Bacteriochlorophyll/imidazole and chlorophyll/imidazole complexes are negatively charged in an apolar environment

Article abstract of DOI:10.1016/S0009-2614(00)01117-9

600 MHz ¹⁵N-NMR and 2D homonuclear ¹H-¹H and heteronuclear ¹H-¹⁵N-NMR data for bacteriochlorophyll a/imidazole and chlorophyll a/imidazole have been recorded. Unambiguous assignments of the ¹⁵N signals of the two nitrogens of imidazole ligated to the Mg of the bacteriochlorophyll a or chlorophyll a were obtained. It follows that the imidazole in complex is deprotonated, which implies that the chlorophyll/imidazole carries a full negative charge in the apolar solvent environment. This information is potentially useful in characterizing the nature of the magnesium-histidine interaction and the charge state of chlorophylls coordinated by histidine in photosynthetic pigment protein complexes.

Full text of DOI:10.1016/S0009-2614(00)01117-9

1
0 November 2000
Chemical Physics Letters 330 (2000) 325±330
www.elsevier.nl/locate/cplett
Bacteriochlorophyll/imidazole and chlorophyll/imidazole
complexes are negatively charged in an apolar environment
a,b
Alia , J. Matysik , C. Erkelens , F.B. Hulsbergen , P. Gast , J. Lugtenburg ,
a
a
a
b
a
a,
*
H.J.M. de Groot
a
Leiden Institute of Chemistry, Gorlaeus Laboratory, Leiden University, P.O. Box 9502, 2300 RA Leiden, Netherlands
b
Department of Biophysics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, Netherlands
Received 25 April 2000; in ®nal form 21 August 2000
Abstract
1
5
1
1
1
15
6
00 MHz N-NMR and 2D homonuclear H± H and heteronuclear H± N-NMR data for bacteriochlorophyll
1
5
a/imidazole and chlorophyll a/imidazole have been recorded. Unambiguous assignments of the N signals of the two
nitrogens of imidazole ligated to the Mg of the bacteriochlorophyll a or chlorophyll a were obtained. It follows that the
imidazole in complex is deprotonated, which implies that the chlorophyll/imidazole carries a full negative charge in the
apolar solvent environment. This information is potentially useful in characterizing the nature of the magnesium±
histidine interaction and the charge state of chlorophylls coordinated by histidine in photosynthetic pigment protein
complexes. Ó 2000 Elsevier Science B.V. All rights reserved.
1
. Introduction
Recently, the ®rst MAS NMR results on the
ground state electronic structure of histidine imid-
azole side chain nitrogens were obtained from
bacterial reaction centers that were labelled with
Interaction of histidine with magnesium has
been suggested in all bacteriochlorophyll-protein
complexes with known structures [1±3]. The spec-
troscopic evidence for this interaction is mainly
based on resonance Raman studies on bacterial
antenna complexes which show that histidine res-
idues in the apoprotein are liganded to Mg at the
centre of the bacteriochlorophyll a (BChl a). These
studies are con®rmed by in vitro Raman experi-
ments on BChl a in the presence of substituted
imidazole [4].
15
15
s- N or p- N histidine [5]. These results were
surprising, since evidence was found that BChl a/
histidine complexes in the reaction centres are
carrying a spinless charge. However, the assign-
15
ment of N chemical shifts of histidines in pig-
ment protein complexes such as reaction centres of
photosynthetic bacteria (BRC) and plants (PSII) is
15
dicult. In particular, the N chemical shifts for
imidazole nitrogens of histidines coordinating with
magnesium of chlorophylls are not known.
To provide a solid basis for the membrane
protein complex studies, a model compound study
is performed. The assignment of the magnesium±
imidazole N-NMR resonance in the chlorophyll
a (Chl a)/imidazole and the BChl a=imidazole
*
Corresponding author. Fax: +071-274537.
E-mail address: ssnmr@chem.leidenuniv.nl (H.J.M. de
15
Groot).
0009-2614/00/$ - see front matter Ó 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 9 - 2 6 1 4 ( 0 0 ) 0 1 1 1 7 - 9

