Caged O
2
Reaction of cytochrome
bo
3
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 bo
3

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
molecularstructuresofthecytochromec oxidases from
Paracoccus denitrificans and 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. coli oxidase), 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

D-44780 Bochum, Germany
Fax: + 49 234 32 14626, Tel.: + 49 234 32 24465,
E-mail:
Abbreviations: HPBC, (l-peroxo)(l-hydroxo)bis[bis(bipyridyl)-
cobalt(III)]; BC, bis(2,2¢-bipyridyl)cobalt(II).
*Present address: Organisch-chemisches Institut, Universita
¨
tMu
¨
nster,
Correnstraße 40, D-48149 Mu
¨
nster, Germany.
Present address: Max-Planck-Institut fu
¨
rBiophysik,
Heinrich-Hoffmann-Str. 7, D-60528 Frankfurt/Main, Germany.
(Received 18 January 2002, revised 15 April 2002,
accepted 19 April 2002)
Eur. J. Biochem. 269, 2630–2637 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02944.x
(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

was 73%. IR(KBr): 1088 cm
)1
and 625 cm
)1
(perchlorate),
855 cm
)1
m(O-O). UV/vis: k
max
: 460 nm, 395 nm
(7100
M
)1
Æcm
)1
), 314 nm (43 300
M
)1
cm
)1
), 304 nm and
212 nm.
Synthesis of the HPBC-nitrate salt
Co(NO
3
)
2
Æ6H
2
O (2.33 g) and of 2,2¢-bipyridine (2.5 g) were

Synthesis of the bis(2,2¢ -bipyridyl)cobalt(II)
(BC)-perchlorate salt
Co(NO
3
)
2
Æ6H
2
O(2mLof0.2
M
in water) and 2,2¢-bipyri-
dine (2 mL of 0.4
M
in 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 10 °C. The yellow hexagonal crystals formed
were collected as described above. UV/vis: k
max
:293nm.
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 3
M
NaI solution in 40% (w/v) NaOH and
1 mL of 40% (w/v) MnCl
2

¨
tGo
¨
ttingen, Germany),
DIAMOND
for
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 m
M
KP
i
pH 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

M
)in20m
M
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 lL100m
M
sodium borate, 1 m
M
EDTA, 0.1% b-decylmaltoside, pH 9.0. Again the mixture
was concentrated under Ar and it was finally redissolved by
adding 0.5 lLof10m
M
HPBC in borate buffer. The
cuvette was sealed with another CaF
2
plate, 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 cytochrome
bo
3
The samples were prepared in a workshop-constructed

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 4 °C 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 4 °C, 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

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 10
3
times 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 P2
1
/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

Æcm
)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 (1540
M
)1
Æcm
)1
for 390 nm,  1350
M
)1
Æcm
)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

yields decreased because the
high UV absorbance leads to a pronounced Ôinner filter
effectÕ 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

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.
Ó FEBS 2002 Caged oxygen reaction with cytochrome oxidase (Eur. J. Biochem. 269) 2633
Reaction of HPBC with cytochrome
bo
3
: visible
spectral region
HPBC photochemistry was employed to explore the
electron transfer from fully reduced cytochrome bo
3
oxidase
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 CaF
2
cuvettes at a sample thickness of equal or
less than 5 lm. The visible spectrum of reduced cyto-
chrome bo
3
exhibits a broad Soret peak at 425 nm and

photolysis was checked using bundles of 15 laser flashes.
The spectrum (Fig. 5A) shows a composite of difference
spectra (light ) dark) from cytochrome bo
3
plus caged
oxygen before and after the photoreaction. The initial and
final states of these static spectra could be classified as to
oxidized cytochrome bo
3
/‘oxygen-free HPBC’ (absorbances
deflecting upwards) and to reduced cytochrome bo
3
/‘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
)1
and at 1600/1612 cm
)1
stand 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.

effect of the protein reaction alone.
2634 C. Ludovici et al. (Eur. J. Biochem. 269) Ó FEBS 2002
can 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 bo
3
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
)1
were 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 bo
3
. Figure 6 (top) shows a redox difference
spectrum resulting from the reaction of dithiothreitol-

)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 cytochrome
bo
3
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 l
M

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.
Ó FEBS 2002 Caged oxygen reaction with cytochrome oxidase (Eur. J. Biochem. 269) 2635
[19] by optical spectroscopy, it was demonstrated in this
work that the dioxygen photoreleased by HPBC acts as
electron acceptor of cytochrome bo
3
. 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 bo
3

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