Low-Temperature Kinetics of the Reaction of Oxygen and Solubilized Cytochrome Oxidase
By BRITTON CHANCE,* CARLO SARONIO,*t JOHN S. LEIGH, JR.,*
W. JOHN INGLEDEW$ and TSOO E. KING§
*Johnson Research Foundation, School of Medicine, University ofPennsylvania, Philadelphia,PA 19174, U.S.A.,
tDepartment of
Biochemistry,Medical SciencesInstitute, UniversityofDundee,
Dundee DD1 4HN,Scotland, U.K., and
§DepartmentofChemistry, State Universityof New York, Albany, NY 12222, U.S.A.
(Received 22August1977)
The reactionofsolubilized cytochrome oxidase in the fully reduced state with02atlow temperaturesreveals components withcharacteristics similartothose observed with the membrane-bound oxidase, namely compounds A and B,which are proposedtobe'oxy' and 'peroxy' compoundsrespectively. Similar species areidentified in both solubilized and membrane-boundoxidases; the reaction velocity constant for the reaction with02 and the dissociation constant are decreased 2-3-fold in the solubilized preparation as comparedwith themembrane-boundspecies, owingtodecreasedreactivitytowards02in theformer. The oxidase prepared in the mixed-valence state shows the distinctiveabsorp- tion band characteristicof compound C, identified in the membrane-bound oxidase. The assignment of the a,
f,
yandnear-i.r. absorption bands topossible valencestatesof these compounds is made.The existence ofan 02 compound ofsolubilized cytochromeoxidasewaspostulated by Okunukietal.
(1958), Orii & Okunuki (1963), Wainio (1965), Lemberg & Mansley (1966) and Orii &King (1972).
Initially, the compoundwaspreparedbyoxygenating the dithionite-reduced material, but more rigorous proceduresweresubsequentlydeveloped by Wharton
& Gibson (1968). Thesluggish kinetics of the com-
pound hadraised questionsinthe earlyworkastothe feasibility of its biological function, and rendered the search for thiscompound by rapid kinetic methods futile in both the solubilized (Wharton & Gibson, 1968)andmembrane-bound(Chance, 1965)systems.
Babcock et al. (1976) have examined the magnetic circular dichroism of the'oxygenated compound' and find itto havea 'spectrum identical with that of the restingenzyme,apartfromsomeadditional intensity at452nmwhichweattribute toincomplete reoxida- tion ofcytochrome a2+'.
Gibson&Greenwood (1965) evaluated the kinetic constants forthedisappearance of the reduced form ofcytochromesa3 anda,andfor the appearanceof oxidizedcopper in thereactions oftheoxidase with
02. The velocity constant for the reaction of the
reduced oxidase with02wasdeterminedtobebetween 3x107 and 8x107M-1*S-1, on the basis of the de-
tDeceasedApril1975.
creasein absorbance at445nm. Onlythe ferric and cupric formswereobserved;nospectroscopicspecies characteristic of the intermediate was identified in thesestudies, and Greenwood & Gibson (1967)con-
cluded that 'it is unnecessary therefore to assign a
kinetically significant roletothe oxygenated form of the enzyme'. Gibson & Greenwood (1965) did ob-
serveanon-linearity in the profile of02concentration
versuspseudo-first-ordervelocityconstantathigh 02 concentrationsthatcan nowbeinterpretedintermsof the resultspresentedhereasbeingduetotheformation ofan 02compoundinasmall andspectroscopically undetectable amount. They noted that the hetero- geneityof the oxidase preparation was sufficient to render interpretation of theresults difficult (Green- wood &Gibson, 1967). More recently,inrapid-flow studies at-30°C, Erecinska & Chance(1972)found preliminary evidence foracompound similartothat identified in thepresentpaperas'compoundA'.
Greenwoodetal.(1974) have pursued the study of the 02reaction of the solubleoxidase,and foundthat, after the reaction with 02, the ferricyanide-treated CO-inhibited preparation in a mixed-valence state gives an 'oxygenated' product. Their conditions of preparation weresimilar to those for compound C observed in mitochondria (Chance et al. 1975a,b;
Chance & Leigh, 1977) and reported here for the solubilized oxidase. However, a lower temperature
wasrequired for the trapping technique in the study of the02compounds of the membrane-bound oxidase (Chance et al. 1974, 1975b) and much better charac- terizedspectrahave beenobtained (Chance & Leigh,
1977).
Although the use of the soluble oxidase involves assumptions as to the integrity and homogeneity of the preparations (Greenwood & Gibson, 1967) and about the rolethat themembraneplays in the function ofcytochrome oxidase, some advantages over the membrane-bound oxidase include the more favour- ableopticalproperties and the possibility of studying the oxidase reaction without endogenous electron donors such as cytochromes c andc, and the iron- sulphur proteins.Inaddition, as in the mitochondria, thevalence state of cytochrome a and itsassociated copper atom may be altered by equilibration with ferricyanide in the presence of CO.
Several studies of the CO compound ofsolubilized cytochrome oxidase have appeared recently, and titration with reducing equivalents and redox- equilibrium studies show that the CO compound acceptstwoelectrons (Lindsay & Wilson, 1974;Wever etal., 1977; Rosen etal., 1977). However, oxidative titrations show that the solubilized oxidase donates three electrons (Hartzell & Beinert, 1976). Both haem and copper components ofcytochrome a3 seem to be active in electron-transport reactions of the mixed-valence-state oxidase (Chance & Leigh, 1977).
Materials and Methods Preparations
Phospholipid-sufficient cytochrome oxidase had anactivity as described by Kuboyama et al. (1972) and Yu et al. (1975). The preparation was stored at 20°C. For use, samples of concentrated enzyme arediluted in a medium containing 75% of 0.01 M- phosphate buffer,pH7.5,25%ethylene glycol,50mM- ascorbate and 24uM-cytochrome c, and, if desired,
5,fM-tetramethyl-p-phenylenediamine.
