Role of key residues of obelin in coelenterazine binding and conversion into 2-hydroperoxy adduct

Article abstract of DOI:10.1016/j.jphotobiol.2013.08.012

Bioluminescence of a variety of marine organisms is caused by monomeric Ca²⁺-regulated photoproteins, to which a peroxy-substituted coelenterazine, 2-hydroperoxycoelenterazine, is firmly bound. From the spatial structure the side chains of Tyr138, His175, Trp179, and Tyr190 of obelin are situated within the substrate-binding pocket at hydrogen bond distances with different atoms of the 2-hydroperoxycoelenterazine. Here we characterized several obelin mutants with substitutions of these residues regarding their bioluminescence, coelenterazine binding, and kinetics of active obelin formation. We demonstrate that Tyr138, His175, Trp179, and Tyr190 are all important for coelenterazine activation; substitution of any of these residues leads to significant decrease of the apparent reaction rate. The hydrogen bond network formed by Tyr138, Trp179 and Tyr190 participates in the proper positioning of coelenterazine in the active site and subsequent stabilization of the 2-hydroperoxy adduct of coelenterazine. His175 might serve as a proton shuttle during 2-hydroperoxycoelenterazine formation.

Full text of DOI:10.1016/j.jphotobiol.2013.08.012

Journal of Photochemistry and Photobiology B: Biology 127 (2013) 133–139
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Role of key residues of obelin in coelenterazine binding and conversion
into 2-hydroperoxy adduct
Elena V. Eremeeva a,b,c, Svetlana V. Markova a,c, Willem J.H. van Berkel b, Eugene S. Vysotski a,c,
⇑
a Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia
b Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands
c Laboratory of Bioluminescence Biotechnology, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, Krasnoyarsk 660041, Russia
a r t i c l e i n f o
a b s t r a c t
Bioluminescence of a variety of marine organisms is caused by monomeric Ca2+-regulated photoproteins,
to which a peroxy-substituted coelenterazine, 2-hydroperoxycoelenterazine, is firmly bound. From the
spatial structure the side chains of Tyr138, His175, Trp179, and Tyr190 of obelin are situated within
the substrate-binding pocket at hydrogen bond distances with different atoms of the 2-hydroperoxycoel-
enterazine. Here we characterized several obelin mutants with substitutions of these residues regarding
their bioluminescence, coelenterazine binding, and kinetics of active obelin formation. We demonstrate
that Tyr138, His175, Trp179, and Tyr190 are all important for coelenterazine activation; substitution of
any of these residues leads to significant decrease of the apparent reaction rate. The hydrogen bond net-
work formed by Tyr138, Trp179 and Tyr190 participates in the proper positioning of coelenterazine in the
active site and subsequent stabilization of the 2-hydroperoxy adduct of coelenterazine. His175 might
serve as a proton shuttle during 2-hydroperoxycoelenterazine formation.
Article history:
Received 2 July 2013
Received in revised form 10 August 2013
Accepted 20 August 2013
Available online 28 August 2013
Keywords:
Bioluminescence
Coelenterazine
Obelin
Aequorin
Photoprotein
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
its excited state [7,8]. The excited coelenteramide relaxes to its
ground state with the production of blue light with maxima
Bioluminescence is a widely distributed phenomenon among
marine dwellers [1,2]. Many of these luminous organisms generate
light by oxidation of coelenterazine, an imidazopyrazinone deriva-
tive [3,4]. The chemical mechanism of light emission appears to be
common among coelenterazine utilizing organisms but often dif-
fers in the detailed biochemical process, probably as necessitated
by the behavioral function of bioluminescence [5].
around 465–495 nm depending on the type of photoprotein [9].
Although Ca2+-regulated photoproteins have been detected in
many different marine organisms [3,10], cDNA sequence informa-
tion is only available for five hydromedusan photoproteins, namely
aequorin [11–13], mitrocomin (halistaurin) [14], and clytin (phial-
idin) [15–17] from the jellyfishes Aequorea, Mitrocoma (Halistaura),
and Clytia (Phialidium), and obelins from the hydroids Obelia lon-
gissima [18,19] and Obelia geniculata [20], and for the light-sensi-
tive photoproteins from ctenophore Beroe abyssicola [21],
Mnemiopsis leidyi [22,23], and Bathocyroe fosteri [24]. All Ca2+-reg-
ulated photoproteins contain three calcium-binding consensus se-
quences characteristic of EF-hand Ca2+-binding proteins [25,26].
