The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases
The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases
P. S. Lopes-de-Oliveira
Vadim R. Viviani
Gabriele V. M. Gabriel
Vanessa R. Bevilaqua
A. F. Sim?es
OPEN Firefly luciferases produce yellow-green light under physiological and alkaline conditions, however at acidic pH, higher temperatures or in the presence of heavy metals the color changes to red, a property called pH-sensitivity. Despite many decades of studies, the proton and metal binding sites responsible for pH-sensitivity remain enigmatic. Previously we suggested that the salt bridge E311/ R337 keeps a closed conformation of the luciferin phenolate binding site. Here we further investigated the effect of this salt bridge and mutations of the neighbor residues H310 and E/N354, on metal and pH-sensitivity of firefly luciferases emitting distinct bioluminescence colors (Cratomorphus distinctus: 548 nm; Macrolampis sp2: 569 nm). The substitutions of H310 and E/N354 modulate metal sensitivity, whereas the carboxylate of E311 may work as the catalytic base essential for green bioluminescence and pH-sensitivity. Modeling studies showed that H310, E311 and E354 side-chains coordinate Zinc, constituting the metal binding site and the pH-sensor. Electrostatic potential and pKa calculations suggest that the external couple H310/E354 is affected by pH, whereas E311/R337 make a stabilized internal pair which retains excited oxyluciferin ejected proton near its phenolate group into a high energy state, promoting yellow-green bioluminescence. Protonation or metal binding weaken these electrostatic gates and their ability to retain the excited oxyluciferin released proton near its phenolate, promoting red light emission.
Luciferases are the enzymes which elicit the beautiful yellow-green flashes of fireflies during summer nights
around the word. They catalyze an ATP-dependent oxidation of a benzothiazolic luciferin1,2. Whereas the
normal bioluminescence color produced by firefly luciferases under physiological and alkaline conditions is usually
yellow-green, at lower pH, in the presence of heavy metals such as Zn2+, Ni2+ and Hg2+ or at high
temperatures, the color changes to bright orange-red, a property that has been called pH-sensitivity or pH-dependency3,4.
Although many studies during the last decades have attempted to elucidate the mechanisms of bioluminescence
color determination in beetle luciferases, the specific structural targets and mechanism of pH-sensitivity have
remained elusive. Because firefly luciferases and their genes are widely used as bioanalytical reagents, reporter
genes and biosensors5,6, and more recently are emerging as novel intracellular ratiometric biosensors for pH and
heavy metals ions7,8, there is a lot of interest to better understand the origin of pH-sensitivity.
The color of bioluminescence depends on both the chemical structure of the emitter and on the surrounding
luciferase active site microenvironment. The emitter, excited oxyluciferin, has potentially 6 forms including
tautomers and anionic species9?11. Most recent experimental and theoretical studies indicate that the keto-phenolate
form is the most likely emitter11,12, but the enol-phenolate and enolate-phenolate forms were also proposed as
potential emitters13?15. Three general mechanisms have been quoted to explain bioluminescence color
determination by the luciferase active site7: (I) non-specific solvent and polarizability effects16,17; (II) specific acid-base
and electrostatic interactions of active site residues with excited oxyluciferin9,18 and (III) the conformation of the
active site affecting the rigidity of the microenvironment and the rotation of thiazinic rings of excited
oxyluciferin19. Among the specific interactions, the presence of bases near the thiazinic side of the luciferin binding site
assisting the tautomerization between a keto and enol forms has been originally claimed to explain green to red
bioluminescence color change in firefly luciferases9,13. More recently, interactions influencing the resonance forms
of excited oxyluciferin20, specific acid-base and finally electrostatic effects around oxyluciferin 6? phenol group are
being considered to determine bioluminescence colors18,21,22.
More than 30 beetle luciferases have been already cloned, sequenced and investigated in the past 20 years23?35,
most of them from fireflies. The three-dimensional structures have been solved for the North-American firefly
luciferase Photinus pyralis (Ppy) in the absence of substrates36, in the presence of bromophorm37 and with the
C-terminal trapped in a closed conformation38, for the Japanese L. cruciata firefly luciferase complexed with the
luciferyl-adenylate analogue DLSA in a closed conformation and with oxyluciferin and AMP in an open
conformation39, and finally, for Lampyris turkanensis luciferase40. The luciferin binding-site is well conserved among
different beetle luciferases and consists mainly of the following residues and segments (P. pyralis numeration):
R218, the motif 244HHGF247, the loop 314SGGAPLS320, and the ?-hairpin motif 340YGLTETTS34741?43.
