To elucidate if distortion of the methoxy groups induces this hydrogen-bonding, their ring-C-O vibrations were assigned by use of site-specifically labelled [5-13C]UQ10and [6-13C]UQ10reco
Trang 1Does different orientation of the methoxy groups of ubiquinone-10
Andre´ Remy1, Rutger B Boers2, Tatiana Egorova-Zachernyuk2, Peter Gast3, Johan Lugtenburg2
and Klaus Gerwert1
1
Lehrstuhl fu¨r Biophysik, Ruhr-Universita¨t Bochum, Germany;2Department of Chemistry, Gorlaeus Laboratories, Leiden University, the Netherlands;3Department of Biophysics, Huygens Laboratory, Leiden University, the Netherlands
The different roles of ubiquinone-10 (UQ10) at the primary
and secondary quinone (QAand QB) binding sites of
Rho-dobacter sphaeroidesR26 reaction centres are governed by
the protein microenvironment The 4C¼O carbonyl group
of QAis unusually strongly hydrogen-bonded, in contrast to
QB This asymmetric binding seems to determine their
dif-ferent functions The asymmetric hydrogen-bonding at QA
can be caused intrinsically by distortion of the methoxy
groups or extrinsically by binding to specific amino-acid side
groups Different X-ray-based structural models show
con-tradictory orientations of the methoxy groups and do
not provide a clear picture To elucidate if distortion of
the methoxy groups induces this hydrogen-bonding, their
(ring-)C-O vibrations were assigned by use of site-specifically
labelled [5-13C]UQ10and [6-13C]UQ10reconstituted at either
the QA or the QB binding site Two infrared bands at
1288 cm)1 and 1264 cm)1were assigned to the methoxy vibrations They did not shift in frequency at either the QAor
QBbinding sites, as compared with unbound UQ10 As the frequencies of these vibrations and their coupling are sensi-tive to the conformations of the methoxy groups, different conformations of the C(5) and C(6) methoxy groups at the
QAand QBbinding sites can now be excluded Both methoxy groups are oriented out of plane at QAand QB Therefore, hydrogen-bonding to His M219 combined with electrostatic interactions with the Fe2+ion seems to determine the strong asymmetric binding of QA
Keywords: electron transfer; Fourier-transform infrared spectroscopy; isotopic labelling; photosynthetic reaction centre; ubiquinone
The photosynthetic reaction centre (RC) of the purple
nonsulphur bacterium Rhodobacter sphaeroides is a
trans-membrane pigment–protein complex, the structure of
which has been determined with up to 2.2 A˚ resolution
[1–4] Upon light excitation, an electron is transferred from
the primary donor P (bacteriochlorophyll a dimer) via a
monomeric bacteriochlorophyll a and a
bacteriopheo-phytin a molecule to the primary quinone QAand finally
to the secondary quinone QB Although ubiquinone-10
(UQ10) is found at QAand QB, the two molecules differ in
function: QAis tightly bound to the RC By accepting one
electron, a semiquinone anion radical QA– is created which
quickly transfers the electron to QB QB is less tightly
bound After the formation of a nonprotonated
semiqui-none anion radical QB–, a second electron and two protons
are accepted here to form a hydroquinone (QBH2), which
is finally released from the RC; for a recent review see [5]
To elucidate the protein–cofactor interactions that deter-mine the different functions of UQ10at QAand QB, Fourier-transform infrared (FTIR) difference spectroscopy has been applied [6–9] By the use of UQ10specifically13C-labelled at the ring positions 1, 2, 3, and 4, the 1C¼O and 4C¼O and 2/3C¼C stretching vibrations of UQ10in the RC have been assigned in the QA–) QA and QB–) QBdifference spectra [10–13] At the QAsite, the mode dominated by the 4C¼O vibration is dramatically downshifted compared with unbound UQ10, indicating unusually strong hydrogen-bonding to the protein environment [10,11] In contrast, the 1C¼O group is only weakly bound to the protein This asymmetric binding is conserved in the charge-separated state [10,11] At the QB site, two fractions of UQ10 are found The minor fraction is loosely bound and almost unaffected by the protein In the major fraction, both C¼O vibrations show symmetric hydrogen-bonding, but weaker than the hydrogen bond of 4C¼O at the QAsite [12,13] These results for the charge-separated state are supported
by EPR [14] and NMR spectroscopy [15]
It is proposed that this difference in binding governs the different roles of UQ10at the QAand QBsites However, the molecular origin of the strong binding of the 4C¼O group is not clear The conformation of the C(5) and C(6) methoxy substituents of UQ may differ at both binding sites as
Correspondence to K Gerwert, Lehrstuhl fu¨r Biophysik,
Ruhr-Universita¨t Bochum, Postfach 102148, 44780 Bochum, Germany.