The reaction medium is saturated with CO before addition of the oxidase. The enzyme (0.5 ml of solution) is then transferredtoane.p.r. tube (3.5mminsidediameter):30min at roomtemperature isallowed forcomplete reduction of the enzyme, as checked by optical spectra taken at room temperature, and the tubes are then stored at 0°C. The ethylene glycol con- centrations used donot have a significant effect on therespiration asmeasured by the platinum micro- electrode(B.Chance &C.Saronio,unpublishedwork).
Protein isdetermined bythe methodofLowryetal.
(1951),withbovineserumalbuminasstandard.
Oxygenationof thesamplesis carriedoutjustabove thefreezing point of the mixture (-25°C), either by vigorous stirring or by generation of 02 from the decomposition of H202 (Chance et al., 1975d). A small amount ofanti-foam (Dow Corning Silicone
Antifoam A, Midland, MI, U.S.A.) is added to the stirring rod to prevent bubble formation during the stirring procedure. The effusion of CO from the deter- gent-solubilized preparation is very rapid, and anti- foammust be used during the stirring procedure to avoidsignificantlossof CO and consequentoxidation ofthe reduced cytochrome oxidase at -25°C. This problem is avoided when a small volume of CO- saturated oxidase is oxygenated by dilution with a largevolumeof 02-saturated ethylene glycol (Chance
&Leigh,1977). The oxygenated samplesaregenerally storedat-80°C in the dark until theexperiment isto be done.
Considerable variations in the oxidase concentra- tion seemed to have little effect on the results, since the02 reactionsarepseudo-first-order with respectto 02. Theabsorbancechanges did not increase linearly with theoxidase concentrations at the higher values (Chance etal.,1972).
Inthestudies of compoundsBand C, prepared at different redox potentials, the preparation (4pM- oxidase, 30% ethylene glycol, 0.2M-mannitol, 5mM- phosphate buffer, pH 7.2) is gassed with argon/
CO (1:1) at 23°C; 30uM-tetramethyl-p-phenylene- diamine is added andreducedbysmalladditions of Na2S204,giving the dye intermediate, Wurster's Blue, at a potential sufficiently negative to reduce the oxidase andcauseit tocombine withCO. Thepoten- tial is then progressively increased by addition of ferricyanide. The samples are then rapidly transferred to the e.p.r. tube by a 1mm catheter and cooled to -20°C, oxygenated as above, and chilled and stored at-80°C.
In a somewhat improved technique(cf. thecharts ofFig.7),thesampleof cytochrome oxidase isequi- librated at-20°C as above in aCO-saturated atmo- sphere.The effluent gas isallowedtoexitthroughthe sampletube and into theopticalcuvette(seebelow) or the e.p.r. tube mentioned above. The sample is then transferred under pressure of CO via a 0.5 m length ofstainless-steel tubing (0.3 ml volume) coiled in a-23°Ccoolingbath andextending into thechilled (-23°C) cuvette. The chilled sample now occupies onlyone-third the volume of the 1mmcuvette(2mm optical path),andinjectionof 0.7mlof02-saturated (2mMat-23°C) chilled ethylene glycol (30%) now completes the filling of the cuvette. Oxygenation is thus effected without the need forvigorousstirring, except to mix the contents of the cuvette, which requires about Ss at -23°C. Mixing is continued after the transfer of thesampletoa-78°Cethanol/
solidCO2mixture and untilfreezingtakesplace.The sampleis then transferred to anappropriatespectro- photometerforspectroscopic (wavelength-scanning) orkinetic(dual-wavelength)studies.
In Fig. 6(a) below, the kinetics werestudiedwith 10,uM-cytochrome oxidase reduced with 5mM- ascorbate and 5uM-tetramethylphenylenediamine.
1978
The oxidase solution (80,uM) is saturated with CO and then diluted at -20°C with 7vol. of aqueous solution containing 30% ethylene glycol that had previously been air-saturated at 23°C. The oxidase preparation andtheair-saturated mediumaremixed for 10s and then transferred to a freezing bath at -78°C. The sample is contained in a 2mm-optical- pathcuvettewithanaperture approx.2cm2, which is thentransferred tothekinetic spectrophotometer, and the darkened sample equilibrated to -97°C with a thermoregulator. When thermal equilibrium is obtained, a 200J flash lamp activates the reaction, leading to the kinetics observed at 591-630nm, 44463 nm and 830-940nm.
The changein pH with temperature has been shown byWilliams-Smithetal.(1977)tobelargelyabolished by the presence of large concentrations of serum albumin (approx. 40mg/ml). Similar effects are observed with mitochondria where the systemequi- librated with Phenol Red at 0°C shows no large shiftof indicatoroncoolingto-30°Cevenwithonly 3.6mg ofmitochondrialprotein/ml. Presumably, the higher protein concentrations (5-25 mg ofprotein/ml) used in these studies avoidsignificant pH shifts with temperature(Chance,1975).
Physical methods
The usual dual-wavelength technique and a split- beam wavelength-scanning spectrophotometer (Chance et al., 1972)were used to record the data of Figs. I and 4. The baseline corrections for the scan- ning spectrophotometer were stored in a digital memory. The kinetics of absorbance changes (Figs.
6a and6b)wererecorded with amulti-channel dual- wavelength apparatus (Chanceetal., 1975e).
Samples poised with redox mediators were also studied in the split-beam split-photolysis apparatus (Chance et al., 1975c; Chance & Leigh, 1977). At -88°C onehalf of the sample tube isflash-photolysed and the absorbance recorded with respect to the unflashed portion. The sample is then chilled to -196°Cand theopticalspectraatthe topandbottom ofthetubesarerecorded(Figs.8aand8b).