In firefly luciferases, several single-point mutations in the luciferin binding site and in other regions located far
from the active site resulted in red mutants41?46, whereas a few of the respective mutations in the pH-insensitive
click beetle and railroadworm luciferases affected the bioluminescence color47?50, indicating that the active sites
of pH-sensitive firefly luciferases is somehow less rigid than that of pH-insensitive luciferases50. Mutation of
the luciferin binding site residues, including histidines H244 and H245, which supposedly could assist
oxyluciferin tautomerization in the thiazolyl part of the active site, resulted in red mutants in firefly luciferases41?43.
However, H245 is invariant and H244 is conserved in beetle luciferases displaying distinct bioluminescence
colors, therefore none of them could play the role of basic residues assisting tautomerization of excited
oxyluciferin13. Furthermore, studies with the 5,5-dimethylluciferin-adenylate indicated that there is no need of C5 proton
abstraction and tautomerization of oxyluciferin for pH-sensitive spectral changes20. Interactions of main-chain
amide bonds with oxyluciferin phenolate44 and the active site conformation, were also proposed to be important
for bioluminescence colors.
Noteworthy, a group of mutations which affected spectra in both pH-sensitive and pH-insensitive luciferases
was found to be clustered in the loops 223?23551,52 and 351?36033,53. The motif 227Y(F/V)GN(T)229 in the loop
223?235 was shown to be critical for bioluminescence colors and pH-sensitivity. In the loop 351?360, mutation of
E354, which is conserved in the great majority of firefly luciferases, affected thermostability54 whereas the natural
substitution E354N was shown to be responsible for the red-shifted, broader and more pH-sensitive
bioluminescence spectrum in Macrolampis sp2 firefly luciferase33. These loops, participate in a network of polar interactions
with E311 and R33720,53 (Fig.?1), in the same bromophorm binding cavity described by Franks et al.37, leading to
a first suggestion that this network could be the pH-sensor of firefly luciferases52. In this amphiphilic cavity, E311
and R337 are located internally close to oxyluciferin phenolate, and the residues H310 and E354 are located more
externally. We recently provided evidences that the salt bridge between E311 and R337 is essential to stabilize a
closed hydrophobic green emitting conformation in firefly luciferases55. However, the specific function of these
residues and the identity of the specific targets responsible for pH- and metal-sensitivity remain unsolved.
Considering that H310, E311 and E354 display both prototropic and metal chelating side-chains, we decided
to investigate whether they are involved in metal and pH-sensitivity. The mutation of H310 and N354 by
other residues with metal chelating groups in Macrolampis firefly luciferase was recently shown to affect the
metal-sensitivity in firefly luciferases8, indicating that this region is indeed critical for metal-sensitivity in firefly
luciferases. Here we compared the effect of natural and artificial substitutions of these residues on metal- and
pH-sensitivities of three Brazilian firefly luciferases displaying different bioluminescence colors (Amydetes
vivianii: 539 nm; Cratomorphus distinctus: 548 nm and Macrolampis sp2: 569 nm; Fig.?2), modelled this region with
Zinc ion, and calculated the associated electrostatic potential and pKas, providing convincing evidences that this
region indeed constitutes the metal binding site and main pH-sensing moiety of firefly luciferases.
Firefly luciferases bioluminescence spectra display different pH-sensitivities. When
comparing the bioluminescence spectra of four firefly luciferases displaying different colors and pH-sensitivities (Fig.?2:
Amydetes vivianii, 538 nm; Cratomorphus distinctus, 548 nm; Photinus pyralis, 558 nm, and Macrolampis, 564 nm),
the bioluminescence spectrum of Amydetes vivianii luciferase was the most blue-shifted with the lowest amount
of red light at pH 8.0, and is the less sensitive to pH, whereas that of Macrolampis sp2 luciferase has the broadest
one with largest amount of red light even at pH 8, with Photinus pyralis and Cratomorphus disctinctus luciferases
displaying intermediate values (Fig.?2). According to the solvent effect on the fluorescence spectra of luciferin
and derivatives16,17, the results indicate that the active site of Amydetes luciferase is the most hydrophobic for both
green and red emitters at pH 8 and 6. Recent quantum yield measurements by Ando et al.56 showed that the pH
affects only the quantum yield of green emission, whereas the quantum yield of red emission is almost unaffected.
Thus, we can assume that in other firefly luciferases too, only the amount of green emitter changes at different
pHs. The results reinforce our previous proposal that in firefly luciferases the emission spectra are determined
mainly by the ratio of green and red emitters52, although both green and red emitters may also experience slightly
different environments in each luciferase, influencing the overall bioluminescence spectrum.
