Caged O2Reaction of cytochrome bo3 oxidase with photochemically released dioxygen from a cobalt peroxo complex Claudia Ludovici, Roland Fro¨hlich*, Karsten Vogtt, Bjo¨rn Mamat† and Mathi
Trang 1Caged O2
Reaction of cytochrome bo3 oxidase with photochemically released dioxygen
from a cobalt peroxo complex
Claudia Ludovici, Roland Fro¨hlich*, Karsten Vogtt, Bjo¨rn Mamat† and Mathias Lu¨bben
Lehrstuhl fu¨r Biophysik, Ruhr-Universita¨t Bochum, Germany
We developed the synthesis of the caged oxygen donor
(l-peroxo)(l-hydroxo)bis[bis(bipyridyl)cobalt(III)] complex
(HPBC) as nitrate salt,which has,compared with the
perchlorate-form described previously [MacArthur,R.,
Sucheta,A.,Chong,F.F & Einarsdottir,O¨ (1995) Proc
Natl Acad Sci USA, 92,8105–8109],greatly enhanced
solubility Now,the quantum efficiency of the photolytical
release of dioxygen was determined to be 0.4 per photon at a
laser wavelength of 308 nm,which was used to observe
biological reactions The X-ray structure of HPBC has been
solved,and the molecular interactions of photochemically
generated oxygen with cytochrome oxidase were
investi-gated with optical and FT-IR spectroscopy: it acts as
acceptor of electrons transferred from prereduced
cyto-chrome bo3,the heme-copper oxidase from Escherichia coli
FT-IR spectra revealed typical absorbance difference chan-ges in the carbonyl region of cytochrome bo3,supported by bandshifts due to solvent isotope exchange and by assign-ment using site-directed mutants IR difference spectra of the photooxidation reaction using the caged oxygen compound, and of the photoreduction reaction using the caged electron donor FMN,have inverted shapes The spectroscopic sig-nals of carboxyl groups are thus equivalent in both reactions: the use of chemically produced oxygen allows the observa-tion of the ongoing molecular changes of cytochrome bo3 oxidase under quasi-physiological conditions
Keywords: cytochrome oxidase; caged compound; FT-IR spectroscopy; oxygen, l-peroxo cobalt complex
Cytochrome oxidases are hetero-oligomeric integral
mem-brane proteins that belong to the superfamily of
heme-copper oxidases [1,2] They are terminal parts of the aerobic
respiratory chains of bacteria and mitochondria,and their
common characteristic is the transfer of electrons from
cytochrome c or ubiquinol to the acceptor substrate,
molecular dioxygen [3] Cytochrome bo3 oxidase of
Escherichia coliis a ubiquinol oxidase It transfers electrons
from the membrane site via heme b to the binuclear reaction
center,which consists of a heme o plus a CuB as redox
carriers The reaction center provides the binding site of
molecular oxygen,which receives electrons and protons
necessary for water formation The electronic energy is
sufficient to drive transmembrane proton transport,which
is tightly coupled to the processes of oxygen reduction and
of water formation [4,5]
X-ray structure data of ubiquinol oxidase from
Escheri-chia coli have been recently published The resolution of
3.5 A˚ allows the reconstruction of the backbone but not of the amino-acid side chain conformations [6] Detailed molecular structures of the cytochrome c oxidases from Paracoccus denitrificansand beef heart mitochondria [6–10] have been determined Due to their extensive sequence similarities these structures could serve as models for the ubiquinol oxidase They allow the prediction of two different proton-translocating channels,called the K- and D-channels The D-channel contains an array of charged or polar amino acids,and is located within two different hydrogen-bonded networks above and below the central Glu286 (numbering according to the subunits I and II of the
E colioxidase),which interacts with the binuclear center [11] Molecular dynamics calculations [12,13] have predicted
a special role of the central Glu286,which could provide the contact between both partial networks FT-IR difference spectroscopy,using either an electrochemical cell [14,15] or photoreduction techniques [16,17] provides information about the orientation of amino-acid side chains and about molecular interactions The photoreduction experiments are designed in such a way,that pre-equilibrated molecules become activated by light to undergo redox changes Out of the many functional groups present in the oxidase,only those affected by the redox transition become visible in FT-IR difference spectra In a previous report,the band signature at 1745 cm)1and at 1735 cm)1occurring in redox FT-IR difference spectra of different heme-copper oxidases has been assigned to Glu286 [17]
In order to study the oxidase reaction with the natural substrate dioxygen at the molecular level with FT-IR spectroscopy,we established a caged dioxygen system that allows O2 release via photolysis Photoactivation of (l-peroxo)(l-hydroxo)bis[bis(bipyridyl)cobalt(III)] complex
Correspondence to M Lu¨bben,Lehrstuhl fu¨r Biophysik,
Ruhr-Universita¨t Bochum,Universita¨tsstr 150,
D-44780 Bochum,Germany
Fax: + 49 234 32 14626,Tel.: + 49 234 32 24465,
E-mail: luebben@bph.ruhr-uni-bochum.de
Abbreviations:
HPBC,(l-peroxo)(l-hydroxo)bis[bis(bipyridyl)-cobalt(III)]; BC,bis(2,2¢-bipyridyl)cobalt(II).
