Proc. Natl. Acad. Sci. USAVol. 89, pp. 6497-6501, July 1992Biophysics

Photoinduced electron transfer from tris(2,2'-bipyridyl)rutheniumto cytochrome c oxidase

(photoreduction/copper ion CUA/cytochrome a)

THOMAS NILSSONDepartment of Biochemistry and Biophysics, Chalmers University of Technology and Goteborg University, S412 % Giteborg, Sweden

Communicated by Harry B. Gray, April 1, 1992

ABSTRACT Flash photolysis has been used to effect elec-tron transfer from tris(2,2'-bipyridyl)ruthenium(I) to cy-tochrome c oxidase in the presence of a sacrificial electrondonor, aniline. The observation that photoreduction occursonly at low ionic strength and high pH indicates that anelectrostatic complex between the ruthenium compound andthe enzyme is the reactive species. The reaction was followed at830, 605, and 445 nm. The initial absorbance changes observedsuggest that the copper ion CUA is the preferred electronacceptor and that electron transfer from the excited rutheniumcomplex takes place in <1 its. Some rapid cytochrome areduction is also observed. Absorbance changes after the initialtransients suggest that the reduced CUA then equilibrates withcytochrome a with a rate constant of 2 x 104 s'. Comparisonof the absorbance changes at 605 and 445 nm and the kineticdifference spectrum in the Soret region indicate that no reduc-tion of cytochrome a3 takes place. With the oxidized enzyme,no further reactions were detected, whereas, in the peroxideand ferryl intermediates, cytochrome a reoxidizes on a milli-second time scale. The reaction appears biphasic in bothintermediates, with rate constants in the range 2 x 102 to 4 x103 s-1. This is considerably slower than the maximal ratesobserved for electron transfer between cytochrome a and thebimetallic site found in earlier work and suggests rate limitationby other processes. The rates obtained for the slower phases areclose to the rate for catalysis of cytochrome c oxidation.

Cytochrome c oxidase catalyzes the final step in the mito-chondrial respiratory chain: the transfer of electrons fromcytochrome c to molecular oxygen. The free energy of thisredox reaction is utilized by the protein to translocate protonsfrom the mitochondrial matrix to the cytosol. A recent reviewof redox-linked proton translocation and electron transfers incytochrome oxidase has been given by Chan and Li (1).The redox-active metal sites of cytochrome oxidase are

cytochromes a and a3 and two copper ions, CUA and CUB.Cytochrome a and CUA are the initial acceptors of electronsfrom cytochrome c, whereas cytochrome a3 and CUB form theactive site for the reduction of oxygen. Since electron trans-fers among these sites are, in general, too fast to be conve-niently investigated by rapid mixing methods, the study ofintramolecular electron transfers has been greatly facilitatedby a photochemical. technique, the flow-flash method intro-duced by Gibson and Greenwood (2). This method can alsobe applied to the study of the redox-induced proton transfersthat take part in proton translocation and oxygen reduction(3, 4). The flow-flash method is, however, applicable only tothe study of reactions after the binding of oxygen to thereduced cytochrome a3-CuB site. The present work was,therefore, undertaken to investigate the possibility of rapid

photoreduction of the fully oxidized enzyme in order to studythe subsequent electron and proton transfers.

Tris(2,2'-bipyridyl)ruthenium(II) has strongly reducingproperties in the excited state (5) and has been used forphotoinduced electron transfer to proteins containing redox-active ruthenium complexes bound at the surface (6). Re-cently, reduction of cytochrome c by a photoactive ruthe-nium complex attached covalently to the protein has alsobeen demonstrated by Pan et al. (7). In the present context,the latter approach has the advantage that limitation of theelectron transfer rate by diffusion of the photogeneratedreductant is avoided. The conditions used for the modifica-tion of cytochrome c are, however, too harsh for use withcytochrome oxidase. Another approach was, therefore,taken.

It is well known that cytochrome oxidase forms a tightelectrostatic complex with the positively charged cy-tochrome c at low ionic strength (8), and so it seemedreasonable to look for photoreduction of the enzyme bytris(2,2'-bipyridyl)ruthenium under conditions expected tofavor electrostatic binding of the ruthenium complex to theprotein-i.e., at low ionic strength and high pH. Promotionof photoinduced electron transfer from bipyridyl complexesto redox proteins by ion pairing has been observed (9). Toavoid thermal back reactions [reoxidation of the enzyme bythe resulting ruthenium(III) complex], aniline was used as asacrificial electron donor as described by Pan et al. (7).