Firefly luciferases display different metal sensitivities. Considering that the structural targets of
sensitivity to pH, metal ions and temperature must be the same, and that firefly luciferases display different degrees
of pH-sensitivity, we compared the effect of Zn2+, as a representative metal ?on, on the bioluminescence spectra
of the above firefly luciferases and their mutants. Similarly to the pH sensitivity, quantum yield measurements
showed that the binding of these metal ions to P. pyralis luciferase induce decrease of the quantum yields of the
green emission component, resulting in the remaining red emission spectra57.
C. distinctus luciferase, similarly to Photinus pyralis luciferase, showed a larger red shift than Macrolampis sp2
luciferase in the presence of Zn2+ (Fig.?3). The luciferase of Amydetes firefly was much less to this metal (results
not shown). Therefore, among these luciferases, that of C. distinctus was the most sensitive to these metal ions.
Effect of E354 and H310 mutations on bioluminescence spectra and pH-sensitivity. Considering
H310 and E354 are located close to E311 and R337, and that natural substitutions and mutations of E354 were
shown to affect bioluminescence spectra and pH-sensitivity, we investigated the effect of mutations by residues
which lack or replace the acid-base side chains of E354 and H310 in firefly luciferases pH-sensitivity. All mutants
of H310 and E354 (H310A, H310C, N354C, N354H, N354E), independently of being substituted by acid-base or
non-acid-base residues, still displayed pH-sensitivity (Fig.?3; Table?1). The wild-type C. distintus luciferase, was
more sensitive to pH than Macrolampis luciferase, which naturally displays the substitution E354N. The most
pH-sensitive mutants of Macrolampis firefly luciferase were N354C> N354E > H310C/N354C > N354H > WT.
Whereas the mutant H310A has little effect on the bioluminescence spectrum of Macrolampis sp2 luciferase at
pH 8.033, the mutation H310A in Cratomorphus luciferase, resulted in a considerable broadening and red-shifting
of the spectrum (results not shown). The absence of effect of H310A in Macrolampis sp2 luciferase, which
naturally has the substitution E354N, and the broadening effect of H310A in Cratomorphus luciferase, which has
E354, supports the existence of a critical stabilizing interaction between H310 and E354 in Cratomorphus
luciferase (and perhaps P. pyralis luciferase) which may stabilize a closed green-emitting conformation, differently
from Macrolampis sp2 firefly luciferase which lacks the interaction and produces a broader and red-shifted
emission. Furthermore, the mutation of H310R in Macrolampis sp2 luciferase, which inserts a permanent positive
charge in this region, was previously shown to result in a very broad and red-shifted spectrum20, indicating that a
positive charge at this position change the distribution of emission toward the red.
Altogether, these results indicate that although mutations the positions 310 and 354 influence spectral
distribution, they are not determinants of pH-sensitivity.
H310 and N354 are metal-sensitive sites. Considering that substitutions of E354 affect the
bioluminescence spectrum and pH-sensitivity of firefly luciferases, we decided to investigate whether this position is
also involved with metal sensitivity. The mutant Mac-N354E in Macrolampis firefly luciferase displayed a larger
red-shift in presence of Zn2+, similar to that observed for the wild-type C. distinctus luciferase that naturally
displays the substitution N354E (Fig.?3; Table?1). The reverse mutant, E354N in C. distinctus luciferase, had opposite
effect, undergoing a smaller red shift, similar to that observed for the wild-type Macrolampis sp2 that displays
N354 at this position. These results indicate that the position 354 is indeed important for metal sensitivity.
Since in Cratomorphus distinctus and Photinus pyralis luciferases E354 is at hydrogen bonding distance to
H310, potentially making a salt bridge, and histidines have strong affinity to divalent metals, we also investigated
whether the mutation of H310 affects metal sensitivity. The mutant H310A8, in which the metal chelating
imidazole side-chain is removed, indeed was much less sensitive to metal ions, indicating that H310 is also important
to bind metal ions such as Zn2+. These results prompted us to investigate the effect of other substitutions of
H310 and N354 by residues with metal chelating side-chains such as His, Cys or Glu on the metal-sensitivity
of Macrolampis firefly luciferase, resulting in a recently published paper in which we produced mutants with
different sensitivities to Zn2+, Hg2+ Cd2+ and Ni2+ which could be used to ratiometrically estimate these metal
Altogether, the above results clearly indicate that H310 and especially E354 are important sites for
metal-sensitivity. However, the fact that the mutation of these residues by residues with non-chelating side-chains
does not completely abolish metal-sensitivity, indicate that these residues are not the only targets for metal
binding, and that there must be at least another metal?binding group in the neighborhood.