Apophotoproteins expressed in Escherichia coli can be converted
to active photoproteins by incubating them with coelenterazine
under Ca2+-free conditions in the presence of O2 and reducing
agents [27].
The Ca2+-regulated photoproteins constitute one specific class
of coelenterazine utilizing molecules. They comprise monomeric
polypeptides with a molecular mass of ꢀ22 kDa to which a per-
oxy-substituted coelenterazine, 2-hydroperoxycoelenterazine, is
tightly but non-covalently bound. Since Ca2+-regulated photopro-
teins contain a stable oxygenated reaction intermediate, they are
capable of emitting light in proportion to the concentration of pro-
tein unlike the luciferases where the amount of light emitted is
proportional to the concentration of the substrate luciferin [6]. Bio-
luminescence is triggered upon binding of Ca2+, which induces the
oxidative decarboxylation of 2-hydroperoxycoelenterazine with
generation of protein-bound reaction product, coelenteramide, in
The main application of Ca2+-regulated photoproteins originates
from their ability to emit light upon Ca2+ binding. Photoproteins
have been successfully used to estimate the intracellular Ca2+ con-
centration under steady-state conditions and to study the role of
calcium transients in the regulation of cellular function in different
types of cells [28]. The cloning of cDNAs encoding apophotopro-
teins has granted a new approach in photoprotein applications.
The recombinant apophotoprotein expressed intracellularly forms
⇑
Corresponding author at: Photobiology Laboratory, Institute of Biophysics,
Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia. Tel.: +7
3912494430; fax: +7 391433400.
E-mail address: eugene.vysotski@gmail.com (E.S. Vysotski).
1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2013.08.012

134
E.V. Eremeeva et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 133–139
the active photoprotein upon binding with coelenterazine, which is
added externally and diffuses into the cell. Thus, such cells have, in
effect, a ‘‘built-in’’ calcium indicator. This approach is highly
valuable because it does not require laborious procedures like
microinjection or liposome-mediated transfer and now is widely
used [29–31]. The success of such photoprotein applications,
however, depends on various factors among which are the rate
and efficacy of the generation of active photoprotein from apo-
photoprotein, coelenterazine, and oxygen, as well as any influence
of the cellular environment on these processes.
In the past decade the crystal structures of photoproteins
aequorin [32], obelin [33,34], and clytin [35] as well as obelin li-
gand-dependent conformation states [36–38] have been deter-
mined. Based on these findings and mutagenesis studies of some
substrate-binding cavity residues [39–42] significant insight has
been obtained into the bioluminescence mechanism [9,38,42,43].
At the same time much less is known about the molecular mecha-
nism of active photoprotein complex formation from apophotopro-
tein, coelenterazine and oxygen (Scheme 1). Previous studies
focused primarily on the efficiency of regeneration of wild type
apo-aequorin with coelenterazine derivatives as well as the effects
of temperature, pH, incubation time and some additives on this
process [27,44–46]. It was shown, for example, that dithiothreitol
(DTT) or b-mercaptoethanol are required to reduce disulfide bonds
in the recombinant apo-aequorin [47]. Furthermore, it was deter-
mined that the binding of coelenterazine to apophotoprotein oc-
curs within milliseconds [48], in contrast to the formation of
active photoprotein complex [27]. This finding evidently showed
that the rate-limiting step of active photoprotein formation is the
conversion of coelenterazine into its peroxy derivative.
atoms of the 2-hydroperoxycoelenterazine (Fig. 1). Recently, it
was shown that the hydrogen bond network formed by His175–
Trp179–Tyr190 triad participates in positioning and stabilizing
the 2-hydroperoxy adduct of coelenterazine and that His175 is
critical for the bioluminescence function [49]. In active obelin
His175, Trp179, and Tyr190 are located near the C2 atom of coel-
enterazine, which is the exact position for oxygen to react with
the substrate. Thus, it looks reasonable to assume that these
residues could be involved in the process of active photoprotein
complex formation. Tyr138 is hydrogen bonded to the N1 atom
of 2-hydroperoxycoelenterazine (Fig. 1) and potentially might
effect the charge distribution in the imidazopyrazinone ring of
the coelenterazine molecule via polarizing the N1-nitrogen and
thus the efficiency of the coelenterazine reaction with oxygen.