It was found that, under the conditions described, flashphotolysis can produce rapid reduction of both CUA andcytochrome a. CUA seems to be the preferred site ofreductionand is reduced within 1 Us. The finding that the absorbancechanges observed initially are followed by increases in ab-sorbance at 605, 445, and 830 nm suggests an electron transferfrom CUA to cytochrome a. The rate constant obtained for thelatter changes is 2 x 104 s-. This is in the same range as therate for equilibration between CuA and cytochrome a asdetermined by Morgan et al. (10) who used a perturbationmethod and by Kobayashi et al. (11) who used pulse radiol-ysis to inject electrons into the enzyme. The final electronacceptor in the oxidized enzyme seems to be cytochrome a,whereas, in the peroxy and ferryl intermediates, furtherelectron transfer to the bimetallic site is also observed. Thistakes place on a considerably longer time scale than the initialreduction of CUA and cytochrome a.

MATERIALS AND METHODSMaterials. Mitochondria were prepared from bovine hearts

as described by Smith (12). Cytochrome oxidase was isolatedby sequential extraction of the mitochondrial membraneswith Triton X-100 followed by hydroxyapatite chromatogra-phy and exchange of Triton X-100 to dodecyl maltosideessentially as described by Brandt et al. (13). The observationthat this preparation exhibited rapid binding of cyanide to theoxidized enzyme, an absorbance maximum at 423 nm for theSoret band, and very little of the g' = 12 electron paramag-

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netic resonance signal suggests that it is composed mostly ofthe "rapid" form of the enzyme (14). Before use in flashphotolysis experiments, the enzyme was exchanged into 5mM Tris/acetic acid buffer (pH 8.1) containing 0.1% dodecylmaltoside by dilution and reconcentration. Tris(bipyridyl)ru-thenium dichloride was from Aldrich, aniline was fromMerck, and dodecyl maltoside from Boehringer Mannheim.Catalase was from Sigma and superoxide dismutase fromyeast was a generous gift from Ulf Nilsson (Department ofPhysiology, Goteborg University). All other chemicals wereof analytical grade.

Preparation of the Peroxy Intermediate (Compound P).Compound P was obtained by oxygenation of the CO-mixedvalence form of the enzyme. Oxidized enzyme (10 LM) in asealed cuvette was degassed and then allowed to react withCO until the formation ofthe mixed-valence CO complex wascomplete (-12 h). Then oxygen was admitted and a differ-ence spectrum between the product and the oxidized enzymewas recorded. The yield of compound P was >90% asdetermined from the absorbance at the peak at 607 nm in thedifference spectrum and a Ae(607-630 nm) of 11 mM-1 cm-1(15).

Preparation of the Ferryl Intermediate (Compound F).Compound F was prepared by reaction of the oxidizedenzyme with excess hydrogen peroxide as described (16).Oxidized enzyme (12 ,uM) was incubated with hydrogenperoxide (5 mM) and the reaction was followed by differencespectroscopy. After 30 min, the characteristic spectrum ofcompound F (15) with a broad peak at 582 nm and a minorpeak at about 530 nm had developed. There was no peakpresent at 607 nm and the spectrum was stable for at leastanother 20 min. The yield of compound F was 70-80%6 asdetermined from the absorbance difference at 582 nm and aAe(582-630 nm) of 5 mM-1-cm-1 (15).

Flash Photolysis Monitored at 605 or 445 nm. Monitoringlight obtained from a 250-W tungsten/halogen lamp waspassed through a heat filter and an interference filter (445 or605 nm) or a monochromator and then focused on the samplecuvette (10 x 10 mm). The transmitted light was focused onthe entrance slit of a double monochromator and detected atthe exit slit by a photomultiplier (Hamamatsu R269; Middle-sex, NJ) operated as described by Porter and West (17). Theoutput was amplified (Hamamatsu C1053 preamplifier fol-lowed by a Tektronix AM502 differential amplifier; offsetvoltage was provided by a Tektronix PS505-1 voltage supply)and recorded with a transient digitizer (Biomation 2805;Santa Clara, CA) interfaced to a microcomputer. A frequen-cy-doubled neodymium-yttrium/aluminum garnet (Nd-YAG) laser (Quantel, Santa Clara, CA) was used for photol-ysis. The wavelength was 532 nm, the duration of the pulsewas 9 ns, and the total energy was -200 mJ (unless otherwisestated). The photolysis light was directed at 900 to themonitoring beam.