*Present address: Organisch-chemisches Institut,Universita¨t Mu¨nster,
Correnstraße 40,D-48149 Mu¨nster,Germany.
Present address: Max-Planck-Institut fu¨r Biophysik,
Heinrich-Hoffmann-Str 7,D-60528 Frankfurt/Main,Germany.
(Received 18 January 2002,revised 15 April 2002,
accepted 19 April 2002)
Trang 2(HPBC) has been described previously,but the reported
chemical (a perchlorate salt) had rather low solubility and
the photochemical conditions were very unfavorable
[18,19] Due to the strong IR absorbance of water, it is
desirable that the FT-IR samples consist of thin and
highly concentrated protein films Hence a highly soluble
and stable HPBC complex had to be used in order to
release enough dioxygen to circumvent the possible
problem of substrate limitation In this study,we describe
the synthesis of a highly soluble HPBC salt and its
(photo)chemical characterization,and we demonstrate the
validity of the caged oxygen complex (HPBC-nitrate salt)
as a suitable probe for FT-IR spectroscopic studies of
cytochrome bo3
M A T E R I A L S A N D M E T H O D S
Synthesis of the HPBC-perchlorate salt
Solutions were prepared of 2.33 g of Co(NO3)2Æ6H2O in
20 mL of water and of 2.5 g of 2,2¢-bipyridine in 20 mL
ethanol The pH was adjusted with 1MNaOH to 9.2 The
following reaction steps were carried out in the dark Under
stirring,oxygen was streamed into the reaction vessel for
10 min The pH of the mixture was readjusted to 9.6,and
oxygen streaming was continued for another 20 min For
crystallization,2.2 mL of a 6Msodium perchlorate solution
in 50% (v/v) of aqueous ethanol was added and the mixture
was kept for 16 at 10C Black crystals were filtered with
suction,washed with ice-cold ethanol and dried under
vacuum for at least 3 h The molar yield of solvent-free salt
was 73% IR(KBr): 1088 cm)1and 625 cm)1(perchlorate),
855 cm)1 m(O-O) UV/vis: kmax: 460 nm,395 nm
(7100M )1Æcm)1),314 nm (43 300M )1cm)1),304 nm and
212 nm
Synthesis of the HPBC-nitrate salt
Co(NO3)2Æ6H2O (2.33 g) and of 2,2¢-bipyridine (2.5 g) were
both dissolved separately in 30 mL ethanol The following
steps where performed in the dark The two solutions were
combined and 170 mg solid NaOH,dissolved in 215 lL of
water,was added A stream of oxygen was bubbled into
the liquid for 30 s The reaction mixture was shaken at
37C for each 5 min at 100,50 and 25 revolutions per min
on a rotary platform; for crystallization it was kept at
30C for at least 12 h in the dark Crystals were collected
on a sintered glass funnel,washed with 20 mL of ethanol
and dried for 6 h by desiccation The product (yield: 70%)
was stored at )20 C IR(KBr): 1380 cm)1 (nitrate),
855 cm)1 m(O-O) UV/vis: kmax: 460 nm,395 nm
(7100M )1cm)1),314 nm (43 300M )1Æcm)1),304 nm and
212 nm
Synthesis of the bis(2,2¢-bipyridyl)cobalt(II)
(BC)-perchlorate salt
Co(NO3)2Æ6H2O (2 mL of 0.2M in water) and
2,2¢-bipyri-dine (2 mL of 0.4Min ethanol) were mixed and 6 sodium
perchlorate solution in 50% (v/v) of aqueous ethanol was
added to a final concentration of 2 and the mixture was kept
for 16 h at 10C The yellow hexagonal crystals formed
were collected as described above UV/vis: k : 293 nm
Determination of the molar yield of photolytical oxygen release
HPBC-nitrate salt (20 mg) and EDTA (100 mg) were placed into a stoppered glass vessel of a total volume of about 120 mL It was filled to the edge with bidistilled water,1 mL of a 3MNaI solution in 40% (w/v) NaOH and
1 mL of 40% (w/v) MnCl2solution were added Another reaction vessel without added HPBC-nitrate salt served as reference To attain completion of the photolytic reaction, the compound (which was kept in the non-UV-transmitting glass bottle) had to be irradiated with visible light emitted by
a workshop-made lamp arrangement for several hours Measurement of dissolved plus photolytically liberated oxygen was chemically determined in sample and reference mixtures according to the Winkler titration technique [20]
