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.     

Oxidation of sulphide by cytochrome aa3

The effectiveness of H2S as an inhibitor of cytochrome c oxidase increase (Ki decreases) with sulphide concentration. A spectroscopic change in cytochrome aa3 is induced aerobically by sulphide at the same rate as that calculated for inhibition. The initial spectroscopic product is not inhibited, but an 'oxygenated' (oxyferri) form of the enzyme. Stoichiometric sulphide addition to cytochrome aa3 under anaerobic conditions produces another low-spin form of the enzyme; subsequent admission of oxygen gives rise to the 607 nm compound. At high enzyme levels sulphide itself acts as a substrate measured polarographically, with an oxygen uptake proportional to the amount of sulphide added. Binding of sulphide to ferric enzyme probably causes reduction at the oxygen-sensitive a3-Cu centre, which is followed aerobically by reoxidation to the oxyferri state via the 607 nm intermediate. A stable sulphide complex is formed only after the reduction of cytochrome a; but once formed this inhibited species is retained if cytochrome a is reoxidized.     

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.     

Effect of pH on the spectrum of cytochrome c oxidase hydrogen peroxide complex

Hydrogen peroxide binding to ferric cytochrome c oxidase in proteoliposomes brings about a red-shift of the enzyme Soret band and increased absorption in the visible range with two prominent peaks at approx. 570 and 607 nm. The molar absorptivity of the H2O2-induced difference spectrum is virtually pH-independent in the Soret band and at 570 nm, whereas the peak at 607 nm increases approx. 3-fold upon alkalinization in a narrow pH range 6.0-7.2, the effect being reversible. The pH profile of this transition indicates ionization of two acid-base groups with close pK values of 6.7. The lineshape of the peroxide compound difference spectrum is found to respond to pH changes inside the proteoliposomes. It is suggested that peroxide-complexed enzyme can undergo a pH-dependent transition to a form with increased extinction at 605-607 nm, possibly corresponding to the 420 nm (or 'pulsed') conformer of the ferric cytochrome oxidase formed as an early product of the enzyme oxidation. Accordingly, relaxation of the '420 nm' form to the resting state would be linked to an uptake of two protons from the M-aqueous phase. This protolytic reaction might be a partial step of the cytochrome oxidase proton pumping mechanism or it could serve to regulate interconversion between the active 'pulsed' and less active 'resting' states of the enzyme in the membrane.     

Siroheme: a prosthetic group of the Neurospora crassa assimilatory nitrite reductase.

The Neurospora crassa assimilatory nitrite reductase (EC 1.6.6.4) catalyzes the NADPH-dependent reduction of nitrite to ammonia, a 6-electron transfer reaction. Highly purified preparations of this enzyme exhibit absorption spectra which suggest the presence of a heme component (wavelength maxima for oxidized senzyme: 390 and 578 nm). There is a close correspondence between nitrite reductase activity and absorbance at 400 nm when partially purified nitrite reductase preparations are subjected to sucrose gradient centrifugation. In addition, a role for an iron component in the formation of active nitrite reductase is indicated by the fact that nitrate-induced production of nitrite reductase activity in Neurospora mycelia in vivo requires the presence of iron in the induction medium. The heme chromophore present in Neurospora nitrite reductase preparations is reducible by NADPH. Complete reduction, however, requires the presence of added FAD. The NADPH-nitrite reductase activity of the enzyme is also dependent upon addition of FAD. A spectrally unique complex is formed between the heme chromophore and nitrite (or a reduction product thereof) when nitrite is added to NADPH-reducted enzyme. Carbon monoxide forms a complex with the heme chromophore of nitrite reductase with an intense alpha-band maximum at 590 nm and a beta-band of lower intensity at 550 nm. CO is an inhibitor of NADPH-nitrite reductase activity. Spectrophotometrically detectable CO complex formation and Co inhibition of enzyme activity share the following properties...     

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.     

Purification and properties of a cytochrome b560-d complex, a terminal oxidase of the aerobic respiratory chain of Photobacterium phosphoreum

A cytochrome b560-d complex, a terminal oxidase in the respiratory chain of Photobacterium phosphoreum grown under aerobic conditions, was purified to near homogeneity. The purified oxidase complex is composed of equimolar amounts of two polypeptides with molecular weights of 41,000 and 54,000, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains 10.2 nmol of protoheme and 22.5 nmol of iron/mg of protein. The enzyme is a "cytochrome bd-type oxidase," showing absorption peaks at 560 and 625 nm in its reduced minus oxidized difference spectrum at 77K. This oxidase combined with CO, and its CO difference spectrum at room temperature in the Soret region showed a peak at 418 nm and a trough at 434 nm. In addition, a trough at 560 nm (cytochrome b), and a trough at 620 nm and a peak at 639 nm (cytochrome d) were observed in the CO-binding spectrum. This cytochrome b560-d complex catalyzed the oxidation of ubiquinol-1 and ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride or phenazine methosulfate. The oxidase activity required phospholipids and was inhibited by the respiratory inhibitors, KCN and NaN3, and the divalent cation, ZnSO4. Formation of a membrane potential by the cytochrome b560-d complex reconstituted into liposomes was observed with the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide, on the addition of ubiquinol-1, showing that the enzyme provided a coupling site for oxidative phosphorylation.     