Flash Photolysis Monitored at 830 rn. Monitoring light at830 nm was obtained from a diode laser and detected using aphotodiode as described by Hoganson et al. (18), except thatthe detector was covered with an 830-nm interference filter.Photolysis conditions were the same as above. All photolysisexperiments were carried out at room temperature (22°C).

RESULTSFig. 1A shows the absorbance changes at 605 nm induced byflash photolysis of tris(2,2'-bipyridyl)ruthenium and cy-tochrome oxidase in the presence of aniline at low ionicstrength. Photolysis results in an absorbance increase takingplace in two kinetically distinct steps: an almost instanta-neous increase followed by a slower phase. The initial phaseis not resolved because of scattered light from the laser flashand the strong luminescence of the ruthenium complex at 605

uL0(0

0 0.05 0.1Time, ms

0.15 0.2

FIG. 1. Absorbance changes at 605 nm induced by flash photol-ysis of cytochrome oxidase in the presence of tris(2,2'-bipyridyl)ru-thenium and aniline. Trace A was recorded from a sample of 18 ,uMenzyme, 80 ,uM tris(2,2'-bipyridyl)ruthenium dichloride, and 10 mManiline in 5 mM Tris/acetic acid buffer (pH 8.1) containing 0.1%dodecyl maltoside. Trace B was obtained from a sample of the samecomposition except that 0.5 M NaCl was added. Both traces areaverages of 20 transients.

nm (life time, 0.6 ,us; ref. 5). For the second phase, a rateconstant of 2.1 x 104 s-1 was obtained. The contribution ofthe slower phase to the total amplitude was found to be"85%. At increased ionic strength (Fig. 1B), no flash-induced absorbance changes were observed. Lowering thepH to 7 also abolished the signal (data not shown).

Similar time courses were observed at 445 nm (Fig. 2). Thetime resolution is not sufficient for determination of the rateconstant of the rapid phase in the trace obtained at low ionicstrength. For the slower phase, the rate constant obtainedwas 2.3 x 104 s-1 and its amplitude was 480% of the totalamplitude.The data shown in Figs. 1 and 2 were collected using

different enzyme concentrations to obtain good signal-to-noise ratios at both wavelengths. Since the resulting differ-ence in the optical density at the wavelength ofthe laser flashmay cause different yields ofthe excited ruthenium complex,

AA =0.005

A

0 0.05 0.1 0.15 0.2

Time, ms

FIG. 2. Absorbance changes at 445 nm induced by flash photol-ysis of cytochrome oxidase in the presence of tris(2,2'-bipyridyl)ru-thenium and aniline. Both traces were recorded under the conditionsgiven in Fig. 1 except that the concentration of cytochrome oxidasewas 8 izM.

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data at 605 nm were also collected from a sample composedas in Fig. 2 for comparison of the absorbance changes at 605and 445 nm. From these results, a value for AA605/A44A (totalamplitudes) of -0.3 was obtained. The value expected forreduction of cytochrome a only is 0.27-0.36, whereas thecorresponding figure for cytochrome a3 is 0.043-0.073 (19,20). It thus seems likely that the major part ofthe signal is dueto cytochrome a reduction. The wavelength dependence ofthe absorbance change in the Soret region is shown in thekinetic difference spectrum in Fig. 3. Comparison with thestatic difference spectra of cytochromes a and a3 (20) sup-ports the conclusion that cytochrome a is reduced.By using a AE605(red-ox) of 16 mM-1'cm-1 for cytochrome

a (19), it can be calculated that the yield of reduced cy-tochrome a after a flash corresponds to -3% of the totalprotein. The observation that the yield was roughly propor-tional to the flash energy and did not increase upon a 10-foldincrease in the concentrations of cytochrome oxidase andruthenium complex (using a 1-mm cell) suggests that it islimited by the yield of the excited ruthenium complex ratherthan incomplete binding of the complex to the protein.The finding that the rate constant for the second phase is

