Observation of a novel transient ferryl complex with reduced CuB in cytochrome c oxidase

The reaction between mixed-valence (MV) cytochrome c oxidase from beef heart with H2O2 was investigated using the flow-flash technique with a high concentration of H2O2 (1 M) to ensure a fast bimolecular interaction with the enzyme. Under anaerobic conditions the reaction exhibits 3 apparent phases. The first phase (tau congruent with 25 micros) results from the binding of one molecule of H2O2 to reduced heme a3 and the formation of an intermediate which is heme a3 oxoferryl (Fe4+=O2-) with reduced CuB (plus water). During the second phase (tau congruent with 90 micros), the electron transfer from CuB+ to the heme oxoferryl takes place, yielding the oxidized form of cytochrome oxidase (heme a3 Fe3+ and CuB2+, plus hydroxide). During the third phase (tau congruent with 4 ms), an additional molecule of H2O2 binds to the oxidized form of the enzyme and forms compound P, similar to the product observed upon the reaction of the mixed-valence (i.e., two-electron reduced) form of the enzyme with dioxygen. Thus, within about 30 ms the reaction of the mixed-valence form of the enzyme with H2O2 yields the same compound P as does the reaction with dioxygen, as indicated by the final absorbance at 436 nm, which is the same in both cases. This experimental approach allows the investigation of the form of cytochrome c oxidase which has the heme a3 oxoferryl intermediate but with reduced CuB. This state of the enzyme cannot be obtained from the reaction with dioxygen and is potentially useful to address questions concerning the role of the redox state in CuB in the proton pumping mechanism.     

Heme P460 of hydroxylamine oxidoreductase of Nitrosomonas. Reaction with CO and H2O2

Hydroxylamine oxidoreductase (HAO) of Nitrosomonas catalyzes the dehydrogenation of NH2OH and subsequent addition of oxygen to form nitrite. HAO contains c hemes and the CO-binding heme P460 in a 7:1 ratio; dehydrogenation of NH2OH involves passage of electrons to P460 and then c hemes. We now report that electrons rapidly pass from c hemes of HAO to the P460 center and then to H2O2. This conclusion is supported by (a) inhibition of c heme oxidation with CO and (b) loss of H2O2-oxidizability of ferrous c hemes following specific destruction of heme P460. Reaction of ferrous P460 with H2O2 is rate-limiting. Activation of dioxygen for N-oxidation by ferrous HAO may involve the two-electron reduction of O2 by P460. The reaction of ferrous HAO with H2O2 was studied as it may reveal aspects of the mechanism of activation of dioxygen. Reaction of ferrous heme P460 with CO is slow and with low affinity as compared with other hemoproteins. Values for reaction of CO with enzyme were: k1, 1.1 X 10(-3) M-1 s-1 and Kd, 12 microM.     

The site and mechanism of dioxygen reduction in bovine heart cytochrome c oxidase

The site and mechanism of dioxygen reduction in cytochrome c oxidase from bovine heart muscle have been investigated. The rate of cytochrome c2+ oxidation by O2 is shown to be affected by several factors: 1) pH, with optima at 5.65 and 6.0, 2) temperature between 0 and 29 degrees C, with E alpha = 13 kcal mol-1, 3) D2O exchange, with a reduction in rate of 50% or more at the pH optima, and 4) the addition of ethylene glycol or glycerol, which significantly lowers the rate. The extremely narrow (delta vCO approximately 4 cm-1) infrared stretch bands at approximately 1964 and approximately 1959 cm-1 for liganded CO are only slightly affected by factors 1-4 or by changes in the oxidation state of metals other than the heme alpha 3 iron. These results indicate a stable, unusually immobile O2 reduction site well-isolated from the external medium, a characteristic expected to be important for oxidase function. Precise stereochemical positioning of hydrogen donors adjacent to O2 liganded to heme alpha 3 iron can be expected in order to achieve the optimization of the time/distance relationships required for enzyme catalysis. These findings support a novel mechanism of O2 reduction via a hydroperoxide intermediate within a reaction pocket that experiences little change in conformation during the hydrogen and electron transfer steps.     