Results CompoundA
Thekinetic andspectral propertiesof thecompound formed by flash photolysis of the CO compound of the reduced solubilized oxidase are shown in Fig. 1.
Fig. l(b) illustrates the kinetics of the reaction.* The lmM-02 concentration used here is the maximum that can be obtained with the stirring technique described above, and the half-time is 1.0s at-860C.
*Thereference number for each experiment is included in theFigure legends, so that further information may be obtainedonrequestfrom the authors(B.C.).
This may be compared withthereactionof themem- brane-bound oxidase, which has a half-time of 1.0s at -100°C (Chance et al. 1974, 1975a). Fig. l(b) indicates that a compound of approximatelyth.- same absorbance is formed byflash photolysis of the CO compound in the presence as in the absence of02 (note that the levels a and c are nearlyequal). The resulting compound cannot be dissociated by light- intensities adequate tophotolyse the CO compound completely (approx. 10 mJ at 591 nm; Stark & Chance, 1969).
Low-temperaturetrapping of the reaction product of Fig. l(b) is also possible if, for example, the material at point c of Fig. 1(b)is chilled rapidly tothe temperatureofliquidN2 (chilled isopentane maybe used to accelerate the chilling); at -1960C, the reaction proceeds no further. In this case, thebaseline stored in the memory of the spectrophotometer correspondstotheabsorbance of a reduced sampleat -196°C. In the absence of 02, point b after photolysis represents the spectrum of reduced cytochrome a32+. The spectrum of the reaction product at pointc is subtracted from that at point b to give the difference spectrum of what we have termed 'compound A' (Chanceetal., 1974, 1975a,b) with a peak at 591nm and a trough at 611 nm. The difference spectrum corresponding to point b minus point a is recorded separately and its amplitude is 25% less than that displayedhere for compound A. The difference may be greater than that shown here, since maximal occupancy of compound A with 02 is scarcely reached at thehighest 02partial pressures obtained byusingthestirringtechnique.
Byaddingvarious amounts of02 with the stirring technique, it is possible to obtain and analyse the resultsasthe equilibrium and kinetics of compound A, asshown in Fig. 2. In these calculations, the02 concentration is taken to be that at the moment of freeze-trapping.Theequilibrium is plotted according to the logarithmic form of the mass-law equation, where the slope corresponds to the stoicheiometric coefficient, and the value at equal concentrations of the free and bound forms corresponds to the dis- sociation constant. A line corresponding to a 1:1 stoicheiometry of 02 to iron and a dissociation constant,Kd,of34O0M-02at-86'Chas been drawn onFig.2andfits thedata within the accuracy of the experiment;it may be compared with the somewhat smaller values for the membrane-bound oxidase. The reversible equilibrium for the combination of cyto- chromeoxidase(a3) and02 atlow temperatures can be written as:
02
+Cu2+a32_
-Cu+a32' .02
kk- o
CompoundA Cu'+a33+.022- (1) CompoundB
(a)
0.027A
(b)
T
570 590 610
Wavelength (nm)
630
I
I
t
I I I I IX__14
LDLflash 1-
500ms
Fig. 1. Differencespectrum (a) and kinetics offormation(b) of compoundAinsolubilizedcytochromtieoxidase Thebaseline for spectrum(a) correspondstopoint b in the kineticdiagram,andwasobtained inaseparateexperiment from which 02 was absent. Thespecies representedinthedifference spectrumcorrespondstothedifference between pointcandpoint b,asindicated in the kinetictrace(temperature for02addition,-25°C; for kinetic recording, -86°C;
forspectral recording, -196°C). Other conditions were:40M-solubilizedcytochrome oxidase, 5,uM-cytochromec, 5mM-ascorbate, 5puM-tetramethylphenylenediamine, 20% ethyleneglycol,10mM-phosphate buffer, pH 7.4, 1mM-CO (saturatedat roomtemperature), 1mM-02 addedat-25°C. LDL,Liquid dyelaser. Furtherinformation may be ob- tainedby use ofreference number1508-lv (see footnotetop.789).
10-3
log [°2I(mm)I
^ 3
1
en @_2
Ca
co Co
3
:>
(b)
0.5 1.0 1.5 2.0 [021 (mM)
Fig.2.Equilibriumandkineticproperties oftheformation of compoundAofsolubilizedcytochromeoxidase (a)Mass-lawplotin log-logco-ordinates fordeterminingthestoicheiometry and equilibriumconstants;(b) kinetic plotfordetermining k+1,k-,and theequilibriumconstant.ConditionsweregenerallyasinFig. 1,with flashactivation occurringat-80°C.The 02concentrationwasvariedovertherangeindicatedbyaddition ofH202inthepresenceof catalase(Chanceetal., 1975d).Reference number CS-10.
592-630nm Absorbance increase
-1.0-
e
2-o
4000
+1.0 - 10-4
9%I iI1-
Thesecond-order constant for the forward reaction andan evaluation of the first-order constant forthe reverse reaction are givenby the plot of Fig. 2(b), of the pseudo-first-order velocity constant versus the 02 concentration on a linear scale.k+1 correspondsto 1.6x 103M-I-s1 at-86°C; thusk-1 corresponds to approx. 0.55s-1 at thistemperature,andKdfromthe kinetic plot agrees well with that from the equilibrium titration(340,UM).Theinclusion of thesecond step of eqn.(1) is based on data that follow in the paragraphs below.