Structure of the metal binding site. Thus, in order to see whether the divalent heavy metal ions can
bind to H310, E354 and surrounding region, modeling studies of beetle luciferases docked with Zn2+ were done
(Fig.?4). Indeed, Zn+2 complexed with H310 N and carbonyl groups, with the side-chains of?N354 in Macrolampis
firefly luciferase or E354 in Cratomorphus firefly luciferase,?and furthermore with E311 carboxylate, supporting
the experimental results, showing that H310, E311 and E/N354 constitute the metal binding site.
Role of the salt bridge between E311 and R337. Previously, we showed that the salt bridge between
E311 and R337 is essential to stabilize the closed hydrophobic conformation in firefly luciferases, and that
mutations which removed the charge or decreased the size of the side-chains, resulted in red light emission in firefly
luciferases54. In order to investigate whether the salt bridge between E311 and R337 has just a conformational
effect on bioluminescence color, or whether these residues display additional catalytic effects on bioluminescence
color, we inverted the identity of these residues by constructing the double mutant E311R/R337E. Such double
mutant is expected to keep the salt bridge, and therefore a closed conformation. If the color of bioluminescence
was related exclusively to the maintenance of the closed conformation, we would expect that the double mutant
would also produce yellow-green light at pH 8.0, such as the wild-type enzyme. However, the double?mutant
produced weak red bioluminescence (601 nm; Table?1). These results clearly indicate that green emission does not
depend exclusively on a closed conformation kept by the salt bridge, but rather on additional specific interactions
that such residues may play, especially E311.
E311 is the main proton binding site. The above structural and site-directed mutagenesis results
indicate that E311 display a key role in bioluminescence color. The mutants E311A and E311Q, in which the negative
charge was removed, besides being the most red-shifted ones, are totally pH-insensitive and completely lost the
bioluminescence activity at pH 6.0. The mutant E311R completely lost the activity, indicating that charge reversal
is incompatible at this position. Only the mutants E311D and R337K, in which the side chains were shortened,
but the charge preserved, still displayed a considerable degree of pH-sensitivity: at pH 6.0 E311D spectrum shifts
about 17 nm to the red and R337K about 10 nm (Fig.?5). These results indicate that E311 negative charge is
essential for green-yellow bioluminescence and also for pH-sensitivity in firefly luciferases. Furthermore, in contrast
to R337 mutants, which independently of charge removal or reversal still produce weak red bioluminescence
activity, the charge reversal of E311 (E311R), or removal (E311Q and E311A) under acidic pH, completely
abolished the bioluminescent activity. Altogether, these results provide compelling evidences that E311 is the
primary base responsible for green bioluminescence, and for proton and metal binding responsible for pH-?and
metal-sensitivityies, playing also a catalytic role in bioluminescence.
Bioluminescence spectra of mutants with 6? Amino-luciferin. Previously we showed that the
6?-amino-luciferin, which has the 6? hydroxil group substituted by an amino group, is a pH-insensitive luciferin
analog which is useful to probe the oxyluciferin phenolate binding site microenvironment polarity21. Therefore,
in order to check whether the mutations of H310 and N354 cause substantial polarity changes, we analyzed the
bioluminescence spectra of this analog with mutants of these residues. However, the bioluminescence spectra
peaks for all the mutants were very close to each other (Table?1), indicating that the microenvironment polarity is
quite similar and is not considerably affected by these mutations.
pKa analysis of the side-chains of pH-sensor. In order to better understand the involvement of the
above residues and their mutants in pH-sensitivity, we calculated the pKa of the identified residues in the
wild-type luciferases and mutants, and correlated them with the electrostatic potential and bioluminescence
spectra at different pHs and in the presence of metals (Fig.?6; Table?2 Supplemental Materials).
Among the wild-type residues, H310 was the one which displayed the largest shift of pKa value in relation
to the reported value in water (~7.0), displaying a much more basic value (7.85 for Macrolampis luciferase and
8.95 for Cratomorphus luciferase). The pKa values for E311 and R337 were 4.53 and 13, respectively, close to the
expected values in water.
As expected, the mutation of these residues affected the pKa of nearby side-chains. Only R337 pKa (13.0) was
insensitive to mutations of nearby residues. Among the side-chains that had their pKa values affected by nearby
mutations, again the imidazolium side-chain of H310 was the most sensitive site, changing several pH units upon
mutations of E311 and R337, whereas the pKa of E311 carboxylate had its pKa only slightly changed upon the
mutations of H310 and N354, but much less upon mutation of R337. The largest effects on the pKa of H310 were
observed upon the mutations E311A and E311Q which removed the negative charge and decreased the value
more than 3 pH units, below 5.4, whereas the mutation E311D, which preserved the negative charge, but with
shorter side chain, also considerably lowered the pKa of H310. Altogether, these results are fully consistent with
the need of the negative charge of E311 carboxylate to counterbalance the positive charge of R337 guanidinium
group. Upon the removal of the negative charge of the E311 carboxylate by the mutations E311A or E311Q, the
H310 imidazolium group becomes much more acidic, apparently to loose the extra positive charge in the
neighborhood of the unshielded R337 guanidinium ion. Mutation of R337K, which preserved the positive charge but
shortened the side chain, also had considerable lowering effect of H310 pKa. In agreement with the above results,
charge reversal upon the mutation R337E resulted in a considerable increase of pKa of H310, which is consistent
with the need of an additional neighbor positive charge in H310 to counterbalance the extra negative charge of
E337. Altogether the results clearly indicate that all these side-chains are close enough to influence each other,
especially E311 and R337 strongly influencing H310.