Carbon monoxide-driven reduction of ferric heme and heme proteins

Oxidized cytochrome c oxidase in a carbon monoxide atmosphere slowly becomes reduced as shown by changes in its visible spectra and its reactivity toward oxygen. The "auto-reduction" of cytochrome c oxidase by this procedure has been used to prepare mixed valence hybrids. We have found that this process is a general phenomenon for oxygen-binding heme proteins, and even for isolated hemin in basic aqueous solution. This reductive reaction may have physiological significance. It also explains why oxygen-binding heme proteins become oxidized much more slowly and appear to be more stable when they are kept under a CO atmosphere. Oxidized alpha and beta chains of human hemoglobin become reduced under CO much more slowly than does cytochrome c oxidase, where the CO-binding heme is coupled with another electron accepting metal center. By observing the reaction in both the forward and reverse direction, we have concluded that the heme is reduced by an equivalent of the water-gas shift reaction (CO + H2O----CO2 + 2e- + 2H+). The reaction does not require molecular oxygen. However, when the CO-driven reduction of cytochrome c oxidase occurs in the presence of oxygen, there is a competition between CO and oxygen for the reduced heme and copper of cytochrome alpha 3. Under certain conditions when both CO and oxygen are present, a peroxide adduct derived from oxygen reduction can be observed. This "607 nm complex," described in 1981 by Nicholls and Chanady (Nicholls, P., and Chanady, G. (1981) Biochim. Biophys. Acta 634, 256-265), forms and decays with kinetics in accord with the rate constants for CO dissociation, oxygen association and reduction, and dissociation of the peroxide adduct. In the absence of oxygen, if a mixture of cytochrome c and cytochrome c oxidase is incubated under a CO atmosphere, auto-reduction of the cytochrome c as well as of the cytochrome c oxidase occurs. By our proposed mechanism this involves a redistribution of electrons from cytochrome alpha 3 to cytochrome alpha and cytochrome c.     

Oxidation of Methanol,Formaldehyde and Formate by a Candida Species

1. The oxidation of methanol to carbon dioxide by Candida N–16 grown on methanol was investigated. The presence of enzymes which catalyze the following reaction was found in the cell-free extract of the yeast employed; CH₃OH→HCHO→HCOOH→CO₂. 2. Methanol was oxidized to formaldehyde by an alcohol oxidase. The reaction was as follows; CH₃OH+O₂→HCHO+H₂O₂. The alcohol oxidase was crystallized after purification by ammonium sulfate-precipitation and column chromatography using DEAE-Sephadex A-50. A prosthetic group of the enzyme was proved to be FAD. The enzyme possessed a broad specificity for alcohols such as methanol, ethanol, n-propanol, n-butanol and n-amylalcohol. The enzyme was inducibly formed only by the addition of methanol. 3. The oxidation of formaldehyde to formate was catalyzed by a NAD-linked dehydrogenase dependent on GSH. 4. Formate was oxidized by a NAD-linked dehydrogenase. 5. Catalase was also found in the extract, and methanol was chemically oxidized by the reaction of catalase and hydrogen peroxide which was generated by the alcohol oxidase system. 6. The oxidation pathway from methanol to carbon dioxide was also found in other methanol-utilizing yeasts such as Candida N-17, Saccharomyces H-1 and Torulopsis M-1.     

Dioxygen activation by putidamonooxin. The oxygen species formed and released under uncoupling conditions

In the presence of substrates not favourable for hydroxylation, more than 80% of the dioxygen consumed by purified, reconstituted 4-methoxybenzoate monooxygenase appears in the reaction mixture as hydrogen peroxide. We have investigated whether under these conditions (a) reduced putidamonooxin, the oxygenase of this enzyme system, either autoxidizes in the presence of dioxygen, with liberation of superoxide anion radicals which then disproportionate to H2O2 and O2, or (b) dioxygen is reduced by two sequential single-electron steps leading to the active oxygen species that forms hydrogen peroxide directly when inactivated by protonation. Quantitative estimation of O-2 radicals, with either succinylated ferricytochrome c or epinephrine used as O-2 scavengers, revealed that only about 6% of the total electron flux channelled via putidamonooxin to dioxygen led to the monovalent reduction on dioxygen. This means that not more than 3% of the hydrogen peroxide found under uncoupling conditions arises from the rapid bimolecular disproportionation of initially formed O-2 radicals. Inconsistent results were obtained when lactoperoxidase was used as an O-2 trap. Our measurements indicate that the conversion of lactoperoxidase into compound III is an inappropriate method of detecting any O-2 radicals that may be found by the uncoupled 4-methoxybenzoate monooxygenase. The stoichiometry of about 1:1 for O2 uptake: H2O2 formation indicates that under uncoupling conditions H2O is virtually not formed. The role of [FeO2]+ as the active oxygenating species of putidamonooxin is discussed.     