Crystallization and X-ray structure determination For crystal structure determination,data sets were collected with a Nonius KappaCCD diffractometer,equipped with a rotating anode generator Nonius FR591 The following computer programs were used for different steps data recording and evaluation: COLLECT for data collection (Nonius BV),DENZO-SMNfor data reduction [21],SORTAV
for absorption correction [22,23], SHELXS-7 for structure solution [24], SHELXL-7 for structure refinement (G M Sheldrick,Universita¨t Go¨ttingen,Germany),DIAMONDfor the graphic display of structures (K Brandenburg,Univer-sita¨t Freiburg,Germany)
Determination of quantum yield of oxygen release The quantum yield of the photorelease of molecular oxygen from HPBC was determined after quantification of the photon flux emitted by a Xe lamp at different wavelengths
by means of the chemical actinometer compound Aber-chrome 540 [25] The samples were placed in 1-cm stirred cuvettes and were irradiated with monochromatic light for defined time intervals to correct for wavelength-dependent emission intensities The numbers of incident photons and the photolytic turnover were quantified by static UV/vis spectroscopy by measurement of the absorbance changes of Aberchrome 540 dissolved in toluene at 494 nm and of HPBC dissolved in 100 mMKPipH 7.4 at 293 nm At high concentrations of HPBC,the absorbance change at 390 nm was also used to quantify the photolytic yield
Preparation of duroquinol-reduced samples for visible spectroscopy and FT-IR spectroscopy and recording of UV/vis spectra
Cytochrome bo3was expressed using the vector pHCL [17] and purified using Ni-agarose chromatography exactly as described previously [26] Optical absorbance spectra of cytochrome bo3 in the presence of caged oxygen were performed using workshop-made CaF2cuvettes constructed for FT-IR spectroscopy (see below) Sample preparations were carried out under an Ar atmosphere in a plastic container (Atmosbag,Sigma) equipped with grips for better sample handling A small volume (2.5 lL) of a 10 mM
ethanolic solution of 10 mMduroquinol was pipetted on the center of a CaF window,covered perimetrically with a thin
Trang 3layer of grease (Apiezon) After evaporation of the solvent
the duroquinol was redissolved with 2–3 lL of a
concen-trated cytochrome bo3 solution (about 0.3 mM) in 20 mM
Tris/HCl,pH 8.0,0.3% (w/v) b-decylmaltoside The
mix-ture was concentrated in an Ar stream The dried layer was
rehydrated with 3 lL 100 mM sodium borate,1 mM
EDTA,0.1% b-decylmaltoside,pH 9.0 Again the mixture
was concentrated under Ar and it was finally redissolved by
adding 0.5 lL of 10 mM HPBC in borate buffer The
cuvette was sealed with another CaF2plate,and placed into
a metallic sample holder The following cuvette handling
was carried out in the aerobic atmosphere The absorbance
spectra of the mixture before and after irradiation with a
150- Xe arc lamp (Oriel) or LPX 240i excimer laser
(Lambda Physics,Go¨ttingen) were measured with a Hitachi
UV/vis spectrometer
Preparation of thiol-reduced samples
of cytochromebo3
The samples were prepared in a workshop-constructed
glass chamber equipped with plate holders for sample and
counter CaF2 plates (technical details will be described
elsewhere) Volumes of 2.5–3.5 lL of 0.2–0.3 mM
cyto-chrome bo3solution (as above) were pipetted on a greased
CaF2sample plate and mixed with 1 lL of a 20 mMfreshly
prepared dithiothreitol solution in 20 mM Tris,pH 8.0,
0.3% (w/v) b-decylmaltoside,and the mixture was spread
to a spot of 5-mm diameter The chamber was assembled
and a CaF2counter plate,spotted with a 0.5-lL drop of
20 mMHPBC dissolved in glycerol,was placed in position
opposite to the sample plate The chamber was evacuated
for 2 min to a residual pressure of 10–50 mbar to allow
dehydration of the sample and formation of a thin film,