Electrostatic potential calculation. To verify the effects of microenvironment in pH sensor residues,
we calculated the electrostatic and van der Waals potential for each one of four putative residues considering the
solvent effects. It can be observed that the positions 310 and 354 have similar contributions for pH-sensor site,
whereas positions 311 and 337 display opposite potentials (Fig.?6-psloA). Furthermore, taking the total potential
of four residues and the difference of these potentials at pH 6 and 8, unveil a clear relationship between increasing
potential difference and a larger spectral shift, reinforcing the role of these residues on pH sensitivity.
To find the main potential components involved in the spectral change, the electrostatic potential for each
candidate residue was combined in all possible pairs for the three wild-type pH-sensitive proteins of this study
(Amydetes?vivianii, Cratomorphus?distinctus, Macrolampis?sp2 firefly luciferases) and comparatively with the
Phrixotrix?hirtus railroadworm luciferase, which is an extreme model of pH-insensitive luciferase which naturally
emits red light. At pH 8.0, of eleven evaluated regression models (Spectrum function as a function of energy), one
model showed the best fitting: energy as the sum of electrostatic potential of the residue pair 310/354 (R2: 0.99)
(Fig. 6_psloB). The green emitting luciferases of Amydetes and Cratomorphus display a stabilized electrostatic
pair E311/R337, as indicated by favorable micro-environment (lowest potential value, about ?900 kJ/mol). Here,
the pair H310/E354 acts as strong acid-base pair, ultimately receiving the proton of phenolic hydroxyl group of
oxyluciferin. In the more red-shifted Macrolampis luciferase, there is just one strong base (E312; equivalent to
position 311 in Photinus pyralis luciferase) which makes an electrostatic pair with R338 (equivalent position to
337). Absence of the second base at position 355 (equivalent to position 354), increased the microenvironment
potential, decreasing the strength to retain the proton ejected from excited oxyluciferin phenolate in the
neighborhood. The red-emitting luciferase of Phrixotrix does not display the electrostatic pair (308/334 corresponding
to 311/337) due the natural substitution of arginine by leucine (L334 corresponding to R337), and the entrance
of this cavity (T307 and N351 corresponding to the couple H310/E354) do not have bases to assist residue E308
(equivalent to position 311) to trap oxyluciferin phenolate released proton. Not surprisingly, this luciferase
displays the worst microenvironment potential (~?500 kJ/mol), with insensitivity to pH changes. Furthermore,
although the mutation L334R slightly blue-shifts the spectrum of Phrixotrix luciferase, its microenvironment
potential remained almost unaffected (?503 kJ/mol). Taken together, these results indicate that measurements of
the microenvironment potential of pair 310/354 can provide a good estimate about pH sensitivity.
The structural identity of the pH- and metal-sensing moiety, as well as the underlying mechanisms of
pH-sensitivity and bioluminescence colors in firefly luciferases, have remained elusive for decades.
Overall charge Overall charge ?max/[Band.]
pH 8.0 pH 6.0 (nm)**
Macrolampis?sp2 WT 7.85
The divalent metal ions such as Zn2+, Cu2+, Hg2+, Pb2+, Cd2+ clearly fit in this amphyphilic cavity,
coordinating especially with E311 and E354 carboxylates, which are located close to each other (3.5 ?), and also with H310
imidazolium in most firefly luciferases (Fig.?4). The imidazole and carboxylate side chains of residues at positions
310 and 354 are responsible for modulating the sensitivity of Macrolampis and Cratomorphus firefly luciferases
to metal ions such as nickel and zinc8. The interaction of the divalent metal ions with these glutamates in most
firefly luciferases disrupt the salt bridges between E311/R337 and H310/E354, unshielding the positive charges
of H310 and especially of R337, polarizing the environment (Fig.?7). Such excess of positive charges increases the
polarity of the oxyluciferin phenolate binding pocket by creating a hydration shell around R337, resulting in red
light emission. Noteworthy, the mutation H310R in Macrolampis luciferase which naturally lacks E354, partially
simulates the effect of the metal, by inserting an additional positive charge in the neighborhood, and thus
broadening and red-shifting the spectra.
