.     

Oxygenated cytochrome o (Vitreoscilla) formed by treating oxidized cytochrome with superoxide anion

Oxygenated cytochrome o can be formed experimentally in twoways, i) by reaction of reduced cytochrome o with molecularoxygen, or ii) by reaction of oxidized cytochrome o with superoxideanion generated by the action of the xanthine oxidase system.It is thermodynamically feasible for oxidized cytochrome o plusO₂–, and reduced cytochrome o plus O₂ to appear as intermediatesin reactions i) and ii), respectively. Superoxide dismutase completely inhibits the xanthine oxidase-catalyzedconversion of oxidized cytochrome o into the oxygenated formbut it has relatively little effect on the oxygenated cytochromeo formation in the reaction system consisting of NADH, NADH-cytochromeo reductase, and cytochrome o. Thus, if superoxide anion doesplay a significant role in the latter system it must be efficientlycoupled to react with cytochrome o and inaccessible to superoxidedismutase. Direct electron transfer from the reductase to thecytochrome without the involvement of superoxide anion is analternative mechanism. (Received December 16, 1976; )