In this paper we report for the first time the study of the func-
tion of amino acid residues located in the substrate-binding cavity
of Ca2+-regulated photoprotein obelin in the formation of 2-hydro-
peroxycoelenterazine and consequently active obelin. Several
obelin mutants with substitutions of Tyr138, His175, Trp179 and
Tyr190 were characterized regarding their bioluminescence, coel-
enterazine binding and kinetics of active obelin formation.
2. Experimental methods
2.1. Materials
Coelenterazine was obtained from Prolume Ltd. (Pinetop, AZ,
USA). Other chemicals, unless otherwise stated, were from Sig-
ma–Aldrich and the purest grade available.
The substrate-binding cavity of obelin is highly hydrophobic
with addition of several hydrophilic side chains (His22, Tyr138,
His175, and Tyr190) directed internally. The side chains of
Tyr138, His175, Trp179, and Tyr190 are situated within the coelen-
terazine-binding pocket at hydrogen bond distances with certain
2.2. Molecular biology
Site-directed mutagenesis was done on the template pET19-
OL8 E. coli expression plasmid carrying the O. longissima wild type
apo-obelin gene [51]. Mutations resulting in the desired amino
Scheme 1.

E.V. Eremeeva et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 133–139
135
with the instrument. All spectra were taken using a standard
quartz cuvette (1 ꢁ 1 cm) in a 1-mL initial volume with varied
coelenterazine additions in 5- to 10-lL portions up to saturation.
To assess fluorescence quenching only the changes of fluorescence
intensity at 336 nm were used. The fluorescence intensities were
corrected for dilution due to the addition of coelenterazine, for
the methanol influence on Trp fluorescence, scattered light, and
for inner filter effects of protein and added coelenterazine. To eval-
uate the inner filter effects, absorbance measurements were per-
formed at excitation and emission wavelengths and fluorescence
(F) was corrected using the equation:
þA
F ¼ FunceA295 336
ð1Þ
2
where A295 and A336 are absorbance of protein and ligand at excita-
tion and emission wavelengths, respectively, and Func is the uncor-
rected fluorescence.
The apparent dissociation constant of the apophotoprotein-
coelenterazine complex was determined using the quenching of
apophotoprotein Trp fluorescence upon binding to coelenterazine.
Analysis assumes that the fraction of bound ligand is equal to the
ratio of the fluorescence quenching (Q = Fo–Fq) to maximum
quenching (Qmax = Fo–Fqmax), where Fo, Fq, and Fqmax are fluores-
cence intensity at 336 nm measured in the absence of added li-
gand, the quenched fluorescence intensity in the presence of
ligand, and the maximum fluorescence quenching at a saturating
level of ligand, respectively. The apparent dissociation constants
were calculated by fitting the relative fluorescence emission to
Eq. (2), a modified equation compared with the one described else-
where [55]:
Fig. 1. Hydrogen binding mode of 2-hydroperoxycoelenterazine with surrounding
residues in obelin (PDB code 1QV0). Hydrogen bonds (dashed lines) are determined
with the PyMOL program [50].
acid change were carried out using the QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the pro-
tocol supplied with the kit. The plasmids harboring the mutations
were verified by DNA sequencing.
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ðC þ L þ KDÞ ꢂ ðC þ L þ KDÞ ꢂ 4CL
Q
Qmax
¼
ð2Þ
2
2.3. Apophotoprotein preparation
where C, L, and KD are apophotoprotein and coelenterazine concen-
trations, and apparent dissociation constant, respectively.