Despite their importance for metal sensitivity, neither H310 nor E354 can be considered the only proton and
metal binding sites, since H310 and E354 are not strictly conserved in other firefly luciferases which are also
pH-sensitive, and their mutations by non-acid-base and residues do not abolish either pH-and metal-sensitivity.
The residue H310 is substituted by threonine or valine in some firefly and other beetle luciferases. InMacrolampis
firefly luciferase the lack of E354 negative charge upon the natural substitution E354N is responsible for the
observed weaker metal sensitivity and for the naturally increased proportion of red light observed in its
bioluminescence spectrum in relation to the close C. distinctus and P. pyralis firefly?luciferases. Therefore, other groups
could be important for metal binding, including the main chain amide bond of C/S/T314 which has been already
shown to be located near oxyluciferin phenolate and to be important for bioluminescence color modulation44.
The internal salt bridge between E311 and R337 was previously shown to play a critical role in
bioluminescence color, by keeping a closed hydrophobic conformation favorable for green light emission55. Although we
already suggested that these residues could play additional specific functions besides the salt bridge, the effective
contributions of the salt bridge in keeping a closed conformation, or specific interactions of E311 and R337 in
bioluminescence color determination, remained to be elucidated. Here we showed that the exchange of these
residues in the double mutant E311R/R337E, with the aim to keep the salt bridge and a?closed conformation, resulted
in a red emitting mutant, indicating that green light emission does not depends exclusively on a closed active site
conformation, but also on more specific effects of E311 and R337.
The results shown in this manuscript bring compelling evidences that E311 carboxylate is the main base
responsible for green light emission and pH-sensitivity, and the third metal binding site in firefly luciferases. The
acid-base properties of E311 carboxylate, the residual pH-sensitivity upon its substitution by the conservative
aspartate (E311D), the complete abolishment of pH-sensitivity upon substitution by other residues that lack
negative charge (E311A/Q), provide compelling evidences that E311?carboxylate, most likely through a water
molecule, acts as a main base?in bioluminescence color determination. Although, the estimated pKa value of E311
carboxylate in this environment (~4.5) is apparently lower than the expected value for a critical base mediating
the pH effect between 6.0?8.0, the close proximity to oxyluciferin phenol which acts as a strong acid upon
chemiexcitation, may affect the pKa of E311 increasing the sensitivity of this group to external pH-changes.
Furthermore, the complete abolishment of bioluminescence activity upon charge reversal in the mutant
E311R, and in the negative charge lacking mutants E311A and E311Q at pH 6.0, indicate the unforeseen
possibility of a more integral role of E311 carboxylate in bioluminescence catalysis. All beetle luciferases have E311, and
in most of them the negative charge of E311 carboxylate is counterbalanced by the positive charge of guanidinium
group of residue R337 (only Phrixotrix luciferases do not). According to CTIL (charger transfer induced
luminescence) mechanism, during the chemiexcitation step, the phenolic hydroxyl group of the dioxetanone intermediate
must be necessarily deprotonated to give phenolate, in order to induce intramolecular charge transfer, promoting
its decomposition into carbon dioxide and the excited singlet oxyluciferin which decays with emission of light8.
The carboxylate of E311, assisted by a water molecule, may play the essential role of catalytic base for proton
abstraction of the phenolic hydroxyl group of the dioxetanone intermediate, generating excited oxyluciferin
phenolate during the chemiexcitation step. Because excited oxyluciferin phenol is a much stronger acid (pKa < 1.0)
than E311 carboxylate (pKa ~ 4.5), it is likely that E311 carboxylate will work as a base retaining the proton. Under
such circumstances, E311 carboxylate will be protonated and R337 guanidinium ion will in turn counter stabilize
the negative charge of oxyluciferin phenolate. In contrast, in the electropositive microenvironment of the mutants
E311R, or E311A/Q at pH?6.0, the phenol proton abstraction necessary for the generation of the critical phenolate
ion during the dioxetanone intermediate activation could be halted, severely impacting bioluminescence activity.