Fax: + 49 234 321 4626, Tel.: + 49 234 322 4461,
E-mail: gerwert@bph.ruhr-uni-bochum.de
Abbreviations: FTIR, Fourier-transform infrared; IR, infrared;
LDAO, lauryldimethylamine N-oxide; Q A , primary acceptor quinone;
Q B , secondary acceptor quinone; Rb., Rhodobacter;
RC, reaction centre; UQ 10 , ubiquinone-10.
(Received 14 April 2003, revised 25 June 2003, accepted 8 July 2003)
Trang 2predicted by theoretical studies [16–19], and the different
conformations would then lead to a shift in electron density
towards the 4C¼O group, which weakens the 4C¼O bond
order
We present QA–) QAand QB–) QBdifference spectra and
IR spectra of unlabelled and site-specifically 13C-labelled
UQ10at the C5 and C6 positions Thereby, the
correspond-ing (rcorrespond-ing-)C-O vibrations are clearly assigned The
implica-tions for the C(5) and C(6) methoxy conformaimplica-tions at
QAand QBwill be discussed
Materials and methods
UQ10, selectively13C-labelled at positions C5 and C6, was
synthesized [20] RC protein was purified from Rb
sphaero-idesstrain R26 [21] Either the native UQ10was removed
from the QBsite or the native UQ10was removed from the
QA and QB sites [22] The QA and QB contents were
determined by fitting the recombination kinetics at 865 nm
after photobleaching of the primary donor to a sum of two
exponentials: A¼ A1exp(–k1t1) + A2exp(–k2t2) Upon
normalization of the amplitudes A1(fast QA–decay) and A2
(slow QB–decay) (A1+ A2¼ 100%), the fraction of
func-tionally bound secondary quinone was obtained In the case
of QA reconstitution, the occupancy of the QA site was
analysed by measuring the photobleaching at 865 nm before
and after addition of a 100-fold excess of UQ0(¼ 100%
activity) and comparing the amplitudes The QAand QB
contents after reconstitution were always better than 85%
Samples were prepared for the IR measurements as
previously described [10] A 45 lL portion of 40 lMRCs,
dissolved in buffer [10 mMTris/HCl, 1 mMEDTA, 0.025%
(w/v) lauryldimethylamine N-oxide (LDAO), pH 8] was
pipetted on a CaF2window, 10-fold concentrated under a
gentle stream of nitrogen, and mixed with 5 lL 10 mM
sodium ascorbate/20 mM diaminodurene dissolved in the
same buffer as the RCs After further careful drying to a
final volume of 1 lL, the sample was sealed with another
CaF2window and thermostabilized at 283 K or 295 K (for
QA–) QA or QB–) QB difference spectra, respectively) in
the FTIR apparatus The ubiquinones were dissolved in
n-pentane and deposited on a CaF2window After
evapor-ation of n-pentane, the remaining UQ10film was measured
in the IR
IR spectra of unbound ubiquinones, QA–) QAdifference
spectra and QB–) QB difference spectra were recorded as
reported [10,23,24] Spectral resolution was 4 cm)1
Double difference spectra were computed as described
[10] The difference spectra with unlabelled and13C-labelled
UQ10at the QAor QBsites were normalized on the 1800–
1700 cm)1region, which was unaffected by the labelling
The IR spectra of the unbound UQ10were normalized on
the 1500–1350 cm)1region, which was unaffected by the
labelling
Results and discussion
To investigate the influence of the protein
microenviron-ment on UQ10 at the QA and QB binding sites, first the
vibrational modes of the unbound UQ10were determined