similar to values reported earlier for the equilibration be-tween CUA and cytochrome a (10, 11) suggests that it couldbe due to electron transfer from CUA. To investigate thispossibility, data were recorded also at 830 nm where theoxidized form of CUA absorbs. Fig. 4 shows a transientdecrease in absorbance, corresponding to CUA reduction,followed by a slower phase of reoxidation. The initial absor-bance decrease takes place in <1 pus and could not beresolved from the flash artifact. For the slower absorbanceincrease, a rate constant of 1.9 x 104 s-1 was obtained. Theamplitude of the transient corresponds to the reduction of-2.5% of the protein, using a value of 2.0 mM-1 cm-l forAE830(ox-red) for CUA (21).The electron transfers taking place on this short time scale

seem to involve only CUA and cytochrome a. To investigatea possible further distribution of electrons to the oxygen-binding site, the reaction was followed at 445 nm on a longertime scale. In Fig. 5, trace A, it can be seen that the initialabsorbance increase is followed by a small and much slowerdecrease, which suggests that cytochrome a is reoxidized.

8

6

4c0C)

2

0

-2

-4

400 410 420 430 440 450 460Wavelength, nm

FIG. 3. Kinetic difference spectrum (absorbance at 0.25 msminus initial absorbance) of the absorbance changes in the Soretregion. Each point was obtained from an individual sample contain-ing 5 ,uM cytochrome oxidase, 80,uM tris(2,2'-bipyridyl)rutheniumdichloride, and 10 mM aniline and was calculated from the averageof 20 transients. The flash energy was -150 mJ.

< AA=2-10

0 0.05 0.1 0.15 0.2

Time, ms

FIG. 4. Absorbance changes at 830 nm induced by flash photol-ysis of cytochrome oxidase in the presence of tris(2,2'-bipyridyl)ru-thenium and aniline. The sample was composed as in Fig. 1, trace A,and the trace is the average of 100 transients. The flash artifact wasrecorded and subtracted from the trace as described (18). In thepresence of 0.5 M NaCl, no signal was seen (data not shown). Thedifference between the final and preflash absorbance is accounted forby a step change immediately after the flash observed also whencytochrome oxidase was omitted (data not shown).

The slower phase became more apparent as the number offlashes increased. This indicates that it originates from en-zyme molecules having received an electron in a previousflash (double hits).

In the presence of oxygen, it is expected that the additionof more than one electron to a cytochrome oxidase moleculeresults in the formation of enzyme species containing par-tially reduced oxygen bound at the bimetallic site (the so-called peroxy and ferryl intermediates or compounds P andF, respectively; ref. 15). However, compounds P and F arealso readily formed by reaction of the enzyme with hydrogenperoxide (16). In the present experiment, photochemical

LOMt1

0 2 4 6 8

Time, ms

FIG. 5. Absorbance changes at 445 nm recorded on a longer timescale. The samples contained 10 ,uM cytochrome oxidase, 80 gMtris(2,2'-bipyridyl)ruthenium, and 10 mM aniline. Trace A wasobtained from an air-equilibrated sample, whereas the sample usedfor trace B was degassed by repeated evacuation and flushing withpurified argon. The laser energy used was higher than in the otherexperiments (-350 mJ). Both traces are averages of 20 transients.

A~~~~~~~~~~~A

|AA=0.01BA

.--

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generation of hydrogen peroxide would be possible by dis-mutation of superoxide produced by reductive quenching ofthe excited ruthenium complex by molecular oxygen (22).The experiment was, therefore, repeated with superoxidedismutase and catalase present (0.25 mg/ml and 30 ,ug/ml,respectively). The result was identical to that obtained in Fig.5, trace A. In the absence of oxygen, only the initial phasecould be detected (Fig. 5, trace B), and no dependence on thenumber of flashes given was observed.The possibility of the slow phase being the result of

generation of compounds P and F was investigated moredirectly by taking advantage of the findings that these com-pounds can be prepared in high yields by reacting themixed-valence CO complex of the enzyme with oxygen or byincubating the oxidized enzyme with excess hydrogen per-oxide. Flash photolysis of compounds P and F prepared usingthese methods is shown in Fig. 6. It is clear that in thesecompounds, the initial absorbance increase at 445 nm isfollowed by a slower decrease. This is probably due toelectron transfer from the initially reduced cytochrome a tothe bimetallic site, which thus appears more facile in theoxygen intermediates than in the fully oxidized enzyme. Theslower absorbance decrease appears biphasic in both com-pounds. The rate constants obtained for compound P were3.7 X 103 s-1 and 2.3 X 102 s51, with the first phasecontributing -40% of the total amplitude. For compound F,the rate constants were 1.3 x 103 s51 and 3.3 x 102 s-1 withequal contribution from the two phases.From these results, it is likely that the slow phase seen in