At alkaline pH, the electrostatic couple E311/R337, reinforced by the external H310/E354 salt bridge, may
work as a gate which keeps a closed conformation, as well as an electrostatic cushion that keeps the oxyluciferin
ejected proton near its phenolate into a high energy state (Fig.?7). Acidic pH, the presence of heavy metals or
higher temperatures weaken such electrostatic gates, decreasing the force to retain the oxyluciferin excited state
released proton near its phenolate group. The few surrounding organized active site water molecules (Fig.?7)
may escape as hydronium ions and be exchanged with water, polarizing and increasing the mobility of the
microenvironment. Altogether these factors will decrease the energy of excited state resulting in red light
emission. Similarly, in the absence of the external salt bridge between H310 and E354 in some beetle luciferase and
mutants, the more internal?salt bridge between E311 and R337 could be also weakened and the environment
relaxed, increasing the proportion of red light. The lack of these salt bridges is observed in Macrolampis firefly
luciferase which?has E354 substituted by asparagine?and produces a broader bioluminescence spectrum, and in a
more extreme case, Phrixotrix?hirtus railroadworm red emitting luciferase, which also displays asparagine at the
corresponding position 351 and, and?additionally lacks arginine at position 334 (L334 corresponding to R337 of
P. pyralis luciferase), missing both salt bridges corresponding to E311/337 and H310/E354.
Material and Methods
Plasmids and beetle luciferases cDNAs. The cDNAs for Macrolampis sp2 luciferase was originally
inserted in pPro vector (Invitrogen) and then was subcloned into NdeI site of pCold vector (Takara, Japan), the
cDNA of Cratomorphus distincus luciferase in pBluescript vector32, the cDNA of Amydetes vivianii luciferase was
cloned in pSport vector (Invitrogen)35.
Site-directed mutagenesis. The mutants Mac H310A, H310C, N354E, N354C, N354H, E311A, E311Q,
E311D were previously constructed8 by site-directed mutagenesis using an Agilent site-mutagenesis kit (Catalog
200518). The following primers were designed for generating new mutations: (E311R) CT AAT T TG CAC AGA
ATT GCT TCT GG; (R337E) CCA GGT ATA GAA CAA GGA TAT GGG C. To prepare the double mutant
Mac-E311R/R337E we performed mutagenesis using the mutant E311R and the primers Mac-R337E. The
plasmids containing the luciferase cDNAs were amplified using Pfu turbo polymerase and 2 complementary primers
containing the desired mutation, using a thermal cycler (1 cycle 95 ?C; 25 cycles 95 ?C, 30 s; 55 ?C, 1 min and
68 ?C 7 min). After amplification, mutated plasmids containing staggered nicks were generated. The products
were treated with Dpn I in order to digest non-mutated parental plasmids, and used directly to transform E. coli
Luciferase expression and purification. For luciferase expression, transformed E. coli BL21-DE3 cells
were grown in 500?1000 mL of LB medium at 37 ?C up to OD600 = 0.4, and then induced at 18 ?C with 0.4 mM
IPTG during 18 h. Cells were harvested by centrifugation at 2,500 g for 15 min and resuspended in extraction
buffer consisting of 0.10 M sodium phosphate buffer, 1 mM EDTA, 1 mM DTT and 1% Triton X-100, 10% glycerol
and protease inhibitor cocktail (Roche), pH 8.0, lysed by ultrasonication and centrifuged at 15,000 g for 15 min
at 4 ?C. The N-terminal histidine-tagged Macrolampis sp2 luciferase was further purified by agarose-Nickel
affinity chromatography followed by dialysis and anion-exchange chromatography, according to established
procedures54. The concentrations of purified luciferases were between 0.5 and 1mg/mL, and the estimated purity,
according to SDS-PAGE gels were about 90%.
Measurement of luciferase activity. Luciferase bioluminescence intensities were measured using a
AB2200 (ATTO; Tokyo, Japan) luminometer. The assays were performed by mixing 5 ?L of 40 mM ATP/80 mM
MgSO4 with a solution consisting of 5 ?L of luciferase and 85 ?l of 0.5 mM luciferin in 0.10 M Tris-HCl pH 8.0 at
22 ?C. All assays were measured in triplicate.
Bioluminescence spectra. Bioluminescence spectra reported here were recorded in ATTO Lumispectra
spectroluminometer (Tokyo, Japan) with cooled CCD camera whereas the previously reported spectra used the
same equipment or a Hitachi F4500 spectrofluorometer. The bioluminescence spectra reported here using the
CCD-based spectroluminometer are red-shifted in relation to previously reported values measured with a
spectrofluorometer.?Such differences?are likely to be caused?by distortions associated to?the longer scanning time
and photosensitivity correction?in the older?spectrofluorometer in relation to the new equipment. For the in
vitro bioluminescence recorded using the spectroluminometer, 5.0 ?L of luciferases were mixed with 90 ?L of
Homology Modelling. Homology-based models of Phrixotrix hirtus, Pyrearinus termitilluminans, Amydetes
vivianii and Macrolampis sp2. luciferases were constructed using as template the three-dimensional structure of
Luciola cruciata luciferase in the presence of DLSA (PDB code 2D1S) and of oxyluciferin and AMP (PDB code ?