Direct detection of a dioxygen adduct of cytochrome a3 in the mixed valence cytochrome oxidase/dioxygen reaction

Time-resolved resonance Raman spectra have been recorded during the reaction of mixed valence (a3+ a2+(3)) cytochrome oxidase with dioxygen at room temperature. In the spectrum recorded at 10 microseconds subsequent to carbon monoxide photolysis, a mode is observed at 572 cm-1 that shifts to 548 cm-1 when the experiment is repeated with 18O2. The appearance of this mode is dependent upon the laser intensity used and disappears at higher incident energies. The high frequency data in conjunction with the mid-frequency data allow us to assign the 572 cm-1 mode to the Fe-O stretching vibration of the low-spin O2 adduct that forms in the mixed valence cytochrome oxidase/dioxygen reaction. The 572 cm-1 v(Fe2(+)-O2) frequency in the mixed valence enzyme/O2 adduct is essentially identical to the 571 cm-1 frequency we measured for this mode during the reduction of O2 by the fully reduced enzyme (Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1989) J. Am. Chem. Soc. 111, 6439-6440; Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1990) J. Am. Chem. Soc. 112, 1297), which indicates that the O2-bound cytochrome a3 site is independent of the redox state of the cytochrome a/CuA pair. The photolabile oxy intermediate is replaced by photostable low- or intermediate-spin cytochrome a3+(3), with t1/2 congruent to 200 microseconds.     

Amine oxidase from Lathyrus cicera and Phaseolus vulgaris: purification and properties

Cu-Amine oxidases (amine oxygen oxidoreductase deaminating, copper containing E.C. 1.4.3.6.) are found in all forms of life (1). They catalyze the following general reaction: R-CH2-NH2 + O2 + H2O----R-CHO + NH3 + H2O2. Cu-amine oxidases (Cu-AOs) have been extracted from different leguminosae: Pisum sativum (2-3), Lathyrus sativus (4), Lens esculenta (5), Vicia faba (6), Cicer arietinum (7), Glycine max (8) but not from Phaseolus vulgaris. Palavan and Galston (9), in a study of polyamine biosynthesis during developmental stages of Phaseolus vulgaris, did not detect diamine or polyamine oxidase activity in Phaseolus. The present paper describes the purification of Phaseolus vulgaris seedlings amine oxidase (PhSAO) and also compares the properties of this enzyme to the Lathyrus cicera enzyme (LcSAO), obtained with the same method of purification.     

The oxidation of cytochrome c oxidase by hydrogen peroxide

The reaction of H2O2 with mixed-valence and fully reduced cytochrome c oxidase was investigated by photolysis of fully reduced and mixed-valence carboxy-cytochrome c oxidase in the presence of H2O2 under anaerobic conditions. The results showed that H2O2 reacted rapidly (k = (2.5-3.1) X 10(4) M-1 X s-1) with both enzyme species. With the mixed-valence enzyme, the fully oxidised enzyme was reformed. On the time-scale of our experiments, no spectroscopically detectable intermediate was observed. This demonstrates that mixed-valence cytochrome c oxidase is able to use H2O2 as a two-electron acceptor, suggesting that cytochrome c oxidase may under suitable conditions act as a peroxidase. Upon reaction of H2O2 with the fully reduced enzyme, cytochrome a was oxidised before cytochrome a3. From this observation it was possible to estimate that the rate of electron transfer from cytochrome a to a3 is about 0.5-5 s-1.     

Cytochrome oxidase-catalyzed superoxide generation from hydrogen peroxide.