Apo-obelin and its mutants were expressed and purified as pre-
viously reported [49,52]. The apophotoproteins obtained after
extraction with 6 M urea and purification on a DEAE-Sepharose
Fast Flow column were concentrated by Amicon Ultra Centrifugal
Filters (Millipore). To fold apophotoproteins, the concentrated
samples containing 6 M urea were diluted approximately 20-fold
with a solution containing 1 mM EDTA, 20 mM Tris–HCl pH 7.0,
again concentrated, and then washed several times with the same
buffer to remove any impurities of urea and salts. The apophoto-
proteins were centrifuged (20,000g ꢁ 10 min) at 4 °C, incubated
overnight with 10 mM DTT, again centrifuged, and then passed
through a Superdex 200 column (Amersham Bioscience) equili-
brated with freshly prepared 10 mM DTT, 5 mM EDTA, 20 mM
Tris–HCl pH 7.0 to produce monomeric apophotoprotein contain-
ing no disulfide bonds and aggregates [49]. The final preparations
of apophotoproteins were homogeneous according to SDS–PAGE
and gel filtration. The active wild type obelin and its mutants were
produced as described elsewhere [52,53]. The concentrations of
apo-obelin and its mutant were determined using the correspond-
ing molar extinction coefficients at 280 nm calculated with the
ProtParam tool (http://us.expasy.org/tools/protparam-doc.html)
that uses Edelhoch’s method [54].
2.5. Measurements of bioluminescence and kinetics of
apophotoprotein activation with coelenterazine
The total bioluminescence of obelin and its mutants was mea-
sured using a plate luminometer Mithras LB 940 (Berthold, Ger-
many) by injection of 50
lL of 100 mM CaCl2, 100 mM Tris–HCl,
pH 8.8 into the well containing 100
l
L of 5 mM EDTA, 100 mM
Tris–HCl pH 8.8 and the photoprotein aliquot. The biolumines-
cence signal was integrated during 10 s.
At studies of apophotoprotein activation kinetics the light emis-
sion was measured with a BLM 8801 photometer (SCTB ‘‘Nauka’’,
Krasnoyarsk, Russia) equipped with the temperature-stabilized
cuvette block and neutral-density filters with different transmis-
sion coefficients to extend the dynamic range. The kinetics was
studied at concentrations of apophotoprotein and coelenterazine
equal to 50 nM and 750 nM respectively in air-saturated buffer
5 mM EDTA, 10 mM DTT, 20 mM Tris–HCl pH 6.5. Coelenterazine
was added from a concentrated stock solution in methanol. To
eliminate any effect of methanol on apophotoprotein activation,
the methanol concentration in activation buffer never exceeded
1% (v/v). The coelenterazine concentration in the methanol stock
solution was determined spectrophotometrically using the molar
2.4. Fluorescence measurements and determination of apparent
dissociation constant of the apophotoprotein-coelenterazine complex
absorption coefficient
The bioluminescence was triggered by forceful injection of 10 lL
e
435 nm = 9800 Mꢂ1 cmꢂ1 in methanol [10].
Fluorescence measurements were carried out with a Varian
Cary Eclipse spectrofluorimeter (Agilent Technologies, USA) in
5 mM EDTA, 10 mM DTT, 20 mM Tris–HCl pH 7.0 at 20 °C. Excita-
tion was at 295 nm (slit 5 nm). The apo-obelin fluorescence emis-
sion spectra were corrected with the computer program supplied
aliquot of the mixture containing apophotoprotein and coelenter-
azine taken from the activation solution into the cuvette of the
luminometer containing 490
lL of 10 mM CaCl2, 100 mM Tris–
HCl pH 8.8. The measurements were carried out at 20 °C.

136
E.V. Eremeeva et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 133–139
replaced for serine is converted into active photoprotein in the ab-
3. Results
sence of these agents [56,57].
We found that at 20 °C and pH 6.5 the concentration of active
obelin reaches a maximum after 15 min of incubation with coelen-
terazine (Fig. 3), instead of several hours as reported before [27].