FTIR spectra of pure UQ10 are shown in Fig 1 in the
spectral range in which methoxy vibrations are expected
The spectrum of unlabelled UQ10(Fig 1a) agrees with the one published [25] The absorption spectrum of [5-13C]
UQ10is displayed in Fig 1b Isotopic labelling induces a frequency shift of the absorption of the labelled group to lower wave numbers and thereby allows unequivocal band assignment Apart from this, bands of nearby groups, the vibrational modes of which are coupled to the vibrations of the labelled group, may also be shifted In fact, various band shifts of C¼C and C¼O vibrations, which are coupled to the (ring-)C-O vibrations of the methoxy groups, occur (spectral range not shown) This is in agreement with previous assignments of C¼C and C¼O vibrations [10,11] A detailed discussion of the C¼C and C¼O vibrations is beyond the scope of this paper and will be given elsewhere Here we focus
on the methoxy vibrations only The strong bands at 1447,
1434 (shoulder) and 1380 cm)1are almost unaffected by the labelling, whereas the bands at 1287 and 1263 cm)1 are downshifted to 1283 and 1254 cm)1, respectively
This is visualized in the double difference spectrum (Fig 1d) If the spectra of unlabelled and specifically
13C-labelled UQ10 are subtracted as described [10], all unshifted bands should in principle disappear, and only the shifted bands appear as difference signals in the double difference spectrum The two band shifts described above occur at 1288/1277 cm)1and 1264/1252 cm)1 The down-shift from 1264 to 1252 cm)1 is obvious, whereas the
Fig 1 IR absorption spectra of (a) unlabelled UQ 10 , (b) [5- 13 C]UQ 10 , and (c) [6- 13 C]UQ 10 and (d) difference b ) a and (e) difference c ) a Inset: structure of site-specifically labelled UQ 10
Trang 3downshift from 1288 to 1277 cm)1is just above the
resolu-tion However, even though the band is small, the shift is
highly reproducible
The absorption spectrum of [6-13C]UQ10 (Fig 1c) is
similar to that of [5-13C]UQ10(see Fig 1b), but the band
shifts are slightly different The band at 1287 cm)1shifts to
1280 cm)1 [in the double difference spectrum (Fig 1e)
1288/1274 cm)1], and the band at 1264 cm)1 shifts to
1252 cm)1
From the observed frequency shifts caused by site-specific
isotopic labelling, the bands at 1288 and 1263 cm)1were
assigned to C(5) and C(6) methoxy vibrations of UQ10
To assign the methoxy vibrations at the QAbinding site,
QA–) QAdifference spectra of Rb sphaeroides RCs
recon-stituted with unlabelled and site-specifically labelled UQ10
were measured (Fig 2) The differences between the
charge-separated and the ground state absorption selectively
represent the light-induced absorption changes of the
RCs Positive bands belong to the charge-separated state,
and negative signals to the ground state
The QA–) QA difference spectrum of unlabelled UQ10
(Fig 2a) agrees well with the one published [23] The
QA–) QA difference spectrum of [5-13C]UQ10 is displayed
in Fig 2b As for unbound UQ10, various band shifts
of coupled C¼C and C¼O vibrations of QA occur, in
agreement with previous assignments [10,11] (spectral range not shown) As both ground state and charge-separated state contribute to the QA–) QA difference spectra, the coupled C-O– vibration of QA– at 1486 cm)1 [10,11] is also affected This is not obvious in the