A new carbon monoxide-induced complex of cytochrome c oxidase.

Cytochrome c oxidase isolated from ox heart forms a complex in the presence of millimolar concentrations of CO with absorption bands at 606, 565 and 435 nm (difference spectrum), distinct from both ferrocytochrome a and the classical 590nm carbon-monoxyferrocytochrome a3. This species, which closely resembles Compound C, the derivative formed on photolysis and oxygenation of mixed-valence cytochrome a3+a32+CO, may represent a cytochrome a32+CO complex in which the associated ('invisible') copper is still oxidized.     

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.     

Purification and properties of a peroxidase from Halobacterium halobium L-33

A peroxidase was purified from Halobacterium halobium L-33 to an electrophoretically homogeneous state and some of its properties were studied. The enzyme showed an absorption peak at 406 nm in the oxidized form and peaks at 440, 558, and 591 nm in the reduced form. The difference spectrum, reduced + CO minus reduced, of the enzyme showed peaks at 425, 538, and 577 nm and troughs at 444, 562, and 596 nm. These spectral properties were apparently similar to those of "cytochrome a1" except for the occurrence of the peak at 558 nm in the reduced form. The molecular weight of the enzyme was 110,000 and the enzyme possessed one unit of protoheme in the molecule. The activity to oxidize guaiacol in the presence of H2O2 of the peroxidase was about one-twentieth of that of horseradish peroxidase. The enzyme also showed a catalase-activity one-fourth as active as that of liver catalase. The reactions catalyzed by the enzyme were strongly inhibited by KCN.     

Evidence for a proposed intermediate redox state in the CO/CO(2) active site of acetyl-CoA synthase (Carbon monoxide dehydrogenase) from Clostridium thermoaceticum

When samples of the enzyme in the C(red1) state were reduced with Ti(3+) citrate, the C-cluster stabilized in an EPR-silent state. Subsequent treatment with CO or dithionite yielded C(red2). The EPR-silent state formed within 1 min of adding Ti(3+) citrate, while C(red2) formed after 60 min. Ti(3+) citrate appeared to slow the rate by which C(red2) formed from C(red1) and stabilize the C-cluster in the previously proposed C(int) state. This is the first strong evidence for C(int), and it supports the catalytic mechanism that required its existence. This mechanism is analogous to those used by flavins and hydrogenases to convert between n = 2 and n = 1 processes. Ti(3+) citrate had a different effect on enzyme in a CO(2) atmosphere; it shifted reduction potentials of metal centers (relative to those obtained using CO) and did not stabilize C(int). Different redox behavior was also observed when methyl viologen and benzyl viologen were used as reductants. This variability was exploited to prepare enzyme samples in which EPR from C(red2) was present without interfering signals from B(red). The saturation properties of B(red) depended upon the redox state of the enzyme. Three saturation "modes", called Sat1-Sat3, were observed. Sat1 was characterized by a sharp g = 1.94 resonance and low-intensity g = 2. 04 and 1.90 resonances, and was observed in samples poised at slightly negative potentials. Sat2 was characterized by weak intensity from all three resonances, and was strictly associated with intermediate redox states and the presence of CO(2). Sat3 was characterized by strong broad resonances with normalized intensities essentially unchanged relative to nonsaturating conditions, and was observed at the most negative potentials. Each mode probably reflects different spatial relationships among magnetic components in the enzyme.     

Induction of photochemical auto-reduction of cytochrome-c oxidase by an organic peroxide

Cytochrome-c oxidase aa3 (CcO) from Paracoccus denitrificans interacts with tertiary butyl hydroperoxide (t-Bu-O-O-H, TBHP) by forming an adduct as indicated by an absorption shift at 408/432 nm and the induction of photochemical autoreduction. The adduct was stable at room temperature for several days even under aerobic conditions. Upon irradiation (413 nm) of the adduct, a photoproduct, similar to the oxygenated mixed valence species (607 nm form), was formed, as indicated by the 418/442 and 607 nm signals in the absorption-difference spectrum. It is concluded that the adduct formation changes the photochemical properties of heme a3. A molecular model for the binding mechanism of TBHP to CcO and for the photochemistry of heme a3-TBHP adduct is proposed.     

