Evidence for two H2O2-binding sites in ferric cytochrome c oxidase. Indication to the O-cycle?

H2O2 addition to the oxidized cytochrome c oxidase reconstituted in liposomes brings about a red shift of the Soret band of the enzyme and an increased absorption in the visible region with two distinct peaks at approximately 570 and 605 nm. Throughout pH range 6-8.5, the spectral changes at 570 nm and in the Soret band titrate with very similar pH-independent Kd values of 2-3 microM. At the same time, Kd of the peroxide complex measured at 605 nm increases markedly with increased H+ activity reaching the value of 18 +/- 2 microM at pH 6.0. This finding may indicate the presence of two different H2O2-binding sites in the enzyme with different affinity for the ligand at acid pH. The Soret and 570 nm band effects are suggested to report H2O2 coordination to heme iron of alpha 3, whereas the maximum at 605 nm could arise from H2O2 binding to Cu alpha 3 followed by the enzyme transition into the 'pulsed' (or '420/605') conformation. Possible implication of the two H2O2-binding sites for the cytochrome oxidase redox and proton-pumping mechanisms are discussed.     

Near-infrared magnetic and natural circular dichroism of cytochrome c oxidase.

A detailed study is presented of the room-temperature absorption, natural and magnetic circulation-dichroism (c.d. and m.c.d.) spectra of cytochrome c oxidase and a number of its derivatives in the wavelength range 700-1900 nm. The spectra of the reduced enzyme show a strong negative c.d. band peaking at 1100nm arising from low-spin ferrous haem a and a positive m.c.d. peak at 780nm assigned to high-spin ferrous haem a3. Addition of cyanide ion doubles the intensity of the low-spin ferrous haem c.d. band and abolishes reduced carbonmonoxy derivative the haem a32+-CO group shows no c.d. or m.c.d. bands at wavelengths longer than 700nm. A comparison of the m.c.d. spectra of the oxidized and cyanide-bound oxidized forms enables bands characteristic of the high-spin ferric form of haem a33+ to be identified between 700 and 1300nm. At wavelengths longer than 1300nm a broad positive m.c.d. spectrum, peaking at 1600nm, is observed. By comparison with the m.c.d. spectrum of an extracted haem a-bis-imidazole complex this m.c.d. peak is assigned to one low-spin ferric haem, namely haem a3+. On binding of cyanide to the oxidized form of the enzyme a new, weak, m.c.d. signal appears, which is assigned to the low-spin ferric haem a33+-CN species. A reductive titration, with sodium dithionite, of the cyanide-bound form of the enzyme leads to a partially reduced state in which low-spin haem a2+ is detected by means of an intense negative c.d. peak at 1100 nm and low-spin ferric haem a33+-CN gives a sharp positive m.c.d. peak at 1550nm. The c.d. and m.c.d. characteristics of the 830nm absorption band in oxidized cytochrome c oxidase are not typical of type 1 blue cupric centres.     

Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing.

1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

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.     

Characterisation of a near infra-red absorption band of the Escherichia coli quinol oxidase, cytochrome o, which is attributable to the high-spin ferrous haem of the binuclear site.

The bacterial quinol oxidase, cytochrome o, is an enzyme which is highly analogous to the better known cytochrome c oxidase, cytochrome aa3, but with the important difference that it lacks the near infra-red absorbing pigment CuA. In this article we report an absorption band in the near IR spectrum of cytochrome o with a maximal absorption at 758 nm, and which is attributable to the ferrous high-spin haem. The 758 nm band has an extinction coefficient of 0.2-0.3 mM-1.cm-1 at 758-800 nm. This region in cytochrome aa3 is dominated by the CuA absorption. The 758 nm absorption is lost on addition of CO or cyanide to the reduced enzyme. The carbon monoxide compound of cytochrome o also has absorbance bands in the near infra-red, and these may be attributable to a low-spin ferrous haem compound.     

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)     

Cytochrome oxidase of Nitrobacter agilis: isolation by hydrophobic interaction chromatography

Cytochrome oxidase has been purified from Nitrobacter agilis using hydrophobic interaction chromatography. The purified preparation contained 3-5% phospholipid and migrated as a single band during polyacrylamide gel electrophoresis under nondissociating conditions, but appeared as three bands in the presence of sodium dodecyl sulfate and 6 M urea. These three bands corresponded to molecular weights of 37 000, 25 000, and 13 000. The absorption spectra of cytochrome oxidase isolated from Nitrobacter were similar to those reported for a-type cytochrome oxidase from other sources and exhibited absorption maxima at 420 and 600 nm when oxidized and 443 and 606 nm when reduced. The purified enzyme reacted both with horse heart and Nitrobacter cytochrome c. The enzymatic activity depended upon the pH of reaction mixture, with the maximum activity at pH 6.5 and 7.5 for Nitrobacter and horse heart cytochrome c, respectively. The activity of the purified enzyme was inhibited by cyanide, azide, and diethyl dithiocarbamate.     

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.     