QA–) QA difference spectra (Fig 2b,c), but resolved in the double difference spectra (Fig 2d,e) Unexpectedly, a positive signal occurs in the double difference spectra, whereas a QA–vibration should cause a negative one The C-O– vibration of QA–, however, shows highly coupled behaviour on isotopic labelling, as described and dis-cussed previously [10,11,26] Moreover, the present study focuses on the methoxy vibrations, and because of the lack of labelling effects in this region in the spectra of unbound UQ10, any contributions of methoxy vibrations
to this band are most unlikely Two negative bands at
1287 and 1263 cm)1are downshifted due to [5-13C]UQ10,
to 1273 and 1254 cm)1, respectively The double differ-ence spectrum (Fig 2d) shows respective downshifts of these bands from 1288 to 1277 cm)1 and from 1263 to
1254 cm)1 These effects are due to the ground state of
QA In principle, contributions of the semiquinone state
QA– may also occur in the difference spectra, but their frequencies are probably below 1000 cm)1, so they are not observed in this study
The QA–) QA difference spectrum of [6-13C]UQ10 is displayed in Fig 2c This spectrum is similar to that of [5-13C]UQ10at the QAsite (see Fig 2b) As in the case of [5-13C]UQ10, the same band shifts down to 1273 and
1254 cm)1 occur In the double difference spectrum (Fig 2e), the bands at 1287 and 1263 cm)1are downshifted
to 1274 and 1254 cm)1, respectively
Therefore, the bands at 1287/88 and 1263 cm)1 are assigned to C(5) and C(6) methoxy vibrations of UQ10at the QA binding site This assignment agrees with the methoxy vibrations of unbound UQ10
The QB–) QBdifference spectrum of Rb sphaeroides RCs reconstituted with unlabelled UQ10is displayed in Fig 3a It agrees well with the one published [24]
The QB–) QB difference spectrum of [5-13C]UQ10 is shown in Fig 3b As for QA–) QA, band shifts of coupled C¼C, C¼O and C-O– (1479 cm)1) vibrations occur in agreement with former assignments [12,13] (spectral range partially not shown) As for QA–) QA, in the QB–) QB
difference spectrum also two negative bands are down-shifted due to [5-13C]UQ10 from 1290 to 1277 cm)1 and from 1264 to 1253 cm)1 This is better visualized in the double difference spectrum below (Fig 3d) The double difference spectrum shows downshifts from 1289 to
1277 cm)1and from 1265 to 1252 cm)1 The QB–) QB difference spectrum of [6-13C]UQ10 is displayed in Fig 3c This spectrum is similar to that of [5-13C]UQ10at the QBsite (see Fig 3b), and the same band shifts to 1277 and to 1253 cm)1 are seen Also in the double difference spectrum (Fig 3e) the bands at 1288 and 1265 cm)1 are downshifted to 1277 and 1252 cm)1, respectively
Therefore, the bands at 1288/89 and 1265 cm)1 are assigned to C(5) and C(6) methoxy vibrations of UQ10at the QBbinding site This is in agreement with the assignment
of the methoxy vibrations of UQ10at the QAsite and of the unbound UQ
Fig 2 Q A
–
) Q A difference spectra of Rb sphaeroides RCs
reconstitu-ted with (a) unlabelled UQ 10 , (b) [5-13C]UQ 10 , and (c) [6-13C]UQ 10 at the
Q A site and (d) double difference b ) a and (e) double difference c ) a.