The oxygenated complexes of the two catalytically active oxidation-reduction states of L-tryptophan-2,3-dioxygenase.

The oxygenated complexes of the two catalytically active forms of pseudomonad and rat liver L-tryptophan-2,3-dioxygenase (EC 1.13.11.11) have been studied. As was previously reported (ISHIMURA, Y., NORZAKI, M., HAYAISHI, O., TAMURA, M., AND YAMAZAK-I I. (1970) J. Biol. Chem. 245, 3593-3602), we observe that the fully reduced form of pseudomonad tryptophan oxygenase during steady state catalysis exists predominantly as the L-tryptophan ferroheme-O2 enzyme complex (lambdamax = 415 nm, 540 nm, 570 nm). However, during steady state catalysis by a half-reduced form of both the pseudomonad and hepatic enzymes, the predominant species present manifest absorption spectra indicative of ternary complexes in which all the heme exists as ferriheme (Soret, 407 nm), there being no trace of a ferroheme-O2 complex. Carbon monoxide is a competitive inhibitor with respect to molecular oxygen of catalysis by either the half-reduced or fully reduced forms of pseudomonad tryptophan oxygenase. During steady state catalysis in the presence of CO, the fully reduced form of the enzyme exists as a mixture of the oxyferroheme (Soret = 415 nm) and carboxyferroheme (Soret = 421 nm) enzyme complexes. However, if the same experiment is repeated with the half-reduced form of the pseudomonad enzyme, all of the enzyme is in the ferriheme state, even though CO is inhibiting this form of the enzyme to the same degree as it does the fully reduced form. We conclude that for the half-reduced form of pseudomonad tryptophan oxygenase the substrate, O2, and the inhibitor, CO, are not binding to the heme moieties, but are bound elsewhere, presumably to the Cu(I) moieties. Examination of the kinetic mechanisms of the half-reduced and fully reduced forms of pseudomonad tryptophan oxygenase using the inhibitors carbon monoxide and 5-fluorotryptophan confirmed that the fully reduced enzyme binds L-tryptophan before O2 (FORMAN, H., AND FEIGELSON, P. (1971) Biochemistry 10, 760-763) and that for the half-reduced enzyme O2 binds first. In the presence of 5-fluorotryptophan a relatively stable oxyferroheme enzyme complex was generated with the fully reduced form of pseudomonad tryptophan oxygenase. Thus, saturation of the catalytic site alone either with the substrate, L-tryptophan, or the competitive inhibitor, 5-fluorotryptophan, enhances binding of O2 to the ferroheme moieties of the enzyme. The resistance of this complex to photolysis indicates that the bound molecular oxygen is predominantly present as superoxide, O2-minus.     

Binding of ligands and spectral shifts in cytochrome c oxidase

1. On addition of reductant (ascorbate plus NNN'N'-tetramethyl-p-phenylenediamine) to isolated cytochrome c oxidase (ox heart cytochrome aa(3)), in the presence of the inhibitors azide or cyanide, an initial partially reduced species is formed with absorption peaks at 415nm, 445nm and 605nm, which slowly gives rise to the final ;half-reduced' species in whose spectrum the 415nm peak has disappeared and a new absorption is seen at 430-435nm. 2. In the absence of reductant, cyanide forms an initial complex with the enzyme with a spectrum similar to that of the uncombined form, which slowly changes into the ;low-spin' cyanide form with a peak at 432nm. Azide, in absence of reductant, shifts the Soret peak slightly, but the resulting complex, which is probably thermally ;mixed-spin', undergoes no further changes. 3. The Soret-peak shift of oxidized cytochrome a(3) which occurs on reduction of the enzyme in the presence of azide is accompanied by a concurrent blue shift of the ferrous cytochrome a peak from 605nm to 603nm. A partial blue shift of the alpha-peak occurs in the half-reduced sulphide-inhibited enzyme, and a complete blue shift is seen in the analogous complexes with alkyl sulphides [a(2+)a(3) (3+)HSR compounds, where R=CH(3), C(2)H(5) or (CH(3))(2)CH]. 4. Analogous, albeit less readily decipherable, spectroscopic effects with the ligands imidazole and alkyl isocyanides suggest that on reduction of cytochrome a an interaction occurs between the two haem groups involving (i) a high- to low-spin change in cytochrome a(3), and after this, (ii) a change in the molecular environment of the cytochrome a. The latter effect, possibly a decrease in the hydrophobicity of the haem pocket, requires that the ligands on cytochrome a(3) have a bulky and partially hydrophobic character.     

Magnetic circular dichroism study of cytochrome ba3 from Thermus thermophilus: spectral contributions from cytochromes b and a3 and nanosecond spectroscopy of CO photodissociation intermediates.

Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented. The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified. TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex. There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase [Goldbeck, R. A., Dawes, T. D., Einarsdóttir, O., Woodruff, W. H., & Kliger, D. S. (1991) Biophys. J. 60, 125-134]. Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later. Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein. The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3. Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3. The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin.     

A temperature-induced absorption band centered in the region of 666 nm related to the configuration of the active site in frozen cytochrome oxidase.

The existence of a temperature-induced absorption band centred in the region of 666 nm is demonstrated for both membrane-bound and soluble cytochrome oxidase in the frozen state. The 666 nm band is generated solely by an increase in temperature of both fully reduced and mixed valence state cytochrome oxidase in the presence of CO or O2 within the 'pocket' containing the active site; it is not formed in the absence of both CO and O2 from the sample. The formation of the 666 nm band is entirely reversible when the temperature is decreased again and its formation is not dependent on the presence of liganded CO at the sixth coordination site of haem a3 in the low temperature range (below --120 degrees C) prior to photolysis. The shape and intensity of the 666 nm band are not affected by the extent of CO recombination following flash and photolysis and temperature increase and are not affected by changes in the valence states of the four metal centres when the O2 reaction is in progress.     

Spectroscopic and equilibrium properties of the indoleamine 2,3-dioxygenase-tryptophan-O2 ternary complex and of analogous enzyme derivatives. Tryptophan binding to ferrous enzyme adducts with dioxygen, nitric oxide, and carbon monoxide

The dioxygen adduct of the heme protein indoleamine 2,3-dioxygenase has been generated at -30 degrees C in mixed solvents, and spectroscopic and equilibrium studies of its L-tryptophan (substrate) binding properties have been carried out for the first time. Comparative studies have also been performed with the NO and CO adducts of the ferrous enzyme. Under the conditions employed (-30 degrees C), both autoxidation and turnover (L-tryptophan + O2----formylkynurenine) of the ternary complex are effectively suppressed. Structural identification of the ternary complex is based on the 1:1 molar stoichiometry for the substrate-oxygenated enzyme adduct formation (Kd approximately 10(-4) M), the time-dependent linear product formation (turnover) at -20 degrees C, and the quantitative conversion of the complex to the ferrous CO derivative by bubbling with CO. Binding of L-tryptophan to the oxygenated enzyme leads to decreases in the intensities of its major absorption bands (lambda max 415, 541, 576 nm) and to a blue shift of its Soret peak. Interestingly, among the ferrous enzyme derivatives examined, only the substrate-bound oxygenated enzyme exhibits solvent-dependent Soret absorption peak positions, e.g., lambda max 411.5 and 413.5 nm in 65% (v/v) aqueous glycerol and ethylene glycol, respectively. In addition, indole binds to the oxygenated enzyme, causing a red shift of its Soret peak in these solvents only in the presence of substrate (411.5----414 nm and 413.5----414.5 nm, respectively), while similar effects of indole are independent of tryptophan for the other ferrous enzyme derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of monosubstituted hydrazines and diazenes with rat-liver cytochrome P450. Formation of ferrous-diazene and ferric sigma-alkyl complexes

The alkyldiazenes RN = NH (R = CH3 or C2H5) react with reduced microsomal cytochrome P450 leading to complexes exhibiting a Soret peak at 446 nm. Upon oxidation of the [cytochrome P450-Fe(II)(CH3N = NH)] complex with limited amounts of dioxygen, a new complex characterized by a Soret peak at 486 nm is formed. The latter complex was also formed upon slow reaction of methyldiazene with microsomal cytochrome P450-Fe(III) or in situ oxidation of methylhydrazine by limited amounts of O2 or ferricyanide. This complex is rapidly destroyed by O2 or ferricyanide in excess and more slowly by excess dithionite in the presence of CO. Reactions of ethyldiazene or benzyldiazene with cytochrome P450-Fe(III) afforded similar complexes characterized by Soret peaks around 480 nm. These results, when compared to those recently described on reactions of monosubstituted hydrazines RNHNH2 and diazenes RN = NH with hemoglobin and iron-porphyrins, are consistent with a [cytochrome P450-Fe(II)(RN = NH)] structure for the 446-nm-absorbing complexes and a sigma-alkyl cytochrome P450-Fe(III)-R structure for the complexes characterized by a Soret peak around 480 nm. They also suggest a sigma-cytochrome P450-Fe(III)-Ph structure for the complex derived from phenylhydrazine oxidation, recently described in the literature. Finally, they provide the first evidence that cytochrome P450-Fe(III)-R complexes are formed upon microsomal oxidation of alkyl or phenylhydrazines.     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

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.