2D1R) as previously described55, Modeller v9.9 was used to align the sequences (using align2d function) and to
construct 200 three dimensional models of each sequence Visualization and analyses of the best model of each
luciferase were performed using PyMol version 1.4.160.
pKa estimation and system neutralization. Initially, side-chain conformations for pH-sensor residues
were optimized using SCWALL method of Optimize function in YASARA61. To achieve theoretical pKa values
for each residues of studied luciferase we used the routine ?Experiment Neutralization? of software YASARA62.
Initially, the hydrogen bonding network was optimized61. The method to predict pKa is based on a empirical
function that uses hydrogen bonds, Coulomb potential and surface accessible area parameters weighted by
experimental data. Electrostatic terms are calculated using Ewald summation63,64 using force field AMBER03. pKa
values for Asp, Glu, His and Lys residues were predicted on the pH 6.0 and 8.0. During model construction,
protonation states were assigned using these rule: Protonate Asp and Glu if the predicted pKa is higher than the pH;
Protonate His if the predicted pKa is higher than the pH and His is not a hydrogen bond acceptor or deprotonate
otherwise. Cys is deprotonated if the selected pH is higher than 8.7. Deprotonate Lys if the predicted pKa is lower
than the pH. After that, simulation box was filled with water molecules, and ions are placed at the positions to
neutralize highest and lowest electrostatic potential residues. A short MD simulation was ran for the solvent, and
water molecules are subsequently deleted until the water density has reached 1.0 g/ml.
Potential Energy calculation. To calculate the potential energy of the selected residues using
parameters from force field Amber03 and cutoff distance of 8.0A. Energies were calculated for wild-type luciferases of
Amydetes, Cratomorphus, Macrolampis and Phrixotrix. Statistical software R65,66 was used to create a script that
processed energy data for each pH-sensor residue. To check the combination of residues that better explained the
spectrum shift, the energies of four residues were combined in pairs and trios, creating eleven microenvironment
putative potentials. Using linear model that relate emission wavelength of a given luciferase against each
microenvironment potential, it was defined the best combination using the explained variance statistic (R2). The results
are plotted using ggplot2 library67.
The proton and metal binding site responsible for pH- and metal-sensitive bioluminescence color change in
firefly luciferases involves mainly the side-chains of H310, E311 and E354 near the oxyluciferin phenolate binding
site. These residues are involved in two critical salt bridges (E311/R337 and H310/E354) which keep oxyluciferin
phenolate binding site electrostatically closed, promoting green light emission. The carboxylate of E311 is the
main base responsible for green light emission and pH-sensitivity, playing also a critical role as a water
mediated?catalytic for bioluminescence activity base, by assisting proton abstraction of the dioxetanone intermediate
phenol group during the chemiexcitation step. Under physiological and alkaline pH conditions, the stable salt
bridge between the residues E311 and R337, aided by the more external salt bridge between E354 and H310 (in
many firefly luciferases), keep a closed hydrophobic active site environment retaining the excited oxyluciferin
released proton near its phenolate group into a high energy state, promoting green light emission. At lower pH or
upon metal binding to H310, E311 and E354, the salt bridge between E311 carboxylate and R337 guanidinium is
disrupted, reducing the ability of E311 to retain the excited oxyluciferin released proton near its phenolate group,
polarizing the environment and thereof resulting in red light emission.
Vadim R. Viviani idealized the research, prepared mutants H310A, H310R, E311A, E311R, E311R/R337E and
Crt-E354E, recorded the bioluminescence spectra of these mutants, prepared Figures 1 and 2, and wrote the
manuscript. Gabriele V. Gabriel prepared and characterized mutants H310C, N354C, N354H and measured
the effect of metals on the bioluminescence spectra of mutants and prepared Figure 3. Vanessa R. Bevilaqua
obtained and analyzed the bioluminescence spectra for the mutants and wild-type luciferases with
aminoanalogs, analyzed bioluminescence spectra and prepared Figure 5. A. F. Sim?es prepared and characterized
mutants E311D, R337K and R337E. Takashi Hirano synthesized amino-analogs, prepared mechanistic Figure 7
and participated on the discussion of paper. Paulo S. Lopes de Oliveira constructed homology-based models of
beetle luciferases complexed with Zinc, calculated the potentials and pKa of the side-chains of the pH-sensor
residues, prepared Figures 4 and 6, participating on the mechanism proposal.
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-33252-x.
Competing Interests: The authors declare no competing interests.
Publisher?s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. The images or other third party material in this
article are included in the article?s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article?s Creative Commons license and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
To FAPESP ( 2013 /09594- 0 ; 2010 /05426-8), CNPq (401867/2016-1) and CAPES?for financial support .