Inset: structure of site-specifically labelled UQ
Trang 4Assignment of methoxy vibrations
In the spectra presented, the bands at 1288 and 1264 cm)1
show frequency shifts due to labelling at the C5 or C6
position, whereas all the other ring carbon vibrations
show smaller shifts or do not shift at all [10–13,26] The
bands at 1288 and 1264 cm)1can now unambiguously be
assigned to (ring-)C-O vibrations of the C(5) and C(6)
methoxy groups The vibration at 1264 cm)1 has been
assigned to a C-C-ring vibration by normal mode analysis
[27,28] In contrast, Breton et al proposed this band to be
a combined (ring-)C-C vibration and C-O vibration of the
methoxy groups [29] The latter proposal agrees with our
results
We did not observe an isolated vibration, but both
methoxy vibrations were coupled to various (ring-)C-C
vibrations As the band at 1264 cm)1shows larger shifts due
to isotopic labelling of the other ring carbons than the band
at 1288 cm)1[10–13,26], we conclude that the vibration at
1264 cm)1is more strongly coupled than the vibration at
1288 cm)1 Interestingly, on labelling one of the
methoxy-bearing carbons, both bands shift This indicates that the
vibrations of both methoxy groups are strongly coupled and
cannot be distinguished
That these two bands do not shift on exchanging the methoxy substituents into one or two ethoxy groups [30] excludes a significant contribution of the O-CH3vibrations and thus favours the assignment to the (ring-)C-O stretch-ing mode as the dominant mode at 1288 and 1264 cm)1 The C-O-C bending and O-C-H bending vibrations may also contribute to these bands However, the clear shifts show that the (ring-)C-O vibration is the dominating mode,
as expected by normal mode analysis (M Nonella,
P Tavan, personal communication, referring to [31]) In this normal mode analysis work [31], only the C¼C and C¼O vibrations of the quinones in the RC are reported, but the calculations include the methoxy vibrations of the quinones (M Nonella, P Tavan, personal communica-tion), which are useful for the conclusions drawn in this work Therefore reference [31] is quoted in combination with the cross reference to the personal communication to make clear that our conclusions are not only based on the published data [31], but also on the information commu-nicated by M Nonella and P Tavan which complements the published calculations [31]
The two bands at 1450 and 1436 cm)1 have been proposed to arise from CH3 and CH2 deformation vibra-tions of the isoprenoid chain [32] As they disappear in duroquinone, which is lacking the methoxy groups, they have tentatively been assigned to the O-CH3vibration of the methoxy groups [29] Duroquinone, however, lacks not only the methoxy groups, but also the whole isoprenoid chain FTIR difference spectroscopy using specifically labelled
UQ10revealed that only the isoprenoid chain is responsible for both bands at 1450 and 1436 cm)1[33] In addition, our measurements of [5-13C]UQ10 and [6-13C]UQ10 do not show any shift of these bands due to isotopic labelling and thus support the latter assignment
Implications for the binding of UQ10at the QA
and QBbinding sites How can chemically identical molecules take over different functions in the RC? FTIR difference spectroscopy identi-fied a large downshift of the 4C¼O stretching vibration of
QA by 60 cm)1 [10,11], indicating strong asymmetric binding of UQ10at the QAsite, in contrast to symmetric, weaker binding of UQ10at the QBsite [12,13]
To explain the difference in binding, it has been proposed that the conformation of the two methoxy substituents is sterically hindered at the QAsite, and therefore electrostatic and/or steric interactions between one methoxy group and the oxygen at C4 lower the binding order of the carbon C4 and strengthen the downshift of the 4C¼O mode [10,11]
In principle, X-ray-based structural models of the reaction centre of Rb sphaeroides should provide the methoxy orientations of UQ10 at the QA and QB binding sites However, there are a large variety of contradicting confor-mations in the different structural models: in refs [1,2] one methoxy group is shown within the plane of the quinone ring and the other out of the plane, as shown in Fig 4 (upper part), whereas refs [3,34–37] show both methoxy substituents
in out-of-plane conformations (Fig 4, lower part) However, even within one type there are several variations The downwards or upwards orientation within these two classes, in-plane/out-of-plane and out-of-plane/out-of-plane,
Fig 3 Q B–) Q B difference spectra of Rb sphaeroides RCs
reconstitu-ted with (a) unlabelled UQ 10 , (b) [5- 13 C]UQ 10 , and (c) [6- 13 C]UQ 10 at the
Q A site and (d) double difference b ) a and (e) double difference c ) a.