Acomparison of the value of
k+,
for thesolubilized oxidase prepared from ox heart mitochondria with that for k+1 in intact ox heart mitochondria, and corrected to the sametemperature,indicates that the solubilized oxidase reacts more slowly than the membrane-bound oxidase of ox heart (and pigeon heart)mitochondria(Chance etal., 1974, 1975a). In summary,compound Aformed from thesolubilized oxidase is qualitatively similar to but not quantita- tively identicalwiththat formedfrom the membrane- boundoxidase.CompoundB
As the temperature is raised, compound A becomes unstable and is converted intoasecond form in which electrons may be transferred from cyto- chrome oxidase to02. Fig. 3 records the kinetics of the conversion at -105°C and -88°C at
4001UM-02.
The reaction is initiated by flashing the liquid dye laser, and the kinetics at 591 nm follow those of Fig. 1, but are slower, owingto the lower tempera- ture; the amount of compound A is also smaller because of the lower02concentration.The time scale ismorecompressedinFig. 3 than in Fig. 1. After the formation of compound A (upward deflection of traceAat-105°C) there isaslow downward deflec- tion as the absorption band of compoundA slowly disappears; thetracereaches aplateauinabout5min.
We view this transition as the conversion of com- pound A into compound B (Chance et al., 1974, 1975a).
Intrace(C)at 608nmand-105'C, the disappear- anceof the CO compound after the photolysis flash is not clearly shown; instead, a rapid decrease in absorbanceat608nmcharacteristicof theformation ofcompound A is recorded. A further decrease in absorbance at 608 nm occurs due to the formation of compound B, which has less absorbance at this wavelength than either the reduced species, cyto- chromea32+,orcompound A. Trace(B), at 604 nm, is close to the isosbestic point between the CO com- poundandcompound A,sothatitshows absorbance changes similartothose of trace(C)rather thanthose of trace(A).
Recording in the i.r. region is possible, and trace (G) indicates, by a delayed downward deflection,an increase in absorbance that follows the kinetics of
(a) (A)
(B) (C) (G) ~
(A) -j1 min |_
(B) (C) (G)( _
LDLflash
Absorbance decrease L
(A)Cytochromea3, 0.013 591-623nm
I T
(B)Cytochromea3
1
0.013 603-623nmITt
(C) Cytochromea31 0.013 607-623nm
T
(G(Copper 0.0068 830,-940nm
T
Fig.3.Effectof temperatureontheformationanddisappearance of compound A and the formation of compound B Twoexperimentsareillustrated,oneat-105°Cand(a) the other at -88°C (b). Time proceeds from left to right and the wavelengths and thesenseof theabsorbance changes are indicated on the diagram. Conditions were:40pM-solubilized
oxidase, 2,uM-cytochromec, 10mM-ascorbate, 5,uM-tetramethylphenylenediamine,
20%.
ethylene glycol,10mM-phos- phatebuffer, pH7.6, 1.2mM-COadded at 20°C,4004UM-02
added at -25°C. LDL, Liquid dye laser. Reference number 1575.formation ofcompound B. This is interpreted as a partial oxidation of copper in the formation ofcom- pound B andaffords support for eqn. (1).
When the temperature is raised to -88°C, the transformation of compound A into compound B proceeds so much more rapidly that the formation of compound Aisscarcely resolved, and traces (A) and(B)at592 and 603nmshowonlythecompletion of the formation ofcompoundB.Traces(C) and(G) now show similar kinetics. Trace(B) now proceeds moreslowly than traces(C)and(G),possibly owing totheformationofafurther unidentified intermediate (see also Fig. 10of Chanceetal., 1975b).
Themethod of preparing thesampleforwavelength scanning is illustrated in Fig. 4(b), in which the kinetics of formation of compound B measured at 605nmand at -67°C are shown. These kineticsare similartothose oftrace(C)ofFig.3at-88°C. Flash photolysiscauses the usualkinetics,i.e. splittingthe CO compound (point b), followed by the cycle of compound A formation and its conversion into
(a)
compound B, with a half-time of approx. 2s. At point c on the trace, thesample is frozen in liquid N2 (compoundBis stable at thesetemperatures) and the difference between these spectra and that of the reduced oxidase, obtained as described above, is displayed inFig. 4(a). Considerable absorbance has been lostat608nm.There isashoulder in theregion from 580to600nm,which ismorepronounced than in the difference spectrum of compound B of the membrane-bound oxidase (Chance et al., 1974,
1975a).
Fig. 5 illustrates the nature of the consecutive reactions of compound A and compound B (see eqn. 1), showing an O2-dependence of the rate of formation of compound B; the pseudo-first-order velocity constant for the formation of compound B is smaller thanthat of its precursor, compound A. Thus a linear plot of the initial slope of the reaction kinetics versusthe02 concentration gives aninitial linear portion and then a plateau with a saturation valueof1.7sat-65°C(Fig. Sa).Adouble-reciprocal
0.027A
I
550
(b)
570
LDLflash
Ilb
605-630nm
absorbance increase _
590
Wavelength (nm)
7I
610 630
71
t1 z I LT 1
x 11 1 1 .027
:
l __ I - T=-67°C 1s
Fig. 4. (a) DifferencespectrumcorrespondingtocompoundB(the referencespectrumisthereducedoxidase)and (b)kinetics offormation of compoundB
Theexperimental conditions for determining the differencespectrumaregiven in Fig. 3;the baselinecorrespondsto
pointbasdetermined inaseparateexperimentinwhichphotolysisinthe absenceof02occurred. The material used for recordingthespectrumofcompound Bwastrappedat-196°Catpointcatthe end of thetrace.The kineticsfor forma- tion ofcompoundB(Fig. 4b)wererecordedat-67°C. Conditionswere:40OuM-solubilized cytochrome oxidase,2pM- cytochrome c, lOmM-ascorbate, 5pM-tetramethylphenylenediamine, 20%/ ethylene glycol, 5mM-phosphate buffer, pH 7.4;1mM-CO addedat20°C, 200pM-02 addedat-25°C. LDL, Liquid dye laser. Reference number 1508-3v.
f'
0.03 m:lt E ai c
-o 0.02
ea)
_ 0.01 co 0
0 0.5 1.0 \ 0 5 1
[021(mM) K=295,UM
1/1021
(mM-')Fig. 5. Kinetic and equilibrium propertiesofcompoundB, measured at-65°Cby using 20pM-solubilizedcytochromeoxidase (a)Velocity constant for theinitial phase of the formation of compoundBplotted versus the 02 concentration.