This essential shortening of activation time is conditioned by the
method of apophotoprotein preparation. In our studies, the
apo-obelin was pre-incubated with DTT overnight and then its
monomeric form was isolated by gel filtration on a Superdex 200
column. This procedure allows the removal of apophotoprotein
aggregates [58] that are eventually responsible for the slow
kinetics of apophotoprotein activation. It should be noted that
the pre-incubation with DTT has no noticeable effect on the rate
of apo-obelin activation.
In effect, the formation of 2-hydroperoxycoelenterazine might
be considered as a chemical reaction of coelenterazine with molec-
ular oxygen occurring in a protein environment. The apparent rate
of active photoprotein complex formation and consequently the
apparent rate of coelenterazine conversion into peroxy adduct
can be monitored by the increase of bioluminescence activity of
apo-obelin mixed with coelenterazine in the activation buffer
(Fig. 3). For wild type apo-obelin the apparent rate constant was
evaluated to be 3.17 0.11 ꢁ 10ꢂ3 sꢂ1 at 20 °C and pH 6.5.
Although the H175N and Y190F mutants revealed apparent disso-
ciation constants equaling that of wild type apo-obelin the appar-
ent rates of active photoprotein complex formation for these
mutants were around one order of magnitude slower (Table 1). A
similar decrease in the apparent rate of active apophotoprotein
complex formation was observed for Y138F, Y138W and W179F
(Table 1). Noteworthy is that although Y138W revealed the highest
apparent dissociation constant indicating the lowest affinity to
coelenterazine, the apparent rate of active photoprotein complex
formation was found to be the largest among the studied group
(Table 1).
3.1. Coelenterazine binding with mutant apo-obelins
Most obelin mutants with replacement of either His175,
Trp179, or Tyr190 display very low or no bioluminescent activity
[49]. To test the function of these residues in formation of active
photoprotein complex the most active mutant from each group
(H175 N, Y190F, and W179F [49]) was selected. To reveal the func-
tion of Tyr138, mutant Y138F [38] was taken because this mutant
retains decent bioluminescent activity and evidently does not form
a hydrogen bond with the N1 atom of 2-hydroperoxycoelenter-
azine. Additionally, we constructed the Y138 W mutant because,
according to the crystal structure of wild type obelin, there is en-
ough space at position 138 for a Trp side chain and because its
amide nitrogen might form a hydrogen bond with the N1 atom
of 2-hydroperoxycoelenterazine.
Addition of coelenterazine to apo-obelin induces a strong con-
centration-dependent decrease of tryptophan fluorescence [48].
This property was used to determine the apparent dissociation
constants of the mutant obelin-coelenterazine complexes. The
tryptophan fluorescence quenching data were fitted according to
Eq. 2 (Fig. 2).
The apparent dissociation constants for H175N and Y190F mu-
tants turned out to be almost equal to that of wild type apo-obelin
(ꢀ0.2
Y138F, W179F and especially Y138W (Table 1). For the latter mu-
tant, the apparent dissociation constant amounts to 1.81 0.2
lM). A lowered affinity for coelenterazine was observed for
lM
which is almost one order of magnitude higher than for wild type
obelin.
3.2. Kinetics of active obelin complex formation
Recombinant apophotoproteins can be converted to the active
photoproteins by incubating with coelenterazine under calcium-
free conditions in the presence of oxygen and reducing agents like
DTT or b-mercaptoethanol [27]. It was proposed that the reducing
agent is needed to prevent the formation of disulfide bonds in re-
combinant apophotoproteins which might lead to conformational
constraints hindering coelenterazine binding [47]. The thiol-reduc-
ing agents are not strictly required for the formation of active
photoprotein because an aequorin mutant with all three cysteines
4. Discussion
The side chains of His175, Trp179, and Tyr190 of photoprotein
obelin (Fig. 1) are essential for stabilizing the 2-hydroperoxy ad-
duct of coelenterazine and the subsequent Ca2+-regulated light
emitting reaction [49]. Tyr138 is another obelin residue involved
in 2-hydroperoxycoelenterazine binding (Fig. 1). Substitution of
Tyr138 to Phe causes a small long-wavelength shift in biolumines-
Fig. 2. Quenching of intrinsic apophotoprotein fluorescence by coelenterazine. (A) Scans displaying the effect of coelenterazine on apo-Y138F fluorescence measured
between 315 and 375 nm (a) in absence of coelenterazine; (b) and (c) with 1.22 and 9.15 M of coelenterazine, respectively. Spectra are normalized to the maximum of
l
fluorescence without coelenterazine addition. (B) Determination of the apparent dissociation constants of wild type apo-obelin- (d), apo-Y138F- (N), and apo-Y138 W-
coelenterazine (j) complexes using the quenching of intrinsic fluorescence of apophotoproteins upon coelenterazine binding. The concentrations of apophotoproteins were
1.22 lM. All titrations were performed in triplicate.

