The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases

Scientific Reports, Dec 2018

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.

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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 T. Hirano 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. Results 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 concentrations8. 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. Luciferase Macrolampis sp2 H310A H310C H310R E311A E311Q E311D E311R R337K R337E N354E N354C N354H WT E354N E311R/R337E Cratomorphus distinctus Amydetes vivianii 100 62 31 40 0 58 162 1.5 3.6 75 5.7 100 100 100 573 621 590 576 579 587 610 ? 600 601 ? 604 ? 601 613 590 564 568 555 594 601 601 597 598 597 ? 605 602 601 602 597 610 584 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. Discussion 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. pKa R337 pKa E/ N354 Overall charge Overall charge ?max/[Band.] pH 8.0 pH 6.0 (nm)** Luciferase/Mutant Macrolampis?sp2 WT 7.85 H310A H310C H310R E311A E311Q E311D R337K R337E N354E N354C N354H Cratomorphus E354N pKa H310 ? 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. 1 0 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 XL1-Blue cells. 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 1 1 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. Concluding Remarks 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. 1 2 1 3 Author Contributions 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. Additional Information Supplementary information accompanies this paper at Competing Interests: The authors declare no competing interests. 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To view a copy of this license, visit 1 4 To FAPESP ( 2013 /09594- 0 ; 2010 /05426-8), CNPq (401867/2016-1) and CAPES?for financial support .

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Vadim R. Viviani, Gabriele V. M. Gabriel, Vanessa R. Bevilaqua, A. F. Simões, T. Hirano, P. S. Lopes-de-Oliveira. The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases, Scientific Reports, DOI: 10.1038/s41598-018-33252-x