which was re-equilibrated with aqueous vapor from a water
reservoir for 30 s Sample and counter plates were then
pressed together,which resulted in efficient mixing of the
reduced protein with the caged oxygen compound The
cuvette was sealed airtight and kept at 4C until
measurement
Recording of FT-IR spectra
Static IR spectra were recorded with a Bruker 66V/S
spectrometer,evacuated to 8–10 mbar residual pressure
The sample containment,maintained at 4C,was purged
with dry air to minimize absorbance by water vapor A
water-cooled globar was used as source of radiation,which
was measured by a nitrogen-cooled HgCdTe detector,using
a low-pass filter which cut off intensity above 1975 cm)1
The scanner mirror was moved in the single-sided mode to
achieve a scan rate of 100 kHz Spectra were measured at
nominal resolution of 2 cm)1,Mertz phase correction was
adjusted and the Blackman–Harris three-term function was
used for apodization If not otherwise indicated,reference
spectra of 800 coadded scans was recorded The sample
photolysis was initiated by application of 15 flashes (90–
140 mJ) of light with a pulse length of 20–30 ns at 308 nm
from an LPX240i excimer laser (Lambda
Physics,Go¨ttin-gen),and 800 scans were coadded To verify that the redox
reaction of protein molecules was complete,a second
spectrum was recorded as above (without reference
meas-urement) after application of another 15 laser flashes
Double difference calculations were carried out using the
OPUSsoftware In order to normalize distinct spectra,the absorbance difference bands of caged oxygen at 1443 and
1451 cm)1were brought to the same scale
Redox spectra with flavine mononucleotide as caged electron donor
Sample preparation and recording of FTIR spectra was carried out as described previously [16]
Enzyme activity test Quinol oxidase activity using duroquinol as artificial substrate of cytochrome bo3 was performed as described previously [26]
R E S U L T S
Synthesis of the HPBC complex After Skurlatov [27] introduced the dibridged dinuclear complex HPBC,MacArthur used this compound as a very poor photoactivatable donor of dioxygen; the photolytic quantum yield was as low as 0.04 if the irradiation was carried out at 355 nm [18] However,in our hands the published preparation protocol for the HPBC-perchlorate salt yielded a product that was contaminated with up to 70–80% of the mononuclear Co(II) species,BC-perchlorate Therefore we established a highly reproducible procedure,
in which the pure perchlorate salt could be obtained at
> 70% molar yield The final product could be gained readily by precipitation of the perchlorate salt; this implies that low solubility in water is an inherent property of the HPBC-perchlorate salt preparation and is a major limiting factor for the maximum oxygen concentration attainable by photo-release
For FT-IR difference spectroscopy of cytochrome oxid-ases,it is necessary to adjust high levels of molecular dioxygen; thus a derivative with much higher solubility had
to be synthesized To this purpose we prepared the nitrate salt of the HPBC complex,which is about 103times more soluble in water than the perchlorate complex
Crystallization and X-ray structure determination
In order to determine the HPBC-perchlorate and -nitrate structures,crystallization trials were set up by mixing solutions of HPBC-nitrate salt with various different anions such as tetrafluoroborate and perchlorate By use of the precipitation/ether diffusion technique,well-ordered large monoclinic crystals (space group P21/c) suitable for X-ray diffraction (Fig 1) were obtained with perchlorate Both Co centers have octahedral coordination and are connected by l-hydroxy and l-peroxo bridges The bond distances [Co-l(O) 1.868 (± 0.005) A˚ and 1.877 (± 0.004) A˚, respectively, l(O)-l(O) 1.415 (± 0.006) A˚] of the bridging core are very similar to that of the corresponding ethylendi-amine complex [28] except the l(O)–l(O) distance,which is significantly smaller (at the short end of the usual range for binuclear l-peroxo complexes) [29] The cation structures of the HPBC-nitrate salt and the -perchlorate salt complexes were identical; the nitrate complex yielded a somewhat