Fig. 5, trace A, originates in compounds P and F formedduring the course of the photolysis experiment. It is alsopossible that part of the signal arises when a single-electronreduced enzyme receives a second electron, which would beexpected to result in the oxidation of cytochrome a and theformation of compound P.

DISCUSSIONThe need for conditions of low ionic strength and high pH toproduce the observed signals suggests that electron transferstake place only when the ruthenium complex is boundelectrostatically to the protein. This can be rationalized by

A f

AA=0.005

'~~~~~~~~J. I~~~~~~~~~~~A

B

0 2 4 6 8

Time, ms

FIG. 6. Absorbance changes at 445 nm after flash photolysis ofcompounds P (trace A) and F (trace B) in the presence of rutheniumcomplex and aniline. Tris(2,2'-bipyridyl)ruthenium dichloride (80,uM) and aniline (10 mM) were added to compounds P and F. The finalenzyme concentrations were 11 IAM and 10 ;iM, respectively. Bothtraces are averages of 20 transients.

comparing the rate of decay of the excited state with themaximal rate for a bimolecular reaction between excitedruthenium complex and the enzyme. At the highest enzymeconcentration used, -20 1LM, and by assuming a diffusion-limited bimolecular rate constant of 109 M-1s-1 (23), amaximal collisional rate of 2 x 104 s-1 is obtained. Clearly,the observed reduction of CUA in <1 Ius or the initial phaseof cytochrome a reduction cannot be due to a collisionalmechanism. It is also unlikely that the slower phase ofcytochrome a reduction is caused by a bimolecular reactionsince the rate constant obtained is not dependent on theprotein concentration. Furthermore, this phase is coincidentwith oxidation of the initially reduced CUA, which wouldmake electron transfer from the latter a more likely expla-nation.The rate constant obtained here for electron transfer from

CUA to cytochrome a in the fully oxidized enzyme (2 x 104s'1) is similar to that found by Kobayashi et al. (11) frompulse radiolysis and is also close to the rate found by Morganet al. (10) for equilibration between CUA and cytochrome a ina three-electron reduced species with CO bound at thereduced cytochrome a3-CuB site. The equilibrium position is,however, different in the two enzyme forms. The reductionpotentials of cytochrome a and CUA in the fully oxidizedenzyme are -310 and 260 mV, respectively (24), which favorcytochrome a reduction. When the cytochrome a3-CuB siteis reduced, the corresponding reduction potentials are 288and 276 mV (25). From the relaxation rate of 17,000 s-1 andthe equilibrium constant, a forward rate constant (for elec-tron transfer from CUA to cytochrome a) of 10,200 s-1 wascalculated (10). In the fully oxidized enzyme, equilibriumfavors cytochrome a reduction and the apparent rate constantwill be dominated by the forward rate constant. The valueobtained (2 x 104 s-1) indicates that the forward reaction isfaster when the cytochrome a3-CuB site is oxidized. Thehigher reduction potential of cytochrome a with the resultinghigher driving force for electron transfer from CUA is, how-ever, sufficient to explain the different rates obtained. Theresults given here and those in ref. 10, thus, support thehypothesis that electron exchange between CUA and cy-tochrome a is rapid in all states of the enzyme.Reduction of CUA by the excited ruthenium complex is the

earliest detectable step in a majority of the reacting popula-tion. However, the rapid initial absorbance increases at 445and 605 nm suggest that rapid electron transfer from theruthenium complex to cytochrome a also takes place. Thereare two possible explanations for this parallel initial reductionof cytochrome a and CUA: the ruthenium complex may bebound at a single site from which it is capable of transferringan electron to cytochrome a and CUA, or ruthenium com-plexes bound at separate sites may be responsible for thereduction of cytochrome a and CUA. These alternativescannot be distinguished by the present results. It is interest-ing to note, however, that CUA was recently found to be thepreferred acceptor of electrons also from cytochrome c in theelectrostatic complex formed between cytochrome c andcytochrome oxidase at low ionic strength (26).The ratio obtained for the absorbance changes at 605 and