Resonance Raman evidence for low-spin Fe2+ heme a3 in energized cytochrome c oxidase: implications for the inhibition of O2 reduction

Resonance Raman (RR) spectra are reported for reduced submitochondrial particles (SMP) with excitation at 441.6 nm, where Raman bands of the cytochrome c oxidase heme a groups are selectively enhanced. Addition of ATP to energize the membranes induces the formation of a new band at 1644 cm-1 and partial loss of intensity in a band at 1567 cm-1. These changes are modeled by adding cyanide to reduced cytochrome c oxidase and are attributed to partial conversion of cytochrome (cyt) a3 from a high-spin to a low-spin state. This conversion is abolished by addition of excess oligomycin, an ATPase inhibitor, or FCCP, an uncoupler of proton translocation, and is reversed when the ATP is consumed. The observed spin-state conversion is attributed to the binding of an endogenous ligand to the cyt a3 Fe atom. This ligation is suggested to be induced by a local increase in pH and/or by a global conformation change associated with the generation of a transmembrane potential. Since O2 binding requires a vacant coordination site at cyt a3, the ligation of this site must retard O2 reduction and could thus provide a simple mechanism for energy-linked regulation of respiration. No changes in the RR spectrum were observed upon adding Ca2+ or H+ to reduced cytochrome c oxidase. The cyt a3 spin-state change associated with membrane energization is unrelated to the cyt a absorption red shift induced by adding Ca2+ or H+ to cytochrome c oxidase.     

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.     

Comparative receptor study in gamete chemotaxis of the seaweeds Ectocarpus siliculosus and Cutleria multifida. An approach to interspecific communication of algal gametes

The terminal component of the electron transport chain, cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase) was purified from Bacillus subtilis W23. The enzyme was solubilized with alkyglucosides and purified to homogeneity by cytochrome c affinity chromatography. The enzyme showed absorption maxima at 414 nm and 598 nm in the oxidized form and at 443 nm and 601 nm in the reduced form. Upon reaction with carbon monoxide of the reduced purified enzyme the absorption maxima shifted to 431 nm and 598 nm. Sodium dodecylsulfate polyacrylamide gel electrophoresis indicated that the purified enzyme is composed out of three subunits with apparent molecular weights of 57 000, 37 000 and 21 000. This is the first report on a bacterial aa3-type oxidase containing three subunits. The functional properties of the enzyme are comparable with those of the other bacterial cytochrome c oxidases. The reaction catalyzed by this oxidase was strongly inhibited by cyanide, azide and monovalent salts. Furthermore a strong dependence of cytochrome c oxidase activity on negatively charged phospholipids was observed. Crossed immunoelectrophoresis experiments strongly indicated a transmembranal localization of cytochrome c oxidase.     

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.     

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.     

Interaction of PGBx and peroxides with cytochrome c and inhibition of lipid peroxidation

PGBx, a derivative of prostaglandin B1, stimulated the oxidation of cytochrome c in the presence of H2O2. Although the reaction was nonenzymatic, the apparent activation energies of 12 and 4.9 kcal above and below the transition at 21.5 degrees C were similar to those for oxidation by cytochrome oxidase. Depletion of H2O2 and oxidation of cytochrome c followed similar time courses, suggesting that H2O2 was consumed in the reaction. PGBx was a specific requirement, but organic hydroperoxides (ethyl and T-butyl) could replace H2O2. Low concentrations of ethyl or t-butyl hydroperoxide initially stimulated the oxidation of cytochrome c; this stimulation disappeared before completion of the oxidation, but was restored when the hydroperoxide concentration was renewed, suggesting that these hydroperoxides were probably also consumed in the reaction. The concentration of PGBx (8.9 microM) required for half-maximum stimulation of the oxidation was similar to the apparent Kd for its dissociation from oxidized cytochrome c (6.8 microM). Binding data and CD spectra suggested that a 1:1 complex between cytochrome c and PGBx was formed, altering the conformation of the heme region. This conformational change caused a shift of the Soret absorption peak from 410 to 406 nm and may be responsible for the enhanced oxidizability of the cytochrome c by H2O2. Cytochrome c inhibited lipid peroxidation in microsomes, an effect enhanced by the addition of PGBx. In the absence of lipid peroxidation, cytochrome c and PGBx stimulated NADPH oxidation via NADPH-cytochrome c reductase. Thus the inhibition of lipid peroxidation by cytochrome c and PGBx may involve either the removal of hydroperoxides or deviation of electron transfer away from the pathway for lipid peroxidation.     