3
26
Alia et al. / Chemical Physics Letters 330 (2000) 325±330
complex in THF is obtained. The surprising result
of the studies is that due to deprotonation of the
imidazole, both the Chl a=imidazole and BChl
a=imidazole complexes are negatively charged.
azole. The identity and purity of the product were
1
con®rmed by H-NMR by comparison with an
authentic sample of 1-methylimidazole.
2
.3. Isolation of pigments
2. Materials and methods
BChl a and bacteriopheophytin a (BPheo a)
were extracted from cells of Rhodobacter sphaero-
ides (R26) and puri®ed by normal phase HPLC as
described previously [7]. Chl a and Pheophytin a
(Pheo a) were extracted from spinach and puri®ed
using the method described in [8].
1
5
2
.1. Synthesis of [ N
2
] imidazole
The procedure of Gridnev and Mihaltseva [6]
15
was followed for synthesizing ‰ N Šimidazole with
2
slight modi®cations. In brief, 12.2 ml of 40%
aqueous solution of glyoxal, 8.1 ml of 37% aque-
2.4. Complex formation
15
ous solution of formaldehyde, 11.1 g NH Cl and
4
1
9
0 drops of 35% HCl were mixed for 90 min at
5°C. The mixture was ®rst cooled and subse-
For the preparation of complexes of imidazole
or 1-methyl imidazole with BChl a, BPheo a, Chl a
or Pheo a pigments, the doubly enriched imidazole
or 1-methyl imidazole was co-dissolved with one
of the pigments as described by Robert [9]. Imid-
azole concentrations were between 3 and 300 mM
in tetrahydrofuran (10% d8) at 25°C. A pigment
concentration of 4 mM was found to be optimal
and was used for all experiments.
quently KOH was added slowly (in ca. 15 min)
dropwise until a pH ˆ 14 was reached. The ®nal
reaction mixture was evaporated to dryness at 40±
50°C by using a rotary evaporator. The brown
solid product was scraped o€ the wall of the ¯ask
and transferred to a vacuum sublimation appara-
15
tus where the crude [ N
2
]imidazole sublimed away
readily at ca. 125°C under vacuum. The crude
To obtain the imidazole anion, imidazole
(80 mM) was reacted with equimolar concentration
of tetrabutylammonium hydroxide-30 hydrate
15
[
which yielded highly pure white, crystalline
N
2
]imidazole was recrystallized from toluene
15
[
N
the starting ammonium salt.
2
]imidazole. The yield was ꢀ53% based upon
(C16
H
37NO Á 30H
2
O). The resulting mixture was
dissolved in THF (10% d8) and the water was re-
moved by using molecular sieves (3A, 8±10 MESH;
Acros Organics, New Jersey, USA). The complex
of anion imidazole with BChl a was made by ti-
trating the anion imidazole with BChl a in THF.
15
2
.2. Synthesis of [ N ] 1-methylimidazole
2
1
5
0
:5 g ‰ N Šimidazole in 30 ml anhydrous THF
2
was added under inert conditions to a 100 ml,
completely dried three-necked ¯ask, previously
2.5. NMR measurements
¯
ushed with argon, and equipped with a mechan-
ical stirrer, an additional funnel, and a condenser.
.6 ml of 1.6 M butyl lithium was added to the
ask with the help of a syringe under inert con-
All NMR spectra were recorded at 25°C with a
Bruker 600 MHz DMX NMR spectrometer
equipped with a pulsed ®eld gradient accessory
(Bruker, Karlsruhe, Germany). 2D homonuclear
H± H and heteronuclear H± N experiments
were performed with a 5 mm inverse triple high
4
¯
ditions. To the resulting mixture, 1 ml of methyl
iodide was added dropwise and the mixture was
re¯uxed for 3 h at 70 °C. The reaction mixture was
then transferred to a 250 ml round bottom ¯ask
and the solvent was evaporated by using a rotary
evaporator at 40±50°C. The resulting residue was
1
1
1
15
resolution probe with an actively shielded z-gra-
dient coil. For 1D N experiments a 5 mm
15
15
broadband high resolution probe was used
chemical shifts are referenced using an external
N
15
distilled twice which yielded highly pure ‰ N Š
2
15
15
1
-methylimidazole as a colorless liquid. The yield
Šimid-
standard of NH4 NO saturated in water at 22.3
3
15
1
was ꢀ25%-based upon the starting ‰ N
2
ppm in reference to liquid ammonia. H chemical