445 nm and the kinetic difference spectrum shown in Fig. 3suggest that most of the observed absorbance changes aredue to the reduction ofcytochrome a rather than cytochromea3. This is in accord with the results of Kobayashi et al. (11)and also with the equilibrium distribution estimated from thereduction potentials of cytochromes a and a3 as determinedby Blair et al. (27). This is simplified by noting that with anaverage yield of 0.03 electron per enzyme molecule, it is veryunlikely that any molecule receives more than one electronper flash. Then, the equilibrium distribution is given by the"upper asymptotic potentials" (i.e., the potentials that per-tain when all sites except the one under consideration are

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oxidized). In ref. 27, upper asymptotic potentials of -350 and260 mV were obtained for cytochromes a and a3 at pH 8 (theformer value is somewhat higher than that obtained in ref. 24and the latter is the average of values obtained for twobatches of enzyme in ref. 27). With these potentials, equi-librium favors the reduction of cytochrome a over cy-tochrome a3 by a factor of z30.The situation appears very different in compounds P and F.

The results shown in Fig. 6 suggest that, after the rapidreduction of cytochrome a, further electron transfer to thebimetallic site does take place. The reduction potential of thebimetallic site has been estimated to be ~900 mV in com-

pound P and to be ~800 mV in compound F (28), and so thissite is the thermodynamically favored acceptor in thesecompounds. The rates of electron entry into these interme-diates, however, were not determined directly earlier. It istherefore interesting to compare the rates obtained here forelectron transfer from cytochrome a to the bimetallic site tothose observed in other states of the enzyme. In flow-flashexperiments, cytochrome a reoxidizes in two steps with rateconstants of 2.4 x 104 s-1 and 525 s-1 (26). Electron exchangebetween the bimetallic site and cytochrome a can also beextremely rapid: photolysis of the mixed-valence CO com-plex leads to a redistribution of electrons in several steps, thefastest of which takes place at an apparent rate constant of 2x 105 s-1 (29). The presently observed rates and the sloweststep observed in the flow-flash experiment are, therefore,probably not limited by the electron transfer per se but ratherby accompanying reactions at the bimetallic site. Such reac-tions may include proton uptake from the medium, whichtakes place in connection with the reduction of both com-pounds P and F (28) or, in the former, cleavage of theoxygen-oxygen bond. Another possibility is that the widelydifferent rates observed for electron transfer to the bimetallicsite reflect the operation of a mechanism for the gating ofelectrons.The maximal turnover number (kcat) for cytochrome c

oxidation under the conditions used here is -200 s-1 (30).The electron transfers from cytochrome a to the bimetallicsite observed here are thus kinetically competent to be partsof normal catalysis. However, the rate constants obtained forthe slower phases of cytochrome a reoxidation are onlyslightly faster than kcat, which would indicate that they reflectreactions that are close to rate limiting for catalysis.

In conclusion, it has been demonstrated that photoreduc-tion of cytochrome oxidase by noncovalently bound tris(2,2'-bipyridyl)ruthenium is possible under suitable conditions.The rate of electron entry is sufficiently rapid as not to be ratelimiting in studies of other electron and proton transfers thatfollow the initial reduction. This approach should be useful inthe further elucidation of electron and proton transfers in theparts of the catalytic cycle of cytochrome oxidase thatinvolve input of electrons to the enzyme.

I thank Mrs. Ann-Cathrine Smiderot and Mrs. Barbro Bejke fortheir help with the preparation of cytochrome oxidase, Dr. Orjan

Proc. Natt. Acad. Sci. USA 89 (1992) 6501

Hansson and Mr. Mikael Oliveberg for their help with the kineticmeasurements at 830 nm, and Prof. Bo G. Malmstrom for criticallyreading the manuscript. This work was supported by grants from theSwedish Natural Science Research Council, the Knut and AliceWallenberg Foundation, and the Ema and Victor Hasselblad Foun-dation.

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