(b) Double-reciprocal plot of the pseudo-first-order velocity constant (k)versus the 02 concentration. Reference number1508-4v.
plot of this initial portion gives an apparent dis- sociationconstantof295 M(Fig. 5b).
Comparison of the reactivity of the solubilized and membrane-bound oxidases towards 02
Fig. 6 shows recordings of the reactivity towards
02
of the twopreparations
ofcytochrome
oxidase underidentical conditions(210,UM-02at-97°C) with identical time and amplitude scales on the recorders.Nearly identical concentrations ofmembrane-bound and solubilized oxidase are used; slight differences in theconditions for reducingtheoxidase have been described in the Materials and Methods section.The kinetics ofthereactions ofhaema3 ironarerecorded in this chart at two wavelength pairs: trace (A), cytochrome a32+ O2 at 591-630nm, and trace (D), cytochrome aC32+ at 444-463 nm, corresponding respectively tothe decreased absorbance caused by the decomposition of the cytochrome a3*CO at 591nmand to the increased absorbance due to the recombination of cytochrome a32+ and 02 at 444 nm. The kinetics of the copper component are recorded at830-940nm intrace(F).
With the solubilized oxidase (Fig. 6a), theforma- tion of oxycytochrome oxidase is clearly resolved at 591nmand reaches a plateau as the disappearanceof
Fig. 6. Comparison ofthe kinetics ofthe reaction with02Of solubilized(a) and membrane-bound(b)oxidase (a) lO,um-Solubilized oxidase, 5mM-ascorbate and 5,uM-tetramethylphenylenediamine; (b) ox heart mitochondria (5mg of protein/ml), 5mM-succinate.
In both (a) and (b) conditions included: 200mM- mannitol, 5mM-Tris/sulphate, pH 7.4, 1.2mM-CO, 1.76mM-02; temperature, -97°C; optical path,2mm.
Reference number2011-5,6".
(a) (A)
r
(F) (D)
Hm75sH
(b) (A)
(F)
(D)
-
75sAbsorbance decrease
(A)cytochromea3o2 0.027 591-630nm
(D) Cytochromea32+ 10.054
(F)Copper
444-463nm
|0.0034 830-940nm
the reduced oxidaseat444nmproceeds exponentially to a steady state. The 830-940nm trace shows no
discontinuity on photolysis, and only a very slow
progress, which continues asthe chart comes to an
end afterapprox. 10min.
The companion recording (Fig. 6b) is obtained withoxheart mitochondria(approx.5mgofprotein/
ml), in this case reduced with 5mM-succinate, saturated withCO, cooledto-20°C, and mixed with air-saturated reaction medium,asin theexperiment ofFig.6(a). Therateofformation of compoundAat 591nmis aboutthreefold faster than the reactionat
444nm, as judged from the initial slope and half- time. A second distinctive feature is that compoundA doesnotattain itsmaximum concentration, but rather reaches a steady state and then decays, owing to a more rapid conversion into compound B. The absorbance change at 830-940nm is, however, similar; there is no fast reaction on photolysis, and thetracecontinues toprogresstocompletion asthe 10minoftherecordingends.
Insummary, the principal differencesbetween the solubilized and themembrane-bound oxidasesare a
threefoldmore rapidreaction with 02, and a more
rapid electron donation to reduce 02, in the membrane-bound oxidase.
Polyphasic-reaction kinetics
If the temperature is raisedhigh enough for the conversion of compound A into compound B to approach completion, multiple phases of the reaction
arerecorded,asshown in Fig. 7at-30°C. Asabove, the initial fast portion containscomponents assign- able to compounds A (200ms/division) and B (1s/division). Aslowphase of the kineticsat605nm
follows,which isatpresentassignedtocytochromea
oxidation. However, the log plot (Fig. 7c) indicates two readily resolvable reactions, one with a rate constant of2.5s-I withan amplitudeof 20-25%of the total,and the other witharateconstantof0.2s1 with an amplitude of 35-40% of the total. In a
separateArrheniusplot (not shown) thetemperature- dependence of these two phases gives an energy of activation of 88kJ/mol in therange from -50°C to -20°C for the faster phase, and 142kJ/mol in the
rangefrom-40°Cto-20°Cfor the slower phase. The slower phase may be due to heterogeneity in the solubilized oxidase,sincetheeffectsarenotobserved inthemembrane-bound preparation.
(a)
I
LDL flash 200ms
L
0.054 T
(b) tbIL IIIIII
-MI
I
I - Il
I
LDL
t
flash1
0.054
T
1s
20 -b-_cb
10 c-cd
<6
2
0 2 4 6 8
Time(s)
Fig.7.Illustrationofbiphascityin the kineticsofformation ofcompoundBrecordedontwo timescales(aandb)andanalysed inasemi-logarithmic plot (c)
Conditions included: 40,M-solubilized cytochrome oxidase, 2puM-cytochrome c, 1OmM-ascorbate, 20% ethylene glycol, 10mM-phosphate buffer, pH7.4, 1.2mM-CO added at 20°C, 200PM-02 added at -25°C. Temperature was
-30°C.LDL, Liquid dyelaser.Wavelengtbsare605-630nm. Referencenumber1505.