Trang 4higher R value due to disordering of the nitrate groups and
solvent molecules included in the crystal (data not shown)
Spectroscopical and photochemical
properties of HPBC
The perchlorate and nitrate salt of HPBC had identical
optical absorbance spectra with maxima at 212,304,314,
395 nm and a shoulder at 460 nm (Fig 2,insert),which
indicates that it is the cation which determines the optical
properties In contrast to the published extinction
coeffi-cient of 1540M )1Æcm)1 at 390 nm [30],we measured a
value of 7000M )1Æcm)1 based on a molecular mass of
977 gÆmol)1 for the trinitrate salt of the HPBC complex
If it is assumed that possible impurities might contribute
to the weighted mass,an even higher numerical value of
the extinction coefficient is expected Thus the quantities
of molecular oxygen reported to be photoreleased by
others [18] must have been overestimated by a factor of at
least 4,if calculated with the low published extinction
coefficient (1540M )1Æcm)1 for 390 nm, 1350M )1Æcm)1
for 355 nm [18]) HPBC could be photolyzed efficiently by
UV light from different sources,e.g transilluminator
(mercury lamp),Xe lamp or UV laser In all cases,the
end product of the photolytic reaction had absorbance
maxima of 230,293,304 nm,identified to be the mononuclear BC The amount of photolytically released oxygen was ascertained to be 100% by UV/vis spectros-copy As an independent check of molar yield,the production of O2 was determined iodometrically accord-ing to Winkler [20] to about 80%; these data confirm our revision of the published extinction coefficient as pointed out above The experiment demonstrates that the photo-release of dioxygen from HPBC virtually has a stoichi-ometry of 1
The uncaging reaction has to be very efficient in order to make HPBC a useful photo-trigger With the irradiation wavelength of 355 nm a quantum yield of as low as 0.04 was reported previously [18] We expected better photolytic yields at shorter wavelengths due to the higher extinction coefficient of the compound as it becomes evident from Fig 2 A quantum yield of 0.5 was obtained,if the oxygen release was activated by irradiation at 314 nm
To investigate the potential for measuring time-resolved reactions of the released oxygen,transient photoactivation
at 308 nm was probed with a single flash from an eximer laser source Figure 3 displays the dependence of single-shot induced product formation on the total concentra-tion of HPBC The photolysis led to a yield of almost 100% at a concentration of 0.5 mM HPBC This corres-ponds to the same concentration of liberated dioxygen, which caused gas bubble formation due to the limited solubility of oxygen in aqueous medium At higher HPBC concentrations the O2yields decreased because the high UV absorbance leads to a pronounced inner filter effect of the samples Even higher oxygen concentrations could be attained by the use of thinner cuvettes,by lowering of temperature and by variation of the solvent composition
Fig 2 Determination of the quantum yield of photolytic reaction of HPBC and concomitant oxygen release Samples were irradiated with monochromatic light at different times to correct for the wavelength-dependent photon fluxes The numbers of incident photons were determined with a chemical actinometer compound Molecular yields
of HPBC photolysis were quantified spectrophotometrically,these numbers were equivalent to the amounts of oxygen released Inset: optical spectra of HPBC before and after photolysis by continuous irradiation at 314 nm with a Xe lamp at low intensity.