E.V. Eremeeva et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 133–139
137
Table 1
Bioluminescence activity, apparent dissociation constants and apparent rate constants of active photoprotein formation of obelin and its mutants.
Bioluminescence activity (%)
Apparent dissociation constant KD, (
lM)
Apparent rate constant k, ꢁ10ꢂ3 sꢂ1
Obelin WT
Y138F
Y138W
H175N
W179F
Y190F
100.0
60.0
57.0
1.7
67.0
14.3
0.2 0.04
0.65 0.06
1.81 0.2
0.15 0.01
1.2 0.07
0.2 0.01
3.17 0.11
0.23 0.02
0.87 0.07
0.37 0.03
0.43 0.05
0.11 0.01
Fig. 3. Kinetics of active photoprotein complex formation from apophotoprotein, coelenterazine, and molecular oxygen monitored by bioluminescence for monomeric wild
type apo-obelin (d), apo-Y138F (h), apo-Y138W (j), apo-H175H (s), apo-W179F (.) and apo-Y190F (}). Each kinetic curve is normalized to its respective maximum of
bioluminescence activity. Apophotoprotein and coelenterazine concentrations were 50 and 750 nM respectively. The activation experiments were carried out at 20 °C and pH
6.5. All kinetic determinations were performed in triplicate. Apparent rate constants were calculated by one-exponential fitting of kinetic curves.
cence maximum but has no significant impact on bioluminescent
activity [38]. To further our insight into initial active photoprotein
complex formation from apophotoprotein, coelenterazine, and
molecular oxygen, we studied selected Tyr138, His175, Trp179,
and Tyr190 mutants regarding their capacity for coelenterazine
binding and conversion into 2-hydroperoxy adduct.
for the bioluminescence because its replacement to Phe does not
eliminate bioluminescence activity; Y138F obelin mutant pre-
serves about 60% of bioluminescence activity of wild type obelin
[38]. However the Y138F substitution clearly influences both the
affinity of apophotoprotein for coelenterazine and the rate of coel-
enterazine conversion into 2-hydroperoxy adduct. The apparent
dissociation constant of the apo-Y138F-coelenterazine complex is
almost 3 times higher and the apparent rate constant of active
photoprotein complex formation is nearly 14 times lower than that
of wild type obelin (Table 1). For Y138W coelenterazine binding is
one order of magnitude weaker than for wild type obelin, indicat-
ing that appearance of the bulky Trp138 side chain within the sub-
strate binding cavity might create a steric hindrance for effective
coelenterazine binding. The rate of active photoprotein complex
formation for Y138W is only ꢀ3.5 times lower than that of wild
type obelin (Table 1). In case of Y138W, the amide function of
the indole ring possibly forms a hydrogen bond with the N1 atom
of coelenterazine thereby mimicking the hydrogen bond formed by
the hydroxyl group of Tyr138 in wild type obelin (Fig. 1). This par-
ticular hydrogen bond is evidently missed in Y138F, allowing the
conclusion that the capacity of Tyr138 to form this bond plays an
important role in coelenterazine activation.