Inset: structure of site-specifically labelled UQ 10
Trang 5respectively, is found in all permutations within the different
structural models From the contradictory picture of the
different structural models with regard to the methoxy
orientations, we conclude that the resolution of the
X-ray-based structural models of the RC is too low to discriminate
between the different orientations of the methoxy groups
We used FTIR spectroscopy to determine these
orienta-tions The methoxy vibrations assigned surprisingly appear
at almost the same frequencies in the unbound UQ10
(1287 cm)1, 1264 cm)1) and in the protein-bound UQ10at
the QA(1287/88 cm)1, 1263 cm)1) and QB(1288/89 cm)1,
1265 cm)1) binding sites If the conformation of one or both
methoxy groups were greaty affected in the protein-bound
case by steric hindrance or electrostatic interactions, one
would expect a clear shift in frequency compared with the
unbound UQ10 Vibrations of proteins and of their
cofac-tors are very sensitive to changes in conformation [6–9]
There have been no experimental investigations of how
different methoxy group conformations influence the
frequency of the methoxy vibrations
However, the effect of different conformations of the
UQ10 methoxy groups on the IR frequency have been
studied in model compounds [16,17] The calculations show
that the frequencies and the coupling of the (ring-)C-O
methoxy vibrations are sensitive to their orientations As the
same frequency and coupling are observed in unbound
UQ10, at QAand at QB, the methoxy groups must have the
same orientation Therefore, different orientations of UQ10
at QAand QBcan be excluded If one methoxy substituent is
in plane and the other is in an out-of-plane conformation
relative to the quinone ring (conformation A in [2], Fig 4),
the C(5) and C(6) (ring-)C-O modes occur at different
frequencies and they are not coupled (M Nonella, P Tavan,
personal communication, referring to [31], see above)
Therefore, isotopic labelling at either the C(5) or the C(6)
methoxy group would lead to different shifts depending on
the labelling position This is not observed
In contrast, when both methoxy substituents are in out-of-plane positions (conformation Bin [3], Fig 4), the C(5) and C(6) (ring-)C-O vibrations of the methoxy groups are coupled and at the same position (M Nonella, P Tavan, personal communication, referring to [31], see above) Isotopic labelling should lead to identical shifts independent
of the labelled group This is experimentally observed in unbound UQ10as well as at the QAand the QBbinding sites Therefore, we conclude that the same conformation is present in unbound UQ10and in protein-bound UQ10at the
QA and QBbinding sites The agreement with the above calculations indicates that both methoxy substituents are
in an out-of-plane conformation as in [3] Furthermore, these theoretical studies confirm our suggestion that the (ring-)C-O vibration mainly contributes to the assigned methoxy vibrations
This is an example of how IR spectroscopy can give detailed local structural information which complements the data obtained by X-ray crystallography
A further FTIR approach proposes Ile M265 to be constitutive for the electrostatic interaction with UQ10at QA
[38], as mutation of this site to Thr or Ser leads to an upshift
of about 4–5 cm)1of the 4C¼O vibration
It is not the methoxy group orientation, but strong binding to His M219 at QA(Fig 5), and His L190 at QB combined with electrostatic interactions with the Fe2+ion and with further amino-acid side chains in the QAbinding niche (e.g Ile M265) that may explain the strong binding of
UQ10 at the QA site Site-directed mutagenesis of these groups should provide a clear answer
Acknowledgements
Drs M Nonella and P Tavan are acknowledged for providing unpublished information about normal mode analysis studies on ubiquinones This work was financially supported by the Deutsche Forschungsgemeinschaft (SFB480-C3).
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