Alia et al. / Chemical Physics Letters 330 (2000) 325±330
327
shifts were calibrated using the THF signal at 1.73
ppm down®eld from tetramethylsilane (TMS) as
an internal standard.
Fig. 1a (Scheme 1). The signal from free imidazole
is observed as a single resonance at 210 ppm due to
the tautomerization reaction, which is very fast at
ambient temperature [12]. Two additional reso-
nances are observed at 255 and 197 ppm, respec-
tively.
The response at 255 ppm is most easily assigned
to a lone pair imidazole (>N:). This is in agree-
ment with the >N: shifts reported in the literature
[13]. In that case, the signal at 197 ppm should be
assigned to the other nitrogen of imidazole coor-
dinating the magnesium of BChl a (>N±Mg). A
reversed assignment is unlikely, since the signal at
1
15
The H± N correlations were determined by
measuring heteronuclear multiple-bond correla-
tion (HMBC) [10] spectra at 25°C. Two dimen-
1
1
sional gradient selected
H± H correlation
spectroscopy (COSY) [11] spectra of the complex
were recorded with a relaxation delay of 1 s. The
NMR spectra were processed with X-WINNMR
(
Bruker) on Silicon Graphic Indy workstations.
3. Results and discussion
197 ppm is well outside the range expected for
protonated imidazole nitrogen (>NH). The range
for imidazole nitrogens is between 177 ppm for
>NH and 186 ppm, when the proton participates
15
The 600 MHz N-NMR spectrum of the BChl
a/imidazole complex in THF at 25°C is shown in
in a strong hydrogen bond ꢁ>NH±X† [13]. As
shown in Fig. 1b, co-dissolving imidazole with
BPheo a only yields the response at 210 ppm from
the imidazole in tautomeric equilibrium. Since
2‡
BPheo a does not contain an Mg ion in its
center, the data in Fig. 1b suggest that Mg is im-
portant for ligation of imidazole with BChl a.
Hence, the resonance 197 and 255 ppm, respec-
tively, arise solely from the bound imidazole.
These results also con®rm that the chemical shift
at 197 ppm arise from nitrogen coordinated with
magnesium.
15
Fig. 1. The 600 MHz one-dimensional N-NMR spectrum of
imidazole (enriched to 95% N at both nitrogens) complexed
15
with BChl a (A) or BPheo (B) in THF (10% d8) at 25°C. For the
N-NMR spectrum 5000 scans with 0.3 s repetition time were
15
measured. The numbering system of the imidazole ring is in-
dicated in Scheme 1.
Scheme 1.