The apparent dissociation and rate constants for W179F are 6
times higher and 7 times lower as compared to those of wild type
obelin (Table 1). Decreasing the dimensions of the hydrophobic
side chain at position 179 influences the geometry and polarity
of the substrate-binding cavity leading to a lower affinity for coel-
enterazine. In obelin, the nitrogen atom of the Trp179 ring is
hydrogen bonded with the carbonyl oxygen of 2-hydroperoxycoel-
enterazine (Fig. 1). It is obvious that absence of this hydrogen bond
in W179F decreases the rate of active photoprotein complex
formation.
The apparent dissociation constants for H175N and Y190F mu-
tants were found to be equal to that of wild type obelin. In contrast,
the apparent rate constants of active photoprotein complex forma-
tion for these mutants are almost 10 and 30 times lower (Table 1).
This clearly shows that these residues are less critical for substrate
binding but strictly important for catalyzing the conversion of
coelenterazine into its 2-hydroperoxy derivative. Based on spectro-
scopic studies of the anaerobic complex of apo-obelin with coelen-
terazine [59] and molecular mechanisms suggested for several
cofactor-independent oxidases and oxygenases according to which
an active site histidine is involved in substrate activation with sub-
sequent insertion of oxygen [60], we proposed that His175 in obe-
lin might function as a proton shuttle in the process of active
photoprotein complex formation from apophotoprotein, coelenter-
azine, and molecular oxygen. Since the H175N substitution slows
down the rate of formation of active photoprotein complex we
can reasonably conclude that this histidine indeed assists in the
oxygenation of coelenterazine and might perform the role of pro-
ton shuttle in this process in the same manner as it occurs in case
of cofactor-independent oxidases and oxygenases [60]. Since the
Y190F substitution also reduces the rate of active photoprotein
complex formation but does not influence the affinity to coelenter-
azine and because the side chains of His175 and Tyr190 might
form hydrogen bonds with each other and with coelenterazine
(Fig. 1) we can also suggest that both residues assist to (or partic-
ipate directly in) the oxygenation of coelenterazine into 2-
hydroperoxycoelenterazine.
Studies of the chemiluminescence reaction of imidazopyrazi-
nones with O2 in aprotic solvents containing base have given a
plausible mechanism for the formation of peroxy derivatives of
Although Tyr138 is hydrogen bonded to the N1 atom of 2-
hydroperoxycoelenterazine (Fig. 1) this residue is not essential

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E.V. Eremeeva et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 133–139
Scheme 2.
imidazopyrazinones [61–63]. According to the suggested reaction
mechanism (Scheme 2), the first step is deprotonation of the N7
of imidazopyrazinone with a base yielding the imidazopyrazinone
anion. Then, single electron transfer (SET) from imidazopyrazinone
anion to triplet oxygen affords the imidazopyrazinone radical and
the superoxide anion of oxygen (Oꢃ2ꢂ). The SET process requiring
spin conversion is considered to be rate-limiting. The radical cou-
pling of imidazopyrazinone radical and superoxide anion leads to
the peroxide anion of imidazopyrazinone. In case of biolumines-
cent reactions catalyzed by luciferases, the cyclization of imidazo-
pyrazinone peroxide anion then will take place. The cyclization
gives dioxetanone which decomposes with loss of CO2 generating
the singlet excited state of a product [63].
mation because substitution of any of these residues significantly
reduces the rate of coelenterazine conversion into its 2-hydroper-
oxy adduct.
Acknowledgements
The work was supported by RFBR grant 12-04-00131, by the
Programs of the Government of Russian Federation ‘‘Measures to
Attract Leading Scientists to Russian Educational Institutions’’
(grant 11.G34.31.0058), ‘‘Molecular and Cellular Biology’’ of RAS,
President of Russian Federation ‘‘Leading science school’’ (grant
3951.2012.4). E.V.E. was supported by Wageningen University
Sandwich PhD-Fellowship Program.
The mechanism of 2-hydroperoxycoelenterazine formation dur-
ing conversion of apophotoprotein into photoprotein, in essence,
might be the same with the only distinction: the coelenterazine
peroxide anion resulting from radical coupling must be quickly
protonated to prevent cyclization into dioxetanone [64]. For in-
stance, a similar protonation of a peroxy adduct is operative in fla-
vin-dependent aromatic hydroxylases [65].
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