Fig 1 X-ray structure of the HPBC complex Structure analysis of
the HPBC-perchlorate salt (deposited under accession no CCDC
169345 at the Cambridge Crystallographic Data Centre): Formula
C 40 H 33 N 8 O 3 CoÆ3ClO 4 ÆH 2 O, m ¼ 1107.97,black crystal with
dimen-sions 0.50 · 0.30 · 0.20 mm; a ¼ 23.541(1), b ¼ 16.353(1), c ¼
11.377(1) A˚, b ¼ 96.71(1), V ¼ 4349.8(5) A˚ 3
, q calc ¼ 1.692 g cm)3,
l ¼ 10.31 cm)1,empirical absorption correction via SORTAV
(0.627 £ T £ 0.820), Z ¼ 4,monoclinic,space group P2 1 /c no 14);
k ¼ 0.71073 A˚, T ¼ 198 K, x and / scans,31572 reflections collected
(± h, ± k, ± l),[(sinh)/k] ¼ 0.67 A˚)1,10625 independent (R int ¼
0.038) and 9456 observed reflections [I ‡ 2 r(I )],625 refined
param-eters, R ¼ 0.097, wR 2
¼ 0.267 The maximal residual electron density was 1.90 ( ) 1.04) eÆA˚ )3 in the region of the perchlorate groups; the
perchlorate groups are disordered (disorder was not refined) The
hydrogen on the bridging oxygen was obtained from difference Fourier
calculations,other hydrogens were calculated and refined riding.
Trang 5Reaction of HPBC with cytochromebo3: visible
spectral region
HPBC photochemistry was employed to explore the
electron transfer from fully reduced cytochrome bo3oxidase
to photo-released dioxygen; the reaction was monitored by
optical absorbance spectroscopy Because of the intention
to eventually study the interactions by IR spectroscopy (see
below),the experiments were carried out in FT-IR
spectro-meter-type CaF2cuvettes at a sample thickness of equal or
less than 5 lm The visible spectrum of reduced
cyto-chrome bo3 exhibits a broad Soret peak at 425 nm and
bands at 530 and 560 nm in the dark Upon irradiation with
a Xe lamp (Fig 4) or with an excimer laser after 15 laser
flashes at 308 nm with an intensity of 90–140 mJ per pulse
(data not shown),typical oxidized spectra are found with
the Soret peak shifted to 409 nm and absorbance loss at
higher wavelengths (Fig 4) The integrity of the protein
sample after irradiation was also checked with SDS/PAGE
If HPBC is irradiated in the absence of protein,no pronounced peaks contributed within the investigated spectral region before or after photolysis As being a prerequisite for sample stabilization during longer periods in FT-IR experiments,the spectrum of reduced cyto-chrome bo3 oxidase in presence of HPBC remained unchanged in the dark after incubation for 48 h at 4C
It is now possible to observe the oxidation of the fully reduced oxidase in situ through dioxygen release from HPBC after photolysis
Reaction of HPBC with cytochrome
bo3: IR spectral region FT-IR spectra were recorded to study the molecular details
of the reaction of caged oxygen and cytochrome bo3 The protein was reduced with the quasi-natural substrate analog duroquinol The samples reached a stable baseline after 2–4 h at 4C,and the completeness of caged oxygen photolysis was checked using bundles of 15 laser flashes The spectrum (Fig 5A) shows a composite of difference spectra (light) dark) from cytochrome bo3 plus caged oxygen before and after the photoreaction The initial and final states of these static spectra could be classified as to oxidized cytochrome bo3/‘oxygen-free HPBC’ (absorbances deflecting upwards) and to reduced cytochrome bo3 /‘oxy-gen-bound HPBC’ (absorbances deflecting downwards),as assessed by the optical spectra before and after photo-irradiation of the sample cuvette The absorbance peaks of HPBC at 1443/1451 cm)1and at 1600/1612 cm)1stand out clearly Sharp difference bands (at 1657 cm)1,1678 cm)1)
Fig 3 Yield of photolysis after single flash activation by excimer laser
at 308 nm, 160 mJ per pulse The solutions of HPBC were prepared in
FT-IR type sample cuvette with 500-lm thickness Yields were
determined from the absorbance changes at 293 nm.