3
28
Alia et al. / Chemical Physics Letters 330 (2000) 325±330
The resonances from the bound imidazole ring
Ã
Ã
are clearly seen and are designated H , H and
Ã
2
4
H . The strong resonances from the free imid-
5
2 4 5
azole are indicated as H ; H and H . The as-
1
1
signments were con®rmed by a standard H± H
Ã
Ã
COSY experiment (data not shown). The H , H
Ã
2
4
and H signals of bound imidazole were used for
the assignment of two N signals of bound imid-
azole in the HMBC spectrum. The N chemical
shift of free rapidly tautomerizing imidazole at
5
15
15
2
H
2
10 ppm exhibits strong correlations with H and
15
4;5. For the bound imidazole, the N signal at
Ã
Ã
255 ppm exhibits correlations with the H and H
signals and therefore it is assigned to N of
2
4
3
15
bound imidazole. The N signal at 197 ppm ex-
Ã
Ã
hibits a correlation with H and H and it is as-
2
5
1
signed to N which is coordinated to the Mg of
1
5
1
the BChl a. No N± H correlation could be
obtained for any bound imidazole with BPheo a
1
5
1
Fig. 2. 600 MHz N± H HMBC spectrum of imidazole (en-
riched to 95% N at both nitrogens) complexed with BChl a in
15
15
1
THF (10% d8) at 25°C, showing the cross-peaks between pro-
ton and nitrogens of bound imidazole. * resonance of H in the
imidazole ring bound with BChl a.
(data not shown). Thus, the N± H correlation
spectra led us to an unequivocal assignment of
the two nitrogens of imidazole in complex with
BChl a.
1
The same set of experiments was repeated with
the complex of plant Chl a in complex with imid-
azole. The data are summarized in Table 1. The
1
5
To further characterize the N resonance of
bound imidazole, a 2D HMBC data set was
1
5
1
15
measured. Fig. 2 shows the N± H HMBC
spectrum of BChl a/imidazole in THF solution.
The trace on top of the dataset shows the
spectrum from the BChl a/imidazole complex.
N-NMR spectrum of plant Chl-imidazole com-
plex also shows two distinct peaks from the bound
imidazole, at 198 and 257 ppm, respectively. These
chemical shifts are almost the same as the chemical
1
H
Table 1
N Chemical shifts of imidazole and its derivatives complexed with BChl a and Chl a
15
1
5
Compound
Nitrogen type
N chemical shifts
ppm)
(
Imidazole
Imidazole-BChl a complex
Tautomer
>N±Mg
>N:
210
197
255
198
257
249
249
196
255
156
257
156
257
N1
N3
N1
N3
N1
N3
N1
N3
N1
N3
N1
N3
Imidazole-Chl a complex
>N±Mg
>N:
>N:
Anionic imidazole
>N:
>N±Mg
>N:
a
Anionic imidazole -
BChl a complex
1
-Methylimidazole
> N±CH
>N:
3
1
-Methylimidazole
BChl a
> N±CH
>N:
3
+
a
Formed by reacting imidazole with an equimolar concentration of tetrabutylammonium hydroxide.