Cyanide binding to bovine heart cytochrome c oxidase depleted of subunit III by treatment with lauryl maltoside

Subunit III was removed from beef heart cytochrome oxidase by incubation of the isolated enzyme at 25 degrees C for 24 h in lauryl maltoside buffer at a detergent to protein ratio of 10:1 (w:w). During the course of the incubation, the reaction of the enzyme with cyanide was followed by spectrophotometry in the Soret region. The starting material binds cyanide in a multiexponential process with 70% of the reaction occurring during the slow phase of the reaction at an observed rate of 3.85 X 10(-5) S-1 with 1 mM KCN. More of the enzyme binds cyanide during the fast phase of the reaction at an observed rate of 3.8 X 10(-3) S-1 as subunit III is removed by lauryl maltoside. After 24 h of incubation in lauryl maltoside, the enzyme reacts with cyanide completely in a rapid, single exponential process. When the protein from such an incubation is recovered by cytochrome c affinity chromatography and analyzed for its subunit content, subunit III is absent. The position of the Soret maximum of the oxidized enzyme shifts from its maximum at 418 nm in the starting material to 422 nm in the subunit III-depleted enzyme. The subunit III-depleted enzyme binds cyanide completely in a simple bimolecular reaction with a rate constant of 3.8 M-1 S-1. We discuss this result in terms of the possible structural and functional roles for subunit III in the cytochrome oxidase complex.     

Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c.

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.     

A functioning complex of two cytochrome c fragments with deletion of the (39-58) eicosapeptide

Two major fragments of horse heart cytochrome c involving the sequences (1-38) and (60-104) were found to produce a stable complex. The two fragments were devoid of any cytochrome c activity. The complex exhibited a hardly measurable electron transfer capacity with respect to cytochrome c oxidase and missed the 695 nm absorption band. The introduction of tryptophan in position 59 restored the intrinsic activity of the complex to the level of native cytochrome c. This was concluded from the convergence of the Eady-Hofstee plots which extrapolate to the same Vmax at high substrate concentrations. The absorption spectrum of the complex in the ferriform contained a clear absorption band at 695 nm (84% of that found with native cytochrome c). The investigation proves the indispensability of tryptophan in position 59 for the transfer of an electron to cytochrome c oxidase and supports the conclusions of Parr et al. about the existence of two consecutive processes in the folding of the two fragments (vide infra).     

Effect of heat treatment on oxidase activity and proton-pumping capability of proteoliposome-incorporated beef heart cytochrome aa3

By incubating beef heart cytochrome c oxidase at 43-45 degrees C, selective inactivation of the H+-pumping function is possible without affecting cytochrome c oxidase activity; proteoliposomes reconstituted with heated enzyme (43.5 degrees C for 60 min at pH 7.0) showed an apparent H+/e- ratio of only 0.3 and a turnover with cytochrome c plus ferrocyanide as substrate of 20 s-1, while those with the intact enzyme showed an apparent H+/e- ratio somewhat greater than 1.0 and a turnover of 19 s-1. This decrease in the H+/e- ratio could not be attributed to a stimulation of H+ permeability upon heating, since the respiratory control ratio and the magnitude of membrane potential formation remained almost the same in the two cases. A pH-dependent Em (midpoint redox potential) change of cytochrome a in the presence of cyanide was still observed after the heat treatment. Heating induced a small spectral shift in the Soret region of the oxidized (resting) enzyme; the peak of the heated enzyme was at 421 nm, while that of the intact enzyme was at 419 nm. The spectral shift obtained by pulsing the enzyme with oxygen under turnover conditions is also altered.     

Activation by reduction of the resting form of cytochrome c oxidase: tests of different models and evidence for the involvement of CuB

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2.10(4) M-1.s-1. In the absence of cyanide, ferrocytochrome a3 appears at a rate (kobs) of 0.016 s-1. Ferricytochrome a3 maintains its 418 nm Soret maximum until reduced. The rate of a3 reduction is independent of dithionite concentration over a range 0.9 mM-131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a3/cyanide complex occurs at the same rate as a3 reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a3 with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a3 shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a3. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow (kobs approximately 0.02 s) entry of a third electron.(ABSTRACT TRUNCATED AT 400 WORDS)     

The 650 and chromophore in Escherichia coli is an 'oxy-' or oxygenated compound, not the oxidized form of cytochrome oxidase d: an hypothesis

The form of cytochrome d in Escherichia coli and Azotobacter vinelandii that shows an absorption maximum at 648 to 652 nm ('cytochrome d650') is generally regarded as the oxidized form of this terminal oxidase. Membranes from E. coli grown under oxygen-limited conditions, when treated with ferricyanide, do not reveal cytochrome d650, whereas a sharp symmetrical band at 652 nm results from the reaction of the reduced enzyme with O2 at either room temperature or after flash photolysis of the CO-liganded form at -130 degrees C. Electron paramagnetic resonance spectroscopy of cytochrome d650 trapped at -130 degrees C shows that its spectrum is indistinguishable from the CO-liganded form and does not reveal resonances of high spin ferric haem previously attributed to cytochrome d. An hypothesis is proposed in which cytochrome d650 is an early intermediate in the reaction of reduced cytochrome d650 and oxyhaemoglobin is presented and the hypothesis discussed in relation to earlier work, in which the indirect interconversions of reduced cytochrome d and d650 have been explained by proposing the existence of an 'invisible' form. It is suggested that this form could be the oxidized enzyme.