Fig 4 Reaction of cytochrome bo 3 fully reduced by the substrate
dur-oquinol with HPBC Absorbance spectra of reduced cytochrome bo 3 in
presence of HPBC in the dark and after photolysis with Xe lamp were
measured,as described in Materials and methods The corresponding
spectra of HPBC in the absence of protein are included.
Fig 5 Reaction of duroquinol-reduced cytochrome bo 3 with photore-leased dioxygen, monitored by FT-IR spectroscopy (0.85 lmol cytochrome resuspended in 20 m M Tris/HCl pH 8.0, 50 m M NaCl, 0.3% b-decylmaltoside, reduced with 2.5 nmol duroquinol After reduction,4.5 nmol HPBC resolved with 100 m M borate pH 9.0,0.1% b-decylmaltoside,1 m M EDTA was added Photolysis conditions:
110 mJ per pulse,308 nm,XeCl-excimer laser,spectra taken after 15 flashes (A) The (light ) dark) difference spectrum is shown (B) As a control,the same experiment as in (A) was carried out,except that duroquinone instead of duroquinol was added (C) The double difference spectrum between (A) and (B) was calculated,yielding the effect of the protein reaction alone.
Trang 6can be seen also in the amide I region,indicating
conform-ational alterations elicited by the redox transition In the
carbonyl region one can clearly distinguish positive bands at
1745 and at 1696 cm)1 Oxidized cytochrome bo3
equili-brated with duroquinone and HPBC was photolyzed in a
control experiment (Fig 5B) The net reaction by the caged
compound itself could be measured; the difference spectra
looked similar to that obtained by the pure HPBC complex
itself The prominent difference bands at 1443/1451 cm)1
and 1600/1612 cm)1were used to scale the spectra for better
comparison In order to obtain the redox difference spectra
of the protein itself,one has to subtract the background
from the composite spectrum Figure 5C displays the
double difference spectrum (A minus B): above 1690 cm)1
it is dominated solely by the spectral response of the protein
Dithiothreitol can be used as an artificial reductant of
cytochrome bo3 Figure 6 (top) shows a redox difference
spectrum resulting from the reaction of
dithiothreitol-reduced cytochrome bo3 with caged oxygen Absorbance
patterns in the spectral region below 1670 cm)1are variable
to some extent,because of the high absorption in the amide
regions due to variable protein concentration and to the
residual amounts of water The carbonyl region of the
spectrum is mostly unaffected by the HPBC difference
bands (Fig 5B); the uncorrected data yielded the same
difference band pattern if the spectral transition was
recorded with either reductant The use of
dithiothreitol-reduced samples was found to be more practical,because it
allowed the preparation of samples in presence of
atmo-spheric oxygen It is important to adjust for low water
content for two reasons: (a) water is a strong absorber of IR
radiation and (b) a considerable amount of solvent is
mobilized by oxygen bubble formation upon photolysis of
HPBC at higher concentrations,which critically affect the
sample stability
FT-IR spectra of photoreduction and photooxidation
In order to validate the redox difference FT-IR signals
obtained from the oxidation reaction with caged dioxygen,
spectra were compared with those obtained by the reverse
reaction triggered by caged electron FMN [16,17] The difference FT-IR spectrum obtained in Fig 6 (bottom) shows the typical redox pattern expected for the carbonyl region of the oxidase; it has the strong negative band at
1696 cm)1 and the prominent carbonyl feature at 1745/
1735 cm)1,which had been assigned to Glu286 in previous spectra [17] The difference spectra in the carbonyl region, generated by either FMN (reduced) oxidized) or HPBC (oxidized) reduced) appear to be reciprocal,which dem-onstrates the equivalence of informational content from both experiments
Time resolution of the reaction of photochemically released oxygen with cytochromebo3
An efficient caged compound has to provide oxygen very quickly Time-resolved measurements of cytochrome oxid-ase kinetics have been successfully carried out with the flow-flash method,by observation of heme absorbance [31–35] HPBC-released dioxygen has been used to measure the kinetics of optical heme absorbance with reduced cyto-chrome c oxidase [19]