Superoxide dismutase is shown to affect spectral changes observed upon cytochrome c oxidase reaction with H2O2, which indicates a possibility of O2- radicals being formed in the reaction. Using DMPO as a spin trap, generation of superoxide radicals from H2O2 in the presence of cytochrome oxidase is directly demonstrated. The process is inhibited by cyanide and is not observed with a heat-denatured enzyme pointing to a specific reaction in the oxygen-reducing centre of cytochrome c oxidase. The data support a hypothesis on a catalase cycle catalyzed by cytochrome c oxidase in the presence of excess H2O2 (Vygodina and Konstantinov (1988) Ann. NY Acad. Sci., 550, 124-138): (formula: see text)     

Oxidation of reduced cytochrome c oxidase with 18O2. A search for mu-oxo-bridged metal species in the oxidized enzyme

We have measured the increase in 18O content of water produced from single turnover oxidations of anerobically reduced cytochrome c oxidase with 18O2 in order to test the hypothesis that a reduced atom of oxygen, originating from dioxygen, remains bound to oxidized cytochrome c oxidase in the form of a mu-oxo-bridge between two metal components when a single turnover occurs. When water samples produced by oxidizing the reduced enzyme with 18O2 were compared to natural abundance control samples obtained by oxidizing with 16O2, all of the 18O2 reduced in a single turnover could be accounted for in the form of additional H218O produced. We conclude that neither atom of the dioxygen reduced is incorporated into the enzyme as a bridge which is stable in the absence of oxidoreductive reactions on the time scale of several minutes.     

Formation and reduction of a ''peroxy'' intermediate of cytochrome c oxidase by hydrogen peroxide.

In the presence of micromolar concentrations of H2O2, ferric cytochrome c oxidase forms a stable complex characterized by an increased absorption intensity at 606-607 nm with a weaker absorption band in the 560-580 nm region. Higher (millimolar) concentrations of H2O2 result in an enzyme exhibiting a Soret band at 427 nm and an alpha-band of increased intensity in the 589-610 nm region. Addition of H2O2 to ferric cytochrome c oxidase in the presence of cyanide results in absorbance increases at 444nm and 605nm. These changes are not seen if H2O2 is added to the cyanide complex of the ferric enzyme. The results support the idea that direct reaction of H2O2 with ferric cytochrome a 3 produces a 'peroxy' intermediate that is susceptible to further reduction by H2O2 at higher peroxide concentrations. Electron flow through cytochrome a is not involved, and the final product of the reaction is the so-called 'pulsed' or 'oxygenated' ferric form of the enzyme.     

Mitochondrial oxygenation of carbon monoxide.

A variety of mitochondria have been observed to oxidize 13CO to 13CO2 in the presence of dioxygen, and on the basis of earlier studies [Young & Caughey (1986) Biochemistry 25, 152-161; Young (1981) Ph.D. Dissertation, Colorado State University] this activity is attributed to cytochrome c oxidase. Implications of these findings in respect of some aspects of the pathological biochemistry of CO poisoning are discussed.     

Mutations at Asp112 adjacent to the trinuclear Cu center in CueO as the proton donor in the four-electron reduction of dioxygen

Asp112 adjacent to the trinuclear Cu center of a multicopper oxidase, CueO was mutated for Glu, Ala and Asn. Mutations on Asp112 affected not only spectroscopic and magnetic properties derived from the trinuclear Cu center but also enzyme activities. The uncoordinated Asp112 was found to play multiple roles to promote the binding of dioxygen at the trinuclear Cu center and to accelerate the conversion of dioxygen to water molecules by facilitating the supply of H+ to the reaction intermediates.     

The dioxygen cycle. Spectral, kinetic, and thermodynamic characteristics of ferryl and peroxy intermediates observed by reversal of the cytochrome oxidase reaction.

The catalytic mechanism of O2 reduction by cytochrome oxidase was studied in isolated mitochondria and mitoplasts by partial reversal of the reaction. At a high redox potential (Eh) of cytochrome c, high pH, and a high electrochemical proton gradient (delta mu H+) across the inner mitochondrial membrane, the initial ferriccupric state (O) of the oxidized enzyme's bimetallic oxygen reaction center is converted to ferryl (F) and peroxy (P) intermediates, the optical spectroscopic properties of which are reported in detail. This is associated with reversed electron transfer from the bimetallic center to ferricytochrome c. The kinetics of reduction of ferricytochrome c by the reversed electron transfer process are compared with the kinetics of formation of F and P. The results are consistent with transfer of one electron from the ferric-cupric bimetallic center (O) to cytochrome c, yielding the F intermediate, followed by transfer of one electron from the latter to cytochrome c, yielding the P state. In the absence of an effective redox buffer, poising cytochrome c highly oxidized, these primary events are immediately followed by reoxidation of cytochrome c, which is ascribed to forward electron transfer to enzyme molecules still in the O state. This forward reaction also results in accumulation of the P intermediate. Kinetic stimulations of the data predict equilibrium constants for the reversed electron transfer steps, and Em,7 values of approximately 1.1 and 1.2 V may be calculated for the F/O and P/F redox couples, respectively, at delta mu H+ and delta psi equal to zero. Taken together with previously measured Em,7 values, these data indicate that it is the two-electron reduction of bound dioxygen to bound peroxide that is responsible for the irreversibility of the catalytic dioxygen cycle of cell respiration.     