1978
I II I I I III I
--4----
:-la 7~~~~ -1Tlc l T
jI Il
IIl
I II_
I
0.011Absorbance
decreasel
T
93mV
390mV
s, 325mV
550 560 580 600 620 630
Wavelength(nmn)
Fig. 8. Absorptionspectraof theformationofcompoundB atlowmid-potentials (+93 mVinspectrumA,+190mV in spectrumB) and of compound Cat+325 mV inspectrum
C, allat-88°C
Traces(A)-(B) and (A)-(C)arealsoprovided.Ab- sorbance decreases are in an upward direction for spectra(A), (B) and (C), whereas theconventionused insubtraction givesanabsorbance increaseas anup-
ward deflection in thespectra(A)- (B)and(A)- (C).
Thepotentials arereferredto thecalomel electrode and areindicated inthediagram. Conditions inclu- ded: 40,uM-solubilized cytochrome oxidase, 30pM- tetramethylphenylenediamine. Dithionite and ferri- cyanidewereaddedasnecessarytogivethemeasured potentials. Reaction mixtures also included 0.2M- mannitol,30mM-phosphate buffer, pH7.0, and30%
ethyleneglycol.Referencenumber 1833-1,2,4v.
CompoundC
In studiesofthemembrane-bound oxidase, treat- mentof thecomponentsof therespiratorychainwith ferricyanide resulted in the formation of a new
species of intermediate compound that is preceded bycompoundA(A2of Chanceetal., 1975a;Chance
&Leigh, 1977).Thesamereaction hasbeen followed inthe solubilizedoxidase,by usingbothpoisinginan
electrometric titration andferricyanidealone(seethe
Materials and Methods section). Samplesaretrans- ferred from the CO-saturated electrometric titrator, oxygenated at -20°C, and stored at -280C. They are flash-photolysed in the slit-beam split-photolysis spectrophotometer at -88°C so that the spectra of Fig. 8 represent the difference between the flash- photolysed portion of thesample tube and that con- taining the unaltered CO compound. Theabsorbance decrease dueto photolysisatapprox. 587nm is indi- cated as an upward deflection, and the absorbance increase at 608 nm due to theformation of compound C isindicated as adownward deflectioninthe upper setof traces.
In trace(A) ofFig. 8, thepotentialis +93 mV and the electron carriers of the oxidase preparation are reduced. The formationof compound B is recorded andits difference spectrum isplotted with referenceto the COcompound. The moststriking features of the difference spectrum oftrace(A) are thedecrease in absorbance at 585 nmandthe increase in theabsor- bancearound 620nm duetothephotolysisof the CO compound. Theabsorbance at 612nm appearsto be duetothe formationofcompoundB.
In trace (B) the inidicated potential has been in- creased to +19OmV and a significant oxidation of haem a and its associated copper component has occurred. The difference spectrum for the flash- photolysis product measured with respecttothe CO compound (trace B) shows increased absorbance at 600-620nm, as also indicated by the difference spectrum for the two states(trace B-A), where in- creasedabsorbance isupwards.
Intrace(C), the indicatedpotentialis+325mVand haem aandits associated copperareoxidized,where- as haem a3and its associated('invisible') copperre- main reduced (Lindsay & Wilson, 1974; Chance &
Leigh, 1977). Thedifference spectrum oftrace(A)- (C) clearlyshows the 609nm peak characteristic of compound C. The apparent mid-potential for the conversion of compound B into compound C is approx.220mV.
Near-i.r.differencespectraofcompounds A, Band C Theabsorption-difference spectra ofcompoundsB and C with respect to the CO compound of cyto- chrome a3 are shown in Fig. 9. The biochemnical preparations are similar to those ofpreviousexpori- ments, with 5mM-ascorbate and 5,/M-tetramethyl- phenylenediamineused asreductant. Theprocedure forbindingwithCO,coolingto-20°C,oxygenating, further cooling, and freeze-trapping with equilibra- tionat-80°Cis also similartothat described above.
The poise in Fig. 9(a) is about 150mV, and over 260mV inFig. 9(b).
The i.r.-absorption difference spectra show in- creased absorbance forboth compoundB (Fig. 9a) andcompound C(Fig. 9b). These spectra differsig- nificantlyfrom thoseobservedbyGriffiths & Wharton
(a) 0.005
7L8
r %>
782(b)
744
Absorbance increase
650 700 750 800 850 900
Wavelength(nm)
700, 750 8 850s
700 750 800 850
Fig. 9. Absorptiondifferencespectraof compoundsB(a)and C(b)inthe near-i.r.region
In both spectra, conditions included 4,pM-cytochrome oxidase, 30%4 ethylene glycol, 0.12M-mannitol, 3mm-Tris/
sulphate, 20mM-Mops (4-morpholinepropanesulphonic acid) buffer, pH7.0, 1.2mM-CO and 2mM-02. In (a) the systemwaspoisedatalowpotential (-150mV),andin(b)atahigh potential (>260mV).Reference number 1898'.
(1961). Thefully oxidized species givesanabsorption difference spectrum at 830nm, and the blue shift is
approx.80nmforcompoundC andapprox.30nmfor compoundB. If the temperature israised, however, theabsorption difference spectrum ofcompoundB shifts from 790nm tonear830nm for the fullyoxi- dized state. However, there is no evidence that the differencespectrum ofcompoundCshiftsto 830nm withincreasedtemperatureorthat itisacomponent of the 830nm band.Also the 830nm band is present afterferricyanidetreatment,and the results here show
nodisappearanceof absorbanceat830nm in the for- mation of compound C (Chance & Leigh, 1977).