In preliminary experiments,the photoirradiation of the caged compound with a single laser flash led to formation of
a stable absorbance line after 1 ls,which is the time resolution limit of the apparatus used It may be assumed that oxygen is liberated in parallel to the absorbance change
of the caged compound itself The relevant lower time-limit could be estimated by reaction of photo-released oxygen with different heme proteins: if 5 lM duroquinol-reduced cytochrome bo3is exposed to low concentrations of caged oxygen,a flash-induced transient of absorbance decrease at
430 nm is observed,which is indicative of heme oxidation The kinetic traces are complex and exhibit half-lives of about
1 ms,which was evaluated without application of spectral deconvolution analysis Using the oxygenation of myoglo-bin as a different indicator,significant flash-induced absorbance changes were recordable even after only
100 ls This demonstrates that the chemical formation of oxygen from its precursor was definitely not a rate-limiting step in the reaction of cytochrome bo3
D I S C U S S I O N
Absorbance changes in the IR region provide information about individual steps of the partial reactions of cytochrome oxidase (and presumably also of other oxygen-binding proteins) at the molecular level The strong absorbance of the solvent water is a problem inherent to this spectroscopic technique,which has to be carried out with highly concentrated samples layered in very thin aqueous films
In the study of oxidases,this need is in conflict with the requirement of efficient mixing of reactants,such as provided by the stopped-flow type apparatus used in flow-flash experiments The fundamental problem is overcome
by delivery of molecular oxygen via photo-triggering of the organometallic oxygen precursor compound HPBC High concentrations of oxygen could be obtained,because the use
of the HPBC nitrate salt as obtained from our preparation does not have the severe solubility problem of the perchlorate derivative
In an extension of earlier experiments with the heme proteins hemoglobin [18] and with cytochrome c oxidase
Fig 6 Comparison of the redox difference FT-IR spectra of
cyto-chrome bo 3 in the carbonyl region generated by FMN (bottom spectrum)
and by caged dioxygen (top spectrum), using a sample containing 1 nmol
dithiothreitol-treated cytochrome bo 3 and 10 nmol HPBC dissolved in
glycerol.
Trang 7[19] by optical spectroscopy,it was demonstrated in this
work that the dioxygen photoreleased by HPBC acts as
electron acceptor of cytochrome bo3 Pre-reduction of the
enzyme with thiol compounds was the most favorable
sample preparation method Electron transfer of the protein
has been verified optically; molecular changes induced by
the photoreaction were monitored by FT-IR spectroscopy
It was possible to recognize absorbance differences of
carboxyl groups,one of which was assigned to the
conformational change of the side chain of Glu286 from
the catalytic subunit I of cytochrome bo3 Comparisons of
the spectra obtained in this study with redox spectra
measured with the caged electron donor FMN yielded
absorbance patterns of inverted shapes Oxidoreduction of
cytochrome oxidase can thus be observed spectroscopically
in the forward and backward reaction Complementary
information is gained by the reciprocal experiments of heme
oxidation and reduction: The electron flow from heme
centers to oxygen arises exactly as expected; it thus seems
clear,that the dioxygen produced after uncaging behaves
like the natural substrate According to prelimirary
experi-ments it is possible to follow time-resolved absorbance
changes at optical wavelengths with cytochrome bo3 In the
future,we will extend the caged oxygen approach to
investigate the kinetics of oxidase reaction at the molecular
level by IR spectroscopy
A C K N O W L E D G E M E N T S
This work was supported by the Deutsche Forschungsgemeinschaft
grant SFB 394-C6 and the Volkswagen-Stiftung to M L We thank
Prof Klaus Gerwert for his support in providing the FT-IR equipment.
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