Alia et al. / Chemical Physics Letters 330 (2000) 325±330
329
shifts obtained with the BChl a/imidazole complex.
The resonances at 198 and 257 ppm were not ob-
served in a Pheo a/imidazole mixture (data not
of the magnesium-histidine interaction in many
photosynthetic pigment protein complexes. From
our results, it appears that when the imidazole
nitrogen is bound to Mg, the other nitrogen is
deprotonated, and the complex carries a net neg-
ative charge. In a way, the Mg can replace the
role of the proton in a free and neutral imidazole
ring. Mg is the smallest ion that can be put into the
macroaromatic porphyrin-type system, which may
explain why it is possible to generate such a com-
plex of imidazole and chlorophyll in an apolar
environment. This is interesting, considering the
importance of the chlorophyll-imidazole structural
motif in light harvesting and charge separation in
photosynthesis.
1
shown). An unambiguous assignment of N and
N was again made on the basis of cross-peaks
3
2
‡
of these nitrogens with H and H or H in 2D
2
4
5
15
1
N± H-HMBC spectra of the Chl a/imidazole
complex. Resonances at 257 and 198 ppm were
similarly assigned to N containing a lone pair of
electron (>N:) and N ±Mg in Chl a, respectively.
3
1
These results show that the chemical shift dif-
ference between imidazole nitrogens which bear a
hydrogen (>NH) [13] and those which bear no
hydrogen and coordinating with magnesium (>N±
Mg) is about 30 ppm. Our model studies put
a question mark on the assignment inferred by
Zysmilich and McDermott [14]. These authors
suggest that for bacterial reaction centres, the
chemical shift di€erence between the imidazole
nitrogen responses of histidines coordinating with
bacteriochlorophyll and a protonated N±H is
Acknowledgements
This work was partially supported by a PIO-
NIER award to HJMdG from the Netherlands
Organization for Scienti®c Research (NWO). Alia
acknowledges the section earth and life science of
NWO (ALW) for a visiting scientist grant. J.M.
thanks the European Commission for a Marie
Curie fellowship (ERB4001GT972589).
ꢀ
58 ppm.
In a control experiment, the one nitrogen of
imidazole was blocked with a methyl group in
15
methylimidazole. We have co-dissolved ‰ N Š-1-
2
methylimidazole and BChl a. No complex could
be formed between the free nitrogen of 1-methyl
imidazole and the magnesium of BChl a (Table 1).
In another set of control experiments, a stable
complex of imidazole with BChl a was formed in
two steps. First an anionic imidazole was pre-
pared by transferring the proton of the imidazole
nitrogen to a tetrabutylammonium hydroxide
counterion. Subsequently, the anion imidazole
References
[
1] G. MacDermott, S.M. Prince, A.A. Freer, A.M. Haw-
thornthwaite-Lawless, M.Z. Papiz, R.J. Cogdell, N.W.
Isaacs, Nature 374 (1995) 517.
[2] J. Deisenhofer, O. Epp, K. Miki, R. Huber, H. Michel,
Nature 318 (1985) 618.
15
[3] B. Matthews, R.E. Fenna, M.C. Bolognesi, M.F. Schmid,
J.M. Olson, J. Mol. Biol. 131 (1979) 259.
was titrated with BChl a in THF. The N-NMR
spectra of the resulting complex showed again
15
N
[4] B. Robert, M. Lutz, Biochim. Biophys. Acta 807 (1985)
chemical shifts of 197 and 255 ppm, correspond-
ing with nitrogens of imidazole coordinating the
magnesium of BChl a ꢁ>N±Mg) and imidazole
nitrogen not bearing a hydrogen (>N:), respec-
tively (Table 1). It is not unlikely that in case of
normal BChl a/imidazole complex the proton of
the imidazole is transferred to free imidazole
pool.
1
0.
[5] C. Soede-Huijbreghts, J.J. Cappon, G.J. Boender, J. Raap,
P. Gast, A.J. Ho€, J. Lugtenburg, H.J.M. de Groot, in: G.
Garab (Ed.), Photosynthesis Mechanisms and E€ects,
Kluwer Academic Publishers, 1998, p. 759.
[
6] A.A. Grindnev, I.M. Mihalseva, Synth. Commun. 24
(
1994) 1547.
[7] T.A. Egorova-Zachernyuk, A. Remy, A. Schkuropatov,
P. Gast, A. Ho€, K. Gerwert, H.J.M. de Groot, Vib.
Spectrosc. 19 (1999) 347.
Our results provide an assignment of the mag-
[
8] T. Omata, N. Murata, Plant Cell Physiol. 24 (1983)
093.
15
nesium-imidazole N-NMR resonance in Chl a/
imidazole and BChl a/imidazole complex. This can
serve as a useful model for identifying the nature
1
[
9] B. Robert, Ph.D. Thesis, Universit ꢀe Pierre et Marie Curie,
Paris, 1983.

3
30
Alia et al. / Chemical Physics Letters 330 (2000) 325±330
[
[
[
10] A. Bax, M.F. Summers, J. Am. Chem. Soc. 108 (1986) 2093.
11] R.E. Hurd, J. Magn. Reson. 87 (1990) 422.
[13] S.O. Smith, S. Farr-Jones, R.G. Grin, W.W. Bachovchin,
Science 244 (1989) 961.
12] A.N. Nesmeyanov, E.B. Zavelovich, V.N. Babin, N.S.
Kochetkova, E.I. Fedin, Tetrahedron 31 (1975) 1461.
[14] M.G. Zysmilich, A. McDermott, J. Am. Chem. Soc. 118
(1996) 5867.