Discussion
Althoughthesolubilizedoxidase has the disadvan- tagesofheterogeneityandunknown electrondonors, and lacks both energy coupling and physiological controlsonelectrontransport,itcanbe usedathigher concentrations and with less difficulty caused by spectroscopic interference from either fixed or variable-time components than can the membrane- boundoxidase. Althoughthese latter difficulties are largely avoidedatlowtemperatures,where onlyafew componentsof the oxidasearecapable of rapidreac- tion,itisimportanttodeterminewhether the various typesof02compoundsidentified inthe membrane- boundsystemexist in thesolubilizedpreparation, and whetherthekineticsandequilibria of theirreactions
arealteredbythesolubilizationprocedure.
Comparison of the membrane-bound and solubilized oxidases
Thedata of thepresentpaperafforda moreincisive comparisonof theactivityof thesolubilized andmem-
brane-bound oxidases than has beenpossible inpre-
vious work. Here the activities of thetwopreparations havebeentestedunder identical experimental condi-
tionswithidentical read-outsof thereaction kinetics.
Two distinctive differences are observed, namely a
threefold more rapid reaction of the membrane- bound oxidase with 02andagreatertendencytoform compoundB. Thismaybe compared with the earlier data of Chance (1965) and Chance et al. (1964), where the second-order velocity constant for the disappearance of the reduced cytochrome a3 in the membrane-boundoxidasewasfourfold smaller than thecorresponding value forthesolubilizedoxidaseas
measured by Gibson &Greenwood (1965).
It isapparentthat therate-limitingstepsmaydiffer vastly.Inourexperimentsat-90°C, the formation of compoundAisinvolved,asisitstransformation into compoundB. Butathighertemperaturestheaccessi- bilityof02tothe activesitemightbeoneof therate- limiting factors, although the reaction velocity constant is not diffusion-limited in a formal sense.
Although the discrepancymaybe duetoexperimental
errorin theabove-mentionedworks, particularly the inabilitytocomparethetwosystemsunder identical conditionsashas beenpossiblein thepresentpaper,
there nevertheless remains the possibility that the 1978
900
active siteismore'open'in thesolubilized preparation thaninthemembrane-boundoxidase.
Relationbetweenroom-and low-temperature results The consistent result ofkinetic studies with both the membrane-bound and the solubilized oxidase is thecomplete absenceofspectroscopicevidence fora
significant concentration of functional intermediate compounds (Gibson et al., 1965) (see the intro- duction). At -28°C, absorbance changesnear430nm recorded at 2-5ms suggest an intermediate com-
pound at low concentration (Erecinska & Chance, 1972),and theseand other datasuggestnear-maximal concentration at lower temperatures (Chance
etal., 1975a,b). The apparentenergies of activation
arelarger forcompoundBformation than forcom-
pound A formation and thus compound A is less likely to be found at higher temperatures. More recent data show that electron donation from cyto- chromea becomes activated above -40°C (Chance
etal., 1977),and thus the steady-stateconcentrationof compoundBmaybegreatlydecreased above-30°C andespeciallyatroomtemperatures.
Since different electron donorsappear to become functional as the temperature is increased, it is not possibleto predictpreciselywhichones,ifany,exist at significant concentrations at room temperature.
Obviously, the possibility that such intermediates would not exist at room temperature at significant concentrations would testifytothe 'success' ofcyto- chromeoxidaseas a veryefficient 'oxygenreductase' ofvery high 02 affinity (Chance, 1965) and ofvery
greatrapidity of function.
CompoundA
Compound A seems tohave qualitatively similar and quantitatively different properties in the two
preparations.Incommonwith the twopreparations
arethegeneral characteristics ofahighlydissociated light-insensitive product formed by the photolysis ofthe CO compound inthe presenceof various 02 concentrations. Themolecular absorptioncoefficient ofcompound A is similar to that of the CO com-
pound, but isdifficulttodetermine exactly, owingto the possibility that incomplete occupancy has been obtainedintheformation ofcompoundA.Assuming that saturation has been obtained, the molecular absorption coefficientatthe a-band forcompoundA exceeds that for the CO compound by as much as
25%. In theregion of the Soret band, thesituation
may be reversed. But at present, the data are suf- ficiently imprecise (on account of light-scattering correction) to give the exact values of the two
molecularabsorption coefficients.
CompoundB
Compound B appears to have characteristics
similar to those of thecorresponding compound in the membrane-bound system. There are significant differencesin thetransformation fromcompoundA into B, thisbeing less rapid in thesolubilizedthan inthe membrane-bound preparation.Animportant feature of thereactioninvolvingtheformation ofcompound B in the solubilized oxidase is the stoicheiometric inferiority, or even the complete absence, ofcyto- chrome c. Afurthercomplication in thesolubilized oxidase is afforded by the slow reactions, which appearto be duetocomponentsofeither haema or haem a3 that are oxidized much more slowly than are other portions of the oxidase. Twoslow phases are observed, one roughly tenfold slower than the other. Since the slower phase isnot observedin the reaction of the cytochrome c-supplemented mem- brane-boundoxidase with02, it isconsidered tobe duetosomeheterogeneityof thispreparation. Alter- natively, the slow phases may be duetothesluggish reaction of cytochrome a observed at low concen- trations ofcytochrome c (Chance et al.,1977).
Compound C
CompoundCformsdistinctively in thesolubilized oxidase as well as in the membrane-bound oxidase and requires a potential for half-maximal trans- formation from compoundBinto Cofapproximately that ofcytochromea,suggesting that thefundamental change
involved
in the formationof compound C is the oxidation of haem a and its associated copper, haem a3 and itsassociated copperremaining reduced under such conditions owing to the liganding of haem a3 to CO(Lindsay&Wilson, 1974; Chance &Leigh, 1977).