Time-resolved optical absorption studies of cytochrome oxidase dynamics

Time-resolved spectroscopic studies in our laboratory of bovine heart cytochrome c oxidase dynamics are summarized. Intramolecular electron transfer was investigated upon photolysis of CO from the mixed-valence enzyme, by pulse radiolysis, and upon light-induced electron injection into the cytochrome c/cytochrome oxidase complex from a novel photoactivatable dye. The reduction of dioxygen to water was monitored by a gated multichannel analyzer using the CO flow-flash method or a synthetic caged dioxygen carrier. The pH dependence of the intermediate spectra suggests a mechanism of dioxygen reduction more complex than the conventional unidirectional sequential scheme. A branched model is proposed, in which one branch produces the P form and the other branch the F form. The rate of exchange between the two branches is pH-dependent. A cross-linked histidine-phenol was synthesized and characterized to explore the role of the cross-linked His-Tyr cofactor in the function of the enzyme. Time-resolved optical absorption spectra, EPR and FTIR spectra of the compound generated after UV photolysis indicated the presence of a radical residing primarily on the phenoxyl ring. The relevance of these results to cytochrome oxidase function is discussed.     

Kinetics of reduction of cytochrome c oxidase by dithionite and the effect of hydrogen peroxide

The reduction of cytochrome c oxidase by dithionite was reinvestigated with a flow-flash technique and with varied enzyme preparations. Since cytochrome a3 may be defined as the heme in oxidase which can form a photolabile CO adduct in the reduced state, it is possible to follow the time course of cytochrome a3 reduction by monitoring the onset of photosensitivity. The onset of photosensitivity and the overall rate of heme reduction were compared for Yonetani and Hartzell-Beinert preparations of cytochrome c oxidase and for the enzyme isolated from blue marlin and hammerhead shark. For all of these preparations the faster phase of heme reduction, which is dithionite concentration-dependent, is almost completed when the fraction of photosensitive material is still small. We conclude that cytochrome a3 in the resting enzyme is consistently reduced by an intramolecular electron transfer mechanism. To determine if this is true also for the pulsed enzyme, we examined the time course of dithionite reduction of the peroxide complex of the pulsed enzyme. It has been previously shown that pulsed cytochrome c oxidase can interact with H2O2 and form a stable room temperature peroxide adduct (Bickar, D., Bonaventura, J., and Bonaventura, C. (1982) Biochemistry 21, 2661-2666). Rather complex kinetics of heme reduction are observed when dithionite is added to enzyme preparations that contain H2O2. The time courses observed provide unequivocal evidence that H2O2 can, under these conditions, be used by cytochrome c oxidase as an electron acceptor. Experiments carried out in the presence of CO show that a direct dithionite reduction of cytochrome a3 in the peroxide complex of the pulsed enzyme does not occur.     

Photosynthetic water oxidation vs. mitochondrial oxygen reduction: distinct mechanistic parallels

The photosynthetic oxygen evolving complex (PSII-OEC) and the mitochondrial cytochrome c oxidase (CcO) not only catalyze anti-parallel reactions (the OEC oxidizes water to dioxygen, whereas CcO reduces dioxygen to water), they also share a number of uncanny molecular and mechanistic similarities. Both feature a redox-active polymetallic cluster that includes a key tyrosine, and both utilize a two-phase mechanism. In one phase the polymetallic cluster undergoes four sequential one-electron transfers: In the PSII-OEC, four successive photooxidations of the photosystem II reaction center P680 (to P680⁺) allows acceptance of 4 × 1e- from the Mn₄Ca cluster; in CcO, four reduced cytochrome c Fe²⁺ cations donate 4 × 1e- to the bimetallic center. In the second phase for each enzyme, the polymetallic cluster undergoes a single four-electron transfer with the O₂/2 H₂O redox couple. Intriguing mechanistic similarities between these two complex redox enzymes first delineated over a decade ago by Hoganson/Proshlyakov/Babcock et al. are updated and expanded in this article.     

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.