Characteristicsofthe i.r.absorption
The attribution of the near-i.r.-absorption bands ofcytochrome oxidase to copper is now supported byanumber of authors(Griffiths& Wharton, 1961;
Chance, 1966; Aasaetal., 1976; Hartzell &Beinert, 1976; Wever etal., 1977).Of greatest interest in the formation ofcompoundsBand C arethe anomalous positions of their i.r.-absorption bands, 782nm and 744nmrespectively, for the particular conditions of Fig. 9. The characteristics of these absorption difference spectra are such that it is unlikely that the shiftto782nmfrom the usual 830nm position of the copperbandofcytochrome oxidase is due simply to a combination of varying amounts of two species absorbing at 744nm and 830nm. Instead, the data suggest that these are two different maxima for hitherto 'invisible' copper, representing absorption- difference 'signatures' under the two conditions in which haemaand its associated copper are reduced (compound B) or oxidized (compound C). Only in thefully oxidized oxidaseis the absorption-difference
peak of copper at 830nm. The anomalous absorp- tion band at 609nmhas been attributed either to the 'umasking' of the copper associated with haem a3 as a'blue' copper protein with prominent absorption bands at 609 and 740nm as in the copper protein stellacyanine or to a charge-transfer interaction between ferrous haem and cupric copper (Chance
&Leigh, 1977).
References
Aasa, R., Albracht, D. P. F., Falk, K. E., Lanne, B. &
VanngArd,T.(1976) Biochim. Biophys. Acta 442, 260- 272
Babcock, G. T., Vickery, L. E. & Palmer, G. (1976) J.Biol.Chem. 251,7909-7916
Chance, B.(1965)J. Gen. Physiol. 49, 163-195
Chance, B.(1966) in Biochemistry of Copper (Piesach, J., Aisen, P. & Blumberg, W., eds.), pp. 293-301, Academic Press, New York
Chance, B. (1975) Ann. N. Y. Acad. Sci. 244,163-173 Chance, B. & Leigh, J. S., Jr. (1977) Proc. Natl. Acad. Sci.
U.S.A.74,4777-4780
Chance, B., Schindler, F. & Schoener, B. (1964) IUB Symp.31,367-392
Chance, B., Graham, N., Sorge, J. & Legallais, V.(1972) Rev.Sci. Instrum. 43,62-71
Chance, B., Saronio, C. & Leigh, J. S., Jr. (1974) Fed.
Proc.Fed. Am. Soc. Exp.Biol. 33,1289
Chance, B., Saronio, C. & Leigh,J.S.,Jr. (1975a)Proc.
Nati. Acad.Sci. U.S.A.72,1635-1640
Chance, B., Saronio, C. & Leigh, J. S., Jr.(1975b) J. Biol.
Chem. 250,9226-9237
Chance, B., Harmon, J. & Wikstrom,M. F. K.(1975c) in ElectronTransfer Chains and OxidativePhosphorylation (Quagliarello,E., Papa,S., Palmieri, S., Slater, E. C. &
Siliprandi, M., eds.), pp. 81-95, North-Holland, Amsterdam
Chance, B., Graham, N. & Legallais, V. (1975d) Anal.
Biochem.67,552-579
Chance, B., Legallais,V., Sorge,J.&Graham,N.(1975e) Anal.Biochem.66,498-514
Chance, B., Leigh,J. S.,Jr. &Waring, A. (1977) BBA Libr. 14, 7-11
Erecinska, M. & Chance, B. (1972) Arch. Biochem.
Biophys. 151,304-315
Gibson,Q.H.&Greenwood,C.(1965)J. Biot.Chem.240, 2694-2698
Gibson, Q. H.,Greenwood, C., Wharton,D.C.& Palmer, G. (1965) in Oxidases and Related Redox Systems (King, T. E., Mason, H. S. & Morrison, M., eds.), pp.591-621, John Wiley and Sons, New York
Greenwood, C. & Gibson, Q. H. (1967) J. Biol. Chem.
242,1782-1787
Greenwood, C., Wilson, M. T. & Brunori, M. (1974) Biochem. J. 137,205-215
Griffiths, D. E.&Wharton,D.C.(1961) J. Biol. Chem.
236,1857-1862
Hartzell, C. R. & Beinert, H. (1976) Biochim. Biophys.
Acta423, 323-328
Kuboyama, M., Yong, F. C. & King, T. E. (1972) J.
Biol.Chem. 247,6375-6383
Lemberg,R. &Mansley, G. E.(1966)Biochim. Biophys.
Acta118,19-35
Lindsay,J.G. &Wilson,D.F.(1974)FEBSLett. 48,45-49 Lowry, 0. H.,Rosebrough,N.J.,Farr; A. L. & Randall,
R. J.(1951)J.Biol. Chem. 193,265-275
Okunuki, K.,Hagihara,B.,Sekuzu, I. & Horio,T.(1958) Proc.Int.Symp. EnzymeChem., pp. 264-272, Maruzen, Tokyo
Orii,Y.&King,T.E.(1972)FEBSLett. 21,199-202 Orii, Y. &Okunuki, K. (1963)J. Biochem. (Tokyo)53,
489-499
Rosen,S.,Branden, C-R.,Vanngard,T.&Malmstrom, B.
(1977) FEBSLett.74,25-30
Stark,H.&Chance,B.(1969)FEBSLett. 3,287-291 Wainio, W. W. (1965) in Oxidases and Related Redox
Systems (King, T.E., Mason,H. S. &Morrison, M., eds.), pp.622-633, John Wiley and Sons,NewYork Wever, R.,vanDrooge, J. H., Muisjers,A. O., Bakker,
E. P. &vanGelder, B. F.(1977) Eur. J. Biochem.73, 149-154
Wharton,D. C. &Gibson,Q. H.(1968)J.Biol. Chem.243, 702-706
Williams-Smith, D. L., Bray, R. C., Barber, M. J., Tsopanakis,A.D. &Vincent, S. P. (1977) Biochem. J.
167,593-600
Yu, C. A.,Yu, L. &King,T. E. (1975)J. Biol. Chem.
